g Modern · PDF fileZoology Histology Developmental AnatomyIndividual Experiments ... Plant Molecular Biology ... New Experiments from Modern Biology Inc.! IND-29. Electrophoretic

MODERNBIO.COM

ModernBiology

Teaching

Our systems are used to supplement

your Teaching Laboratory MODERNBIO.COM

The Basic Laboratory Programs ................................................................................................6Structure, Isolation, and Function of DNA ......................................................................................6 1. Properties of DNA .............................................................................................................................................6 2. Cell Fractionation and DNA Isolation ................................................................................................................6 3. Gene Function and Cloning in Bacteria ............................................................................................................6Animal Biology and Evolution .......................................................................................................7 1. and 2. Quantifying Amylase Activity ................................................................................................................7 3. Proteases and Factors that In!uence Enzyme Activity..............................................................................................7 4. Serum Enzymes and Disease ............................................................................................................................7 5. Evolution of Serum Proteins .............................................................................................................................7 6. Identifying a Speci"c Enzyme in Chicken Erythrocytes by Enzyme Cytochemistry ..........................................7Chromatographic Separation of Proteins .......................................................................................8 1. Separating Molecules by Gel Filtration .............................................................................................................8 2. The Molecular Weight of Hemoglobin ..............................................................................................................8 3. Binding Speci"city of Serum Albumin .............................................................................................................8 4. Properties of Amylase ......................................................................................................................................8 5. Analysis of a Protein From Egg White ...............................................................................................................8An Introduction to Plant Cell and Molecular Biology ......................................................................9 1. E#ects of Temperature on Cell Respiration .......................................................................................................9 2. Extraction and Analysis of an Enzyme from Wheat .........................................................................................9 3. Location of an Enzyme in Plant Cells and Tissues .............................................................................................9 4. Osmolarity and a Cytological Bioassay .............................................................................................................9 5. Mitotic Activity and Cell Respiration.................................................................................................................9 6. Isolating DNA from Plants ................................................................................................................................9Introduction to Molecular Biology ............................................................................................... 10 101. Electrophoretic Separation of Proteins .......................................................................................................10 102. Genetics and Sickle Cell Anemia .................................................................................................................10 103. Analysis of Serum Proteins .........................................................................................................................10 104. Evolution of Serum Proteins .......................................................................................................................10 105. A Comparison of Human and Bacterial Amylase ...............................................................................................10 106. Protein Fingerprinting ....................................................................................................................... 10 Individual Experiments ............................................................................................................................. 11 IND 24. An Introduction to Electrophoresis.......................................................................................................11 IND-30 Speci"c Binding of Dyes to DNA .................................................................................................... 11Tissue Speci!c Proteins ............................................................................................................... 12 801. Serum Proteins and the Western Press-Blot ...............................................................................................12 802. Identifying Ovalbumin by the Western Press-Blot ...........................................................................................12 803. Tissue-Speci"c Isoenzymes in the Cow ......................................................................................................12 804. Peroxidase Isoenzymes in Corn ..................................................................................................................12 Individual Experiments ............................................................................................................................. 13 IND-4. Development of the Immune System and the Western Press-Blot. ........................................................13 IND-5. Speci"city of Albumin Binding. ...............................................................................................................13Molecular Biology of Proteins ..................................................................................................... 14 201. Molecular Weight Determination ...............................................................................................................14 202. Identifying Sex-Speci"c Proteins ................................................................................................................14 203. Comparing Human and Bacterial Amylase ................................................................................................14 204. Peptide Mapping Analysis ..........................................................................................................................14 205. Protein Evolution and the Western Blot .....................................................................................................15 206. A$nity Chromatography ...........................................................................................................................15 Individual Experiments ............................................................................................................................. 15 201P. Molecular Weight Determination ............................................................................................................16 IND-11P. Contractile Proteins from Cow Heart ..................................................................................................16 IND-25P. Isolation of Chromosomal Proteins .....................................................................................................16 Electrophoresis Package for SDS Polyacrylamide Gels .......................................................................................16 204P. Peptide Mapping Analysis ........................................................................................................................17 205P. Protein Evolution and the Western Blot ....................................................................................................17 206P. A$nity Chromatography ..........................................................................................................................17 IND-27 Composition of the Nuclesome ..............................................................................................................17Molecular Biology of Nucleic Acids ............................................................................................... 18 301. The Length of DNA Molecules ....................................................................................................................18 302. Restriction Nuclease Mapping of DNA........................................................................................................18 303. Plasmid DNA Structure ...............................................................................................................................18 304. Molecular Cloning ......................................................................................................................................18 305. Identifying Satellite Sequences ..................................................................................................................19 306. The Nucleosome Structure of Chromatin ....................................................................................................19 Individual Experiments ......................................................................................................................................19The New Genetics ....................................................................................................................... 20 1001. Anatomy and Evolution of the Genome ...................................................................................................20 1002. Analysis of a Genome Segment ...............................................................................................................20 1003. DNA Fingerprinting (An authentic analysis) .............................................................................................20 1004. Genotype to Phenotype ...........................................................................................................................21 Individual Experiments ......................................................................................................................................21DNA Hybridization Analysis ......................................................................................................... 22

Table of Contents 401. Evolution of the Vertebrate Genome ..........................................................................................................22 402. Application of the Southern Blot Procedure ...............................................................................................22 403. Detecting a Speci"c Sequence in the Mammalian Genome ......................................................................23 Individual Experiments ............................................................................................................................. 23 IND-16. Identifying Viral DNA by Rapid Southern Blotting ................................................................................23Genetic Engineering ................................................................................................................... 24Tissue to Gene ............................................................................................................................ 26Plant Molecular Biology.............................................................................................................. 27Cloning a DNA Segment from Sheep ............................................................................................ 28Mini Programs for DNA Analysis .................................................................................................. 29 IND-9. Producing a Strain of E.coli that Glows in the Dark ....................................................................................29 IND-6. Analysis of a Mutant Hemoglobin Gene .................................................................................................29 IND-7. Ampli"cation of a Hemoglobin Gene by the Polymerase Chain Reaction (PCR) .....................................30 IND-10. PCR Ampli"cation of a Gene for Ribosomal RNA from Di#erent Organisms .........................................30Mini Programs for DNA Analysis .................................................................................................. 30 IND-21. Identifying Genomic and Plasmid DNA Sequences in E.coli by Colony PCR .........................................31 IND-12. Characterization of the Satellite DNA from the Meal Worm..................................................................31 IND-13. Transformation and Analysis of Yeast ....................................................................................................32 IND-15. Bacterial Phenotype .............................................................................................................................32 IND-28. Synthetic Biology: Using Bacterial Computers to Solve the Pancake Problem ......................................32Contemporary Cell Biology ......................................................................................................... 33 701. Enzyme Cytochemistry ...............................................................................................................................33 702. Analysis of a Cell-Surface Receptor ............................................................................................................33 703. The Cell Nucleus ..........................................................................................................................................33Mini Programs in Cell Biology ...................................................................................................... 34 IND-2. Tissue Printing .........................................................................................................................................34 IND-3. The ELISA Immunoassay .........................................................................................................................34 IND-22. Student Designed Research Projects: Characterization of Peroxidases in Plants ..................................35 IND-29. Electrophoretic and Chromatographic Analysis of Photosynthetic Pigments from Blue- Green Algae 35 IND-17. A Rapid Immunological Method to Study Evolution .............................................................................36 IND-14. Enzyme Kinetics ....................................................................................................................................36Zoology Histology Developmental Anatomy ................................................................................ 37 IND-18. An Introduction to Zoology ...................................................................................................................37 IND-19. An Introduction to Vertebrate Histology ...............................................................................................37 IND-20. An Introduction to Developmental Anatomy ........................................................................................37 IND-26. Localizing Tublin by Immunohistochemistry ........................................................................................38The Overview Programs .............................................................................................................. 39 A Molecular Approach to the An Introduction to Electrophoresis ......................................................................39 An Introduction to Molecular Genetics...............................................................................................................39 An Introduction to Electrophoresis .....................................................................................................................39A Laboratory Course in Innovative Biology .................................................................................. 40A Laboratory Course in College-Level Biology ..........................................................................42A Laboratory Course in Cell Biology ........................................................................................43A Laboratory Course in Molecular Biology ...............................................................................44A Comprehensive Laboratory Course in Molecular Biology .......................................................45A Laboratory Course in Cell and Molecular Biology ..................................................................46A Computer Course in DNA and RNA Sequence AnalysisBiology .................................................47Equipment ...........................................................................................................................48 Electrophoresis Equipment in the Teaching Laboratory ............................................................................. 48 Electrophoresis Equipment in the Teaching Laboratory ............................................................................. 49 Electrophoresis Equipment for the Entire Class .......................................................................................... 50 Accessories For Sample Handling .............................................................................................................. 51 Balances and Mixers .................................................................................................................................. 52 Spectrophotometer, Centrifuges and Dry Baths ........................................................................................ 53 Water Baths and Stir Plate ......................................................................................................................... 54 Incubator and Shaker ................................................................................................................................ 55 Microscopes .............................................................................................................................................. 56 Thermal Cycler and Orbital Blotter ............................................................................................................ 57 Small Equipment and Labware ................................................................................................................. 58 Supplies and Expendables ......................................................................................................................... 59 Individual Products and Kits for Teaching and Research ............................................................................ 60 Products for DNA Analysis ......................................................................................................................... 61Ordering Information and Policy ............................................................................................63Order Form ...........................................................................................................................64

On the CoverCover painting of DNA by Dr. Robert T. Schimke, Professor Emeritus, Standford University, Department of Biological Sciences. Read more about the artist on the inside back cover.

1

Mechanical Adjustable Volume PipettorOur mechanical adjustable pipettors o!er accurate and precise sampling and dispensing of liquid volumes. The pipettors come in a variety of ranges to meet your needs. All pipettors have been quality tested according to ISO 8655/DIN 12650 and come with a one year warranty.Features:

Easy to calibrate and maintainEjector collar and tip cone can be removed for easy cleaning and maintenanceDigital display clearly reads volume settingSupplied with full documentation, warranties and certi"cates

PIPETTOR PACKAGE1 Pipettor Stand & 4 Pipettors of your choice

Micropette™ Pipettor StandLinear Pipettor Stand, holds up to 6 pipettors.Item No. 71000085Reg. Price $40.00

$378.00Equipment Packages!

Order Now!

See Page 51 For More Information

For the Value of $512.00

New Experiments from Modern Biology Inc.!IND-29. Electrophoretic and Chromatographic Analysis of Photosynthetic Pigments from Blue-Green Algae $65.28

Cyanobactera, also known as blue-green algae, obtain their energy by photosynthesis using sunlight as their energy source. These organisms have been considered to be the oldest and the most important bacteria on the earth. It is believed that they were responsible for the initial oxygenation of the earth's atmosphere through photosynthesis and it is also felt they were the precursors to the chloroplasts that are found in true algae and plants. In part A of this exercise, students prepare a water-soluble extract from blue green algae and show that it contains the single major protein Phycocyanin by electrophoresis as shown

in the gel below. They also determine the charge of this protein by comparing its electrophoretic mobility to the mobilities of dyes with known charges. In part B, they prepare an alcohol extract and analyze the smaller alcohol soluble pigments by thin layer chromatography in order to identify the chlorophylls and major carotenoid pigments. The results of this two-part study give students practical hands-on experience with isolation of components from cells as well as electrophoresis and thin layer chromatography and introduces them to one of the most important organisms on the earth.

See Page 35 For More Information

IND-30. Speci!c Binding of Dyes to DNA $19.99Proteins that bind to DNA control the processes of gene regulation and DNA replication. The electrophoretic mobility band shift assay is a common technique used to study these proteins and to study speci"c protein-DNA interactions. In this exercise, students use this assay to identify dye molecules that bind to DNA and attempt to determine the mechanism by which these drugs interact with the DNA molecule.

CAT NO. CAPACITY PRICE720080 2-20ul $118720020 5-50µl $118720070 20-200µl $118720060 100 - 1000µl $118

4

1. Individual Experiments Begin

ning

Inter

mediat

eAd

vanc

ed

Cell

Biolo

gyMol

ecula

r Biol

ogy

Gene

tics

Microb

iolog

yAn

imal

Studi

es

Plan

t Stud

iesEv

olutio

n / Im

munol

ogy

Human

Dise

ases

Stude

nt Des

igned

PageNo Electrophoresis Properties of DNA 6 Cell Fractionation and DNA Isolation 6 Gene Function and Cloning in Bacteria 6 Evolution of Serum Proteins 7 Effects of Temperature on Cell Respiration 9 Analysis of an Enzyme from Wheat 9 Producing E. coli that Glows in the Dark 29 Transformation and Analysis of Yeast 32 Bacterial Phenotypes 32 Digestive Enzymes 7 Analysis of a Cell-Surface Receptor 33 The Cell Nucleus 33 Tissue Printing 34 The ELISA Immunoassay 34 Immunological Methods and Evolution 36 Enzyme Kinetics 36 Zoology / Histology 37 Developmental Anatomy 37 Enzyme Cytochemistry 33 Analysis of a Cell-Surface Receptor 33 DNA and RNA Sequence Analysis 47 Evolution of the Vertebrate Genome 22 Localizing Tubulin by Immunohistochemistry 38Non-Denaturing Protein Electrophoresis (Electrophoresis Package 1/8 - Page ) Electrophoretic Separation of Proteins 10 Genetics and Sickle Cell Anemia 10 Analysis of Serum Proteins 10 Binding of Dyes to DNA 11 Human and Bacterial Amylase ௗ 10 Protein Fingerprinting 10 Pigments in Blue Green Algae 35 Protein Evolution and the Western Blot 12 Tissue-Specific Isoenzymes in the Cow 12 Peroxidase Isoenzymes in Corn 12 Immune System and the Western Blot 13 Specificity of Albumin Binding 13SDS-Denaturing Protein Electrophoresis (Electrophoresis Package 2 & 2M - Page ) Molecular Weight Determination 14/16 Human and Bacterial Amylase 14 Peptide Mapping Analysis 14/17 Protein Evolution and the Western Blot 14/17 Affinity Chromatography 15/17 Contractile Proteins from Cow Heart 16 Isolation of Chromosomal Proteins 16/17

Highly Recommended Recommended

Suitability for your Teaching Laboratory

TopicLevel

SDS-Denaturing Protein Electrophoresis (Electrophoresis Package 2 & 2M - Page 15)

Non-Denaturing Protein Electrophoresis (Electrophoresis Package 1/8 - Page 11)

5

DNA Electrophoresis Begin

ning

Inter

mediat

eAd

vanc

ed

Cell

Biolo

gyMol

ecula

r Biol

ogy

Gene

tics

Microb

iolog

yAn

imal

Studi

es

Plan

t Stud

iesEv

olutio

n / Im

munol

ogy

Human

Dise

ases

Stude

nt Des

igned

Page(Electrophoresis Package 3/4 - Page ) The Length of DNA Molecules 18 Restriction Nuclease Mapping of DNA 18 Plasmid DNA Structure 18 Molecular Cloning 18 Identifying Satellite Sequences 19 Nucleosome Structure of Chromatin 17/19 Anatomy and Evolution of the Genome 20 Analysis of a Genome Segment 20 DNA Fingerprinting 20 Genotype to Phenotype 21 Analysis of a Mutant Hemoglobin Gene 29 Amplification of a Hemoglobin Gene by PCR 30 PCR Amplification of Ribosomal RNA Gene 30 Identifying Sequences in E.coli by Colony PCR 31 Satellite DNA from the Meal Worm 31 Application of the Southern Blot Procedure 22 A DNA Sequence in the Mammalian Genomeௗ 23 Identifying Viral DNA by Rapid Southern Blotting 23

2. Experiments with a Related Theme Structure, Isolation, and Function of DNA 6 Animal Biology and Evolution ௗ 7 Chromatographic Separation of Proteins ௗ 8 Plant Cell and Molecular Biology ௗௗ 9 Introduction to Molecular Biology 10 Tissue-Specific Proteins 12 Genetic Engineering 24 Tissue to Gene 26 Molecular Biology of Proteins 14 Molecular Biology of Nucleic Acids 18 Plant Molecular Biologyௗ 27 Cloning a DNA Segment 28 Peroxidases in Plants 35 DNA Hybridization Analysis 22 The New Genetics ௗ 20 Contemporary Cell Biology 33 Human Genetics and Disease 39 Introduction to Molecular Genetics 39 Introduction to Electrophoresis 39 Synthetic Biology 32

3. College-Level Laboratory Courses General Biology 42 An Introduction to Cell and Molecular Biology 46 Cell Biology 43 Molecular Biology 44 Comprehensive Molecular Biology 45 A Laboratory Course in Investigative Biology 40

Highly Recommended Recommended

Suitability for your Teaching Laboratory

TopicLevel

DNA Electrophoresis (Electrophoresis Package 3/4 - Page 19)

6

This program is used to explore some of the major concepts and techniques that have revolutionized the biological sciences during the past two decades. Without the use of hazardous chemicals, students obtain an introduction and practical experience with the structure and isolation of DNA, cell fractionation, and gene cloning procedures.

The Basic Laboratory ProgramsGENERAL FEATURES

The four basic programs are supplied complete with all of the laboratory manuals, chemicals, and accessories needed for beginning students to perform exciting experiments in modern biology. Electrophoresis equipment is not required in all four of the basic programs. All programs are designed to be used by 16 students working in pairs. In most cases, each experiment can be completed within a 90 minute laboratory session.

CAT. NO. DESCRIPTION PRICE

Price List - Basic Laboratory Program 1B1 The Chemical Package for 16 students $149.99

working in pairs plus one student and one instructor manual (The student manual may be reproduced for educational purposes).

B1-BK 8 Student Manuals $48.12 B1-SM 1 Student Manual and 1 Instructor Manual $6.87

Single Experiments O"ered From Basic Program 1B1-1 Properties of DNA $54.48B1-1F Chemical re"ll for (B1-1) Properties of DNA $35.55

Contains DNA solution and DNAse B1-2 Cell Fractionation and DNA Isolation $54.48B1-3 Gene Function and Cloning in Bacteria $73.34

Multi Section Lab Experiments o"ered from Basic Program 1 Each MSL (Multi Section Lab) experiment provides one set of reusable items and four sets

of chemicals which can be used to teach 4 Sections of 16 students working in pairs.

B1-2 MSL Cell Fractionation and DNA Isolation $107.00

EXPERIMENTS1. Properties of DNAThe DNA molecule from a single human chromosome is about 4 cm long and the length of DNA in an individual is about 200 times the distance from the earth to the sun. Isolated DNA in a test tube is also a long, stiff molecule. When alcohol is added to a DNA solution, the DNA fibers precipitate and can be spooled onto a glass rod. This feature of DNA is illustrated in the exercise, which provides enough purified DNA for 16 students working in pairs to perform the experiment. The spooling phenomenon is also used by the student to study the double-stranded nature of DNA and to investigate the effect of breaking DNA into small pieces with the enzyme DNAse. Ethyl alcohol (90-100%) is needed but not provided.

2. Cell Fractionation and DNA IsolationStudents isolate nuclei from calf thymus tissue and examine them microscopically. The DNA is then extracted from the nuclei by a simple procedure that uses a detergent and alcohol. Microscopes and a small centrifuge are desirable but not absolutely necessary for the exercise.

3. Gene Function and Cloning in BacteriaOccasionally, E.coli cells are found in nature that are resistant to the toxic effects of the antibiotic ampicillin. In this exercise, students create such an ampicillin-resistant population of E.coli by introducing into bacterial cells a plasmid that contains an ampicillin-resistance gene. Sufficient sterile materials are supplied for sixteen platings. The introduction of plasmid DNA into bacterial cells is called transformation and has enabled scientists to obtain large quantities of more than 1000 genes including those for human interferon, insulin, and growth hormone. This exercise requires minimal teacher preparation and provides a laboratory experience with one of the most important techniques used in gene-cloning studies.

Materials Provided

*19 Sterile Tubes 8 Tube Racks Nuclear Stain *20 Petri Dishes 8 Glass Vials Sodium Dodecyl *24 Inoculating Loops 8 Glass Rods Sulfate 2 Macropipetors Nuclear Buffer *Nutrient Agar- *30 Sterile Transfer Pipets *Plasmid DNA Ampicillin 10 Small Transfer Pipets Cheese Cloth *Nutrient Broth Calf Thymus DNA *CaCl2 DNAse I E.coli Calf Thymus Tissue Ampicillin*Provided sterile and ready to use

Structure, Isolation, and Function of DNABasic Laboratory Program 1

The programs are intended to introduce advanced secondary and beginning college students to contemporary topics including enzyme action, DNA structure and function, molecular evolution, and DNA cloning. A full-semester course in college-level biology was created from experiments in the programs and is described on page 43 of this catalog.

A waterbath is optional. See page 54.

BASI

C PRO

GRAM

S

7

This collection of experiments was prepared for the novice to help bridge the gap between traditional animal physiology and the biochemistry of cell processes in animals. Following the step-by-step procedures in their laboratory manuals, students will study a variety of topics including the action, properties, and cell location of enzymes, and the evolution of mammalian proteins.

EXPERIMENTS



working in pairs plus one student and one instructor manual (The student manual may be reproduced for educational purposes).

B2-BK 8 Student Manuals $48.12 B2-SM 1 Student Manual and 1 Instructor Manual $6.87B2-CR Chemical Re"ll Package for Basic Program 2 $149.77

Single Experiments O"ered From Basic Program 2B2-5 Evolution of Serum Proteins $56.79

(Previous catalog number was B2-4)

1. and 2. Quantifying Amylase ActivityThis exercise begins with a discussion of the digestive enzymes produced by the salivary glands, stomach, intestine and pancreas. The digestive system of a rodent is provided to familiarize students with anatomical features of the digestive organs of a mammal. Students are then given the opportunity to prepare enzyme extracts from pancreatic tissue and to determine the amount of amylase in the extracts and in their own saliva. This exercise offers practical experience with enzyme extraction procedures and introduces the concept of the calibration curve for determining the levels of a specific enzyme in a complex mixture.

In the figure above, starch was incorporated into an agar gel, and samples containing amylase were placed in sample wells made in the agar. After one day, the starch was stained with iodine. Areas where the starch was degraded by amylase are seen as clear rings around the wells and the diameters of the rings are proportional to the concentration of amylase.

3. Proteases and Factors that Influence Enzyme ActivityStudents contrast the effects of pH on the activities of gastric pepsin and pancreatic chymotrypsin and relate their results to the acidic pH of the stomach and neutral pH of the small intestine. The effects of temperature and thermal denaturation on enzyme activity are also investigated.

4. Serum Enzymes and DiseaseTissue injury often results in the leakage of large amounts of cellular enzymes into the circulatory system and measurements of the amounts and types of enzymes in serum are frequently made for the diagnosis of human disease. For example, inflammation of the pancreas is accompanied by the release of pancreatic amylase and proteases into the blood stream. In this exercise, students determine the levels of amylase and proteases in serum and compare these levels to those found in saliva and pancreatic extracts.

5. Evolution of Serum ProteinsEach protein carries in its amino acid sequence information pertaining to its evolutionary history and origin, and provides clues to the evolutionary history of the organism in which it is found. Indeed, proteins existing today are in effect living fossils.

Animal Biology and EvolutionBasic Laboratory Program 2

Chemical Package Contents 8 Tube Racks Digestive System Potassium Iodine 50 Small Test Tubes (from rat or mouse) Casein Solution 60 Transfer Pipets Pepsin Chymotrypsin 20 Petri Dishes Extraction Buffer Chicken Serum 2 Macropipetors Pancreatic Powder Horse Serum (2 tubes) 16 Large Test Tubes Amylase Cow Serum Antibodies 20 Pasteur Pipets Pancreatic Extract Cow Serum 8 Chicken Blood (4 tubes) Sheep Serum Smears Starch Solution Goat Serum 3 Plastic trays/lids Agar Donkey Serum Methylene Blue Gel Buffer NP-40 Eosin LDH Substrate

A centrifuge is helpful but not necessary

This concept is illustrated in this exercise where students examine the ability of antibodies against cow serum proteins to react with serum proteins from cow, sheep, goat, horse, donkey, and chicken. The results of this study show that sheep and goat are most closely related to cow which is in agreement with classical taxonomy.

6. Identifying a Specific Enzyme in Chicken Erythrocytes by Enzyme CytochemistryThe concept that different enzymes are found in different regions of a cell is illustrated in this exercise. Here, students use the technique of enzyme cytochemistry to show that the enzyme lactate dehydrogenase is located in the cytoplasm of chicken erythrocytes. The exercise introduces students to the basic structure of animal cells and provides an illustration of an important technique used in modern cell biology. Eight chicken blood smears are provided for the experiment and microscopes are required.

BASIC PROGRAMS

8

Chromatographic Separation of ProteinsBasic Laboratory Program 3

Gel filtration is a chromatographic method used to separate molecules of different sizes. This technique is an important tool for isolating and characterizing proteins. In this series of experiments, students use this method along with basic enzyme and antibody assays to study the structure and function of proteins.

EXPERIMENTS 1. Separating Molecules by Gel FiltrationStudents fill their chromatographic columns with packing material and separate three colored molecules of known molecular weights as shown in the photograph. The exercise illustrates how components of a complex mixture can be separated by chromatography and introduces the concept of the standard curve for determining the size of an unknown protein.

2. The Molecular Weight of HemoglobinStudents determine the size of hemoglobin and estimate the number of amino acid residues in this protein.

3. Binding Specificity of Serum AlbuminThe binding of an enzyme to its substrate is only one example of the many specific molecular interactions that occur in biological systems. An analogous binding process occurs with serum albumin which binds certain small molecular weight molecules in blood. In this exercise, students use a gel filtration assay to examine the binding of various dyes to albumin and determine if the native shape of the protein is required for its binding specificity.

4. Properties of Amylase Gel filtration chromatography has the advantage of being able to yield the size of different proteins in a complex mixture. This feature is illustrated in this experiment where students compare the molecular weight of amylase from pancreas and saliva.

Materials Provided



working in pairs plus one student manual and one instructor manual (The student manual may be reproduced for educational purposes.)

B3-BK 8 Student Manuals $48.12B3-SM Sample Student Manual (43 pages) plus one $6.87

instructor manual.B3-CR Chemical Re"ll Package for Basic Program 3 $152.73

Single Experiments O"ered From Basic Program 4B3,1-3 Mini Program in Chromatography $127.90

Includes chemicals and instructions for experiments 1-3. This series can be completed in two

2-3 hour laboratory sessions.

8 Collection Plates70 Small Test Tubes60 Transfer Pipets20 Petri Dishesand2 Macropipetors16 Large Test Tubes20 Pasteur Pipets8 Tube racksCheese clothSDS

Watch a video of this experiment in action at MODERNBIO.COM

5. Analysis of a Protein From Egg WhiteStudents first isolate ovalbumin, a major protein found in egg white. They then use gel filtration and a simple immunological procedure to determine the size of this protein and the amount of it in the egg.

Chemical Package Contents

8 Chromatographic Columns (column tubes, reservoirs, lower fittings, bed supports caps)Column Packing MaterialColumn BufferPhenol RedMyoglobin-Blue DextranCytochrome C-HemoglobinLysis Buffer

Rabbit BloodBromophenol BlueCow Serum Albumin(2)AgarStarch SolutionPotassium IodineAmylasePancreatic ExtractOvalbuminOvalbumin Antibodies

BASI

C PRO

GRAM

S

9

Recent discoveries in the plant sciences have illustrated the potential of modern techniques for the production of genetically engineered plants that are resistant to pathogens, herbicides, and adverse environmental conditions. A solid foundation in basic plant cell and molecular biology is required in order for students to participate in this green revolution. The novel experiments in this program are intended to provide this background. In the program, students extract and characterize enzymes and DNA from plant tissues, localize enzymes in plants at the tissue and cell levels, and analyze fundamental process common to all cells including mitosis, osmosis, and cell respiration. The program is ideal for introducing the beginning student to basic biological principles and plant biology and can serve as a supplement to elementary laboratory courses in botany, plant physiology, and agriculture. Microscopes, slides, cover slips, a tabletop centrifuge, ethyl alcohol, and hydrochloric acid are needed, but not provided.

1. Effects of Temperature on Cell RespirationCell respiration can be viewed as a series of enzyme catalyzed reactions in which carbohydrates, proteins, and fats are broken down to carbon dioxide and water with the release of energy. During the process, hydrogen is removed from the fuel molecules and oxygen is consumed. With this background information, students measure oxygen consumption and hydrogen liberation in germinating barley at different temperatures. The program provides eight calibrated respirometers for measurement of oxygen consumption and the chemicals required to perform a graphic dye reduction assay. The exercise introduces students to a fundamental biological process and provides insight into seed structure and germination.

2. Extraction and Analysis of an Enzyme from Wheat Acid phosphatase is present in many plant tissues where it catalyzes the removal of phosphate groups from macromolecules at low pH. In this exercise, students prepare a cell-free extract from wheat germ and determine the amount of the enzyme present in the extract. The experiment offers practical experience with enzyme extraction procedures and is an excellent introduction to the analysis of enzyme activity and basic enzyme kinetics. A colorimeter is desirable but not absolutely necessary for this exercise.

3. Location of an Enzyme in Plant Cells and TissuesFlowering plants are multicellular organisms and different enzymes are frequently produced in different tissues and in different cell types. This concept is illustrated in the first part of the exercise where students use an enzyme histochemical procedure to show that peroxidase is produced by cells that comprise a layer in the corn seed called the aleurone as shown in the figure below. In the second part of the exercise, a similar procedure is used by the student to show that peroxidase is found in the cell wall of onion epithelial cells.

4. Osmolarity and a Cytological BioassayIn this exercise, osmosis is examined in living onion epithelial cells which are used in a cytological assay to determine the osmolarity of an unknown sucrose solution provided with the program.


Price List - Basic Laboratory Program 4B4 The Chemical Package for 16 students working in $193.12

pairs plus one student and one instructor manual. B4-BK 8 Student Manuals $48.12B4-SM Sample Student Manual plus one instructor manual. $6.87B4-CR Chemical Re"ll Package for Basic Program 4 $152.73

Single Experiments O"ered From Basic Program 4B4-1 E!ects of Temperature on Cell Respiration $64.79B4-2 Extraction and Analysis of an Enzyme from Wheat $63.73

Multi Section Lab Experiments o"ered from Basic Program 1 Each MSL (Multi Section Lab) experiment provides one set of reusable items and four sets

of chemicals which can be used to teach 4 Sections of 16 students working in pairs.B4-1 MSL E!ects of Temperature on Cell Respiration $120.06B4-2 MSL Extraction and Analysis of an Enzyme from Wheat $117.47

Basic Laboratory Program 4An Introduction to Plant Cell and Molecular Biology

The picture shows typical results of the first portion of Experiment 3. Germinating corn seeds were incubated in water, iodine, tetrazolium, or in a peroxidase substrate. Note that the triangular embryo exhibits a high rate of respiration, the aleurone layer contains peroxidase and the starchy endosperm is stained with iodine. Students also locatlities the site of cellular respiration and starch storage as shown.

Chemical Package ContentsCalibrated RespirometersCollection Plates Glass Rods MacropipetsSmall Transfer PipetsLarge Transfer PipetsTetrazoliumBarley SeedsPotassium HydroxideCorn SeedsOnion Bulbs

5. Mitotic Activity and Cell RespirationThe onion root has served as a model system for the study of mitosis, cell elongation, and cell differentiation in plants. In this experiment, students examine various segments of onion roots for cytological differences and then correlate their cytological findings with different respiration rates in the root segments. The exercise provides practical experience with tissue fixing, staining and slide making procedures, and introduces students to the concepts of cell division, the cell cycle, and differentiation in plant systems.

6. Isolating DNA from PlantsA first step in plant recombinant DNA research frequently involves the isolation of DNA from plant tissue. Students perform this procedure in the exercise where they prepare DNA from wheat germ by a simple procedure that uses a detergent, salt, and alcohol.

Hydrogen Peroxide (2 tubes)Potassium IodineEDTA

Tris Buffer (2 tubes)ChloronaptholWheat GermAcid PhosphataseExtraction BufferSucrose SolutionCheese Cloth

Methylene BlueEosinSodium Dodecyl SulfateAmmonium AcetateAcid Phosphatase SubstrateNitrophenol StandardLarge Test Tubes

BASIC PROGRAMS

10

101. Electrophoretic Separation of ProteinsStudents are introduced to the theory of separating proteins according to charge differences using electrophoresis. They then study four proteins and relate differences in their charges to their migration rates in an electric field. Each protein is a different color so that its progress during the separation can easily be followed.

102. Genetics and Sickle Cell AnemiaMany changes in the structure of hemoglobin have arisen by mutations. About one person in 100 carries a mutant hemoglobin gene, and these individuals have abnormal hemoglobin molecules in their blood. One of the most common abnormal hemoglobins is hemoglobin S, which causes sickle cell anemia. When the gene for hemoglobin S is inherited from both parents, all of the hemoglobin in the circulation is hemoglobin S and the individual suffers from severe anemia. When the gene for hemoglobin S is inherited from only one parent, the individual is heterozygous for the condition and has sickle cell trait. Although these individuals rarely have severe anemia, half of their hemoglobin is hemoglobin S and half is normal hemoglobin. In this exercise, students compare the electrophoretic patterns of hemoglobin from a normal individual to hemoglobin S and hemoglobin from a person with sickle cell trait. Students are also given the opportunity to study their own hemoglobin in order to test for hemoglobin variants. Typical results of this exercise are shown above.

103. Analysis of Serum ProteinsSerum contains a variety of small molecules, as well as hundreds of different serum proteins. Serum proteins are frequently separated and characterized by electrophoresis in the clinical laboratory to determine the concentration of various proteins and to detect abnormal protein species. In this exercise, students use electrophoresis to study cow serum and to identify major serum proteins including albumin, transferrin and antibodies. This exercise serves as a good introduction to the function and complexity of proteins in blood.

Staining is not necessary. Watch this experiment in action: MODERNBIO.COM

Introduction to Molecular BiologyStandard Laboratory Program 1

This program stresses the relevance of molecular biology to the study of physiology and human disease. Students first learn how proteins are separated on the basis of charge by electrophoresis. With this information, they perform a series of experiments aimed at illustrating the importance of the technique in biology and medicine. Students examine proteins in biological fluids and relate their findings to physiological and pathological states. They also study specific antigens and enzymes in serum and the abnormal hemoglobin in patients suffering from sickle cell anemia. This fine collection of experiments provides an introduction to electrophoresis and to protein composition, structure, and function in health and disease.

EXPERIMENTS104. Evolution of Serum ProteinsAntibodies can be used to study evolution at the molecular level. In this exercise, immuno-electrophoresis and double diffusion are used to illustrate this important concept. The analysis shows that cow serum proteins are similar to those from sheep and goat but are distinct from serum proteins from horse, donkey and chicken. This exercise acquaints students with basic immunological techniques and serves as an excellent illustration of the utility of these techniques for the study of protein evolution. This exercise requires two 2-3 hour laboratory periods.

105. A Comparison of Human and Bacterial AmylaseA powerful technique used in the modern laboratory involves the detection of specific enzymes in agarose gels following electrophoresis of proteins from serum, tissue extracts and other biological preparations. This procedure makes it possible to study certain properties of individual enzymes in complex protein mixtures. In this exercise, students are introduced to this procedure by comparing the amylase found in bacteria with that in their own saliva.

106. Protein FingerprintingA comparison of specific proteins from different species provides a powerful approach for establishing evolutionary relationships and for identifying organisms. A common approach used for this purpose is protein fingerprinting where electrophoretic properties of specific proteins are analyzed in different species. In this exercise, students use the approach to compare the forms of the enzyme lactate dehydrogenase that are found in serum of different mammals. The photo shows typical results of this graphic experiment. The results illustrate that each species has a characteristic pattern of bands and that the pattern in sheep and goat is similar, while the patterns from cow and especially horse are distinct.

CAT. NO. DESCRIPTION PRICES1 The Chemical Package for 16 students $202.17

working in pairs plus 1 student and 1 instructor manual. S1-BK 8 Student Manuals $63.14S1-SM Sample Student Manual plus one instructor manual. $9.61

Normal and Sickle HemoglobinEXP 102

In this analysis, hemoglobin from a normal individual (gel lanes 1, 4, 5, 8) is compared to hemoglobin S (lanes 3 and 7) and hemoglobin from a person who has sickle cell trait (lanes 2, and 6). Note the two forms of hemoglobin in the individual with sickle cell trait.

1 2 3 4 5 6 7 8

Forms of lactate dehydrogenase in serum - EXP 106

Serum proteins from cow (lanes 1, 5), sheep (lanes 2, 6), goat (lanes 3, 7), and horse (lanes 4, 8) were separated by electrophoresis. The di!erent forms of lactate dehydrogenase were then detected by activity staining.

1 2 3 4 5 6 7 8

PROTEIN ELECTROPHORESIS #NATIVE#

Extra LDH can be purchased to expand this Experiment. See page 60 CAT. 1-10.

PROT

IENS

11

Select the individual experiments in this series that best suit your needs. Each experiment is supplied with the chemicals needed for 16 students working in pairs. Each one also comes with an instructor guide and a single student guide which you may reproduce for educational purposes. Student guides give background information and detailed instructions that are necessary for successful performance and comprehension of the experiments. The instructor guide illustrates the results of the exercise and gives answers to the study questions posed in the student guide. If you chose one or more of the experiments


Cow SerumHorse SerumSheep SerumChicken SerumFilter PaperPasteur PipetsCow Serum AntibodiesPetri DishesTris Buffer SalineEXP-105Starch SolutionPotassium Iodine SolutionSample BufferAlpha-amylaseEXP-106LDH-SubstrateCow SerumSheep SerumGoat SerumHorse SerumElectrophoresis Sample Buffer

EP-1/8AgaroseGel Stain (Coomassie blue)Electrophoresis BufferHemoglobin Elect. BufferEXP-101Cytochrome CHemoglobinSerum AlbuminMyoglobinEXP-102Normal Human HemoglobinSickle Hemoglobin Sickle Trait HemoglobinErythrocyte Lysis Buffer (2)EXP-103Cow SerumCow Gamma GlobulinsCow TransferrinCow Serum AlbuminEXP-104Donkey SerumGoat Serum

IND-24 and Experiment-101 are each in overview program 3 see page 39. Save over $42 by purchasing 03.

PROTIENS

IND 24. AN INTRODUCTION TO ELECTROPHORESIS

Part A. Identifying Unknowns by electrophoresisPart B. Identifying Dyes that Bind to DNA This economical 2-part exercise provides an introduction to

agarose gel electrophoresis. In the first part of the exercise, students identify unknown dye molecules by comparing their electrophoretic migration with the migration of known dyes. In the second part, students identify dye molecules that bind to DNA and determine the mechanism.

EP-l/8 Electrophoresis Package 1/8 $62.42 (For up to 6 individual experiments)

Single Experiments from Standard Program 1 The following experiments include Chemicals and Instructions.EXP-101 Electrophoretic Separation of Proteins $42.44EXP-102 Genetics and Sickle Cell Anemia $46.54EXP-103 Analysis of Serum Proteins $48.86EXP-104 Evolution of Serum Proteins $71.77EXP-105 A Comparison of Human and Bacterial Amylase $47.92EXP-106 Protein Fingerprinting $59.89

below, you should also order Electrophoresis Package 1/8. This Electrophoresis Package provides sufficient agarose, gel stain and electrophoresis buffers for up to 6 of the individual experiments in this series.

INDIVIDUAL EXPERIMENTS

IND30 SPECIFIC BINDING OF DYES TO DNA

Proteins that bind to DNA control the processes of gene regulation and DNA replication. The electrophoretic mobility band shift assay is a common technique used to study these proteins and to study specific protein-DNA interactions. In this exercise, students use this assay to identify dye molecules that bind to DNA and attempt to determine the mechanism by which these drugs interact with the DNA molecule.

CAT. NO. DESCRIPTION PRICEIND-30 Speci"c Binding of Dyes to DNA $19.99

The exercise was designed for 8 groups of students and includes four colorful dye mixtures and DNA. Electrophoresis Package 1/8 is needed but not provided.

CAT. NO. DESCRIPTION PRICEIND-24 An Introduction to Electrophoresis $19.99

12

Electrophoresis is a powerful tool for separating proteins in complex mixtures. However, it is often necessary to identify specific enzymes and structural proteins after electrophoresis so they can be studied in further detail. In this series of experiments, students use electrophoresis in conjunction with novel immunological and enzymatic procedures to study tissue-specific proteins in animals and plants. As in all of our programs, enough materials for 8 groups of students are included. Sufficient materials are provided so that experiments 803 and 804 can be preformed twice: once with the tissues provided with the program and once with tissues of your choice. The PROCELL Electrophoresis Unit, a tabletop centrifuge, and a 37°C waterbath are required.

EXPERIMENTS801. Serum Proteins and the Western Press-BlotWestern blotting is one of the most powerful methods in molecular biology for identifying and characterizing specific proteins in complex protein mixtures. We have now streamlined western-blotting procedures so that the entire analysis can be performed during a single 3-hour, or two 2-hour laboratory sessions. In exercise 801, students use the procedure outlined below to identify albumin, transferrin and gamma globulins in serum and then to study the evolutionary relationships of albumin in vertebrates.

Tissue Specific ProteinsStandard Laboratory Program 8

Corn extracts and peroxidase standards were separated by electrophoresis and the different peroxidase iso-enzymes were then detected by activity staining. A simple procedure is described for the exercise which enables the student to position the sample wells in the middle of the gel. This operation permits the detection of both acidic and basic forms of peroxidase.

Negative Electrode

Positive Electrode

<

<

PROTEIN ELECTROPHORESIS #NATIVE#

Peroxidase Isoenzymes EXP-804

802. Identifying Ovalbumin by the Western Press-Blot Chicken egg white contains a variety of proteins including ovalbumin, conalbumin, ovomucoid and lysozyme. In this exercise, students use the western press-blot procedure to characterize chicken ovalbumin and then to determine if the protein is found in egg whites from turkey and quail.

803. Tissue-Specific Isoenzymes in the CowIsoenzymes are different molecular forms of the same enzyme and five major lactate dehydrogenase (LDH) isoenzymes are found in vertebrate tissues. The amounts of the isoenzymes vary in a tissue specific manner and these differences can be readily detected by localizing LDH activity in an agarose gel after electrophoresis of tissue extracts. In this exercise, students prepare a tissue extract from calf thymus and then compare the LDH isoenzyme profile to those from calf serum, heart and muscle.

804. Peroxidase Isoenzymes in Corn Peroxidase is associated with the plant cell wall and different forms of peroxidase are found in different plant tissues. These tissue-specific differences are revealed by this exercise where students examine the electrophoretic patterns of peroxidase in corn root and shoot extracts.

PROT

IENS

13

IND4. DEVELOPMENT OF THE IMMUNE SYSTEM AND THE WESTERN PRESSBLOT.

In most mammals, antibody production does not occur until after birth. The newborn calf receives antibodies in milk and these antibodies are responsible for passive immunity during early postnatal life. Thus, fetal calf serum is devoid of antibodies, neonatal calf serum contains antibodies of maternal origin while serum from the adult contains large amounts of antibodies made by the mature immune system. This fascinating developmental scheme is illustrated in this exercise. First, students electrophorese serum proteins from fetal, newborn, and adult animals on an agarose gel and the separated proteins are transferred onto a nitrocellulose membrane by the Western Press-Blot procedure shown on the facing page. All proteins on the blot are stained with protein blot stain and then the major class of circulating antibodies (IgG) is detected by using an immunological procedure. The results show that albumin and transferrin are present in sera from all stages while the antibodies are absent in fetal serum but present at high levels in serum from the adult. This analysis can be performed during a single 3-hour, or two 2-hour laboratory sessions. The exercise was designed for 8 groups of students and includes: fetal calf serum, newborn calf serum, adult cow serum, anti-Cow IgG (peroxidase linked), nitrocellulose, blotting paper, protein blot stain, Tris buffer saline (TBS), TBS + NP40, color development buffer, chloronapthol, cow transferrin, cow gamma globulins, cow albumin, hydrogen peroxide, dishes for blot incubation, and gelatin. The PROCELL Electrophoresis Unit and Electrophoresis Package 1/8 are needed but not provided.

CAT. NO. DESCRIPTION PRICEIND-4 Development of the Immune System $103.99

IND5. SPECIFICITY OF ALBUMIN BINDING.The binding of an enzyme to its substrate is only one example of the many specific molecular interactions that occur in biological systems. An analogous binding process occurs with serum albumin which binds certain small molecular weight compounds and serves as a carrier molecule for these compounds in blood. In this exercise, students use an electrophoretic assay to examine the binding of various dyes to albumin. The results of this graphic analysis show that the binding of dyes to albumin is saturable, specific, compatible, and dependent on the native structure of the protein. The exercise is designed such that each of the eight groups of students performs a different experiment. Each group then describes their results and conclusion to the entire class. This exercise is a valuable experience in analyzing data and provides a fine introduction to enzyme kinetics. The analysis can be performed during a single 3-hour or two 2-hour laboratory session and includes: bovine serum albumin, dilution buffer, bromophenol blue, orange G, ponceau S, sodium dodecyl sulfate, and rabbit hemoglobin. Electrophoresis Package 1/8 is needed but not provided.

CAT. NO. DESCRIPTION PRICEIND-5 Speci"city of Albumin Binding $62.39

Ep-1/8AgaroseGel Stain (Coomassie Blue)Electrophoresis BufferExp- 801Goat SerumSheep SerumCow SerumHorse SerumChicken SerumCow TransferrinCow Gamma GlobulinsCow AlbuminAntibodies to CowAlbumin – HRPAnti-Cow GammaGlobulins – HRPNitrocelluloseBlotting PaperProtein Blot StainTris Buffer Saline (TBS)TBS + NP40Color Development BufferChloronapthol

Chemical Package ContentsHydrogen PeroxideDishes for Blot IncubationGelatin

Exp-802Sample BufferChicken Egg WhiteQuail Egg WhiteTurkey Egg WhiteChicken SerumChicken OvalbuminChicken ConalbuminAntibodies to OvalbuminHRP Coupled AntibodiesNitrocelluloseBlotting PaperProtein Blot StainTris Buffer Saline (TBS)TBS + NP40Color Development BufferChloronaptholHydrogen PeroxideDishes for Blot IncubationGelatin

Exp-803Calf ThymusCalf SerumExtraction Buffer (2 Tubes)Sample BufferLDH – SubstrateCalf Heart LDHCalf Muscle LDHEXP 804Horse Radish Peroxidase – BasicHorse Radish Peroxidase – MixtureSample BufferExtraction Buffer (2 Tubes)Corn SeedsHydrogen Peroxide4M TrisChloronaptholCytochome CHemoglobin-Serum Albumin

Price List - Standard Program 8CAT. NO. DESCRIPTION PRICES8 The Chemical Package for 16 students working $235.14

in pairs plus student manual and one instructor manual (The student manual may be reproduced for educational purposes.)

S8-BK 8 Student Manuals $63.14S8-SM Sample Student Manual plus one instructor manual. $9.61

INDIVIDUAL EXPERIMENTSEach of the individual experiments is supplied with the chemicals and laboratory guides needed for 16 students working in pairs. If you chose one or more of the experiments below, you should also order Electrophoresis Package 1/8. Electrophoresis Package 1/8 provides sufficient agarose, gel stain and electrophoresis buffers for up to 6 of the individual experiments in this series and in Standard Laboratory Program 1.

EP-1/8 Electrophoresis Package 1/8 $ 62.42EXP-801 Serum Proteins and the Western Press-Blot $ 96.41EXP-802 Identifying Ovalbumin by the Western Press-Blot $ 96.41EXP-803 Tissue-Speci"c Isoenzymes in the Cow $ 74.87EXP-804 Peroxidase Isoenzymes in Corn $ 68.09

PROTIENS

For fast dependable service:Call toll free: 800-733-6544

FAX: 765-523-3397 Visit: www.modernbio.com

14

A common procedure used for determining the size of proteins in the research laboratory is polyacrylamide gel electrophoresis, where denatured proteins are separated in the presence of the detergent sodium dodecyl sulfate (SDS). However, preparing polyacrylamide gels is laborious and requires a number of toxic chemicals. To avoid these limitations, we provide a special blend of nontoxic agarose that yields excellent resolution of SDS-treated proteins. In this innovative series, students use SDS-agarose gel electrophoresis to perform procedures including molecular weight determinations, peptide mapping analysis, detection of specific enzymes in crude cell extracts, affinity chromatography, and immunological studies using the Western blot procedure.

EXPERIMENTS

Molecular Biology of ProteinsStandard Laboratory Program 2

201. Molecular Weight Determination

A first step in characterizing a protein often involves determining its molecular weight. From this information, different proteins may be compared and the number of amino acid residues in a protein can be determined. Here, students determine the molecular weight of two unknown proteins by comparing their electrophoretic migration with the migration of standard proteins. The protein standards and unknowns have been pre-stained so that your students can follow their progress during the separation as shown below.

PROTEIN ELECTROPHORESIS #DENATURING#

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

PEPTIDE MAPPING ANALYSIS.Serum Albumin from human (lanes 2-4), cow (lanes 6-8), chicken (lanes 10-12), and pigeon (lanes 14-16) were digested with chymotrypsin prior to this separation by electrophoresis. Standard proteins are in lanes 1, 5, 9, and 13.

5 25 45 65ELECTROPHORESIS TIME $MINUTES%

Prestained protein standards of known molecular weights and two proteins of unknown molecular weights were subjected to electrophoresis for the indicated times. The molecular weights of the unknown proteins can be determined by comparing their migration with the migration of the standard proteins.

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

characterized by the procedure described in this exercise. The analysis is performed by incorporating starch into an SDS-agarose gel prior to electrophoresis of the denatured proteins. After electrophoresis, the SDS is removed and the enzyme spontaneously renatures. The location of amylase in the gel is then carried out by staining the starch in the gel with iodine. Zones of enzyme activity are devoid of starch and are seen as clear bands against a background of blue. Using this procedure, students determine the molecular weight of amylase in their own saliva and compare this value to the molecular weight of amylases produced by bacteria

204. Peptide Mapping AnalysisSDS gel electrophoresis is used extensively to separate and identify denatured proteins. However, because this method relies on protein size alone, little information about proteins with the same molecular weight can be obtained. Peptide mapping is one of a number of techniques used to study the relatedness of similarly sized proteins. With this method, proteases are used to

202. Identifying Sex-Specific ProteinsVitellogenin is a protein produced in hen liver under the influence of estrogen. This sex-specific protein enters the circulatory system and is transported to the ovaries where it is broken down into the egg yolk proteins lipovitellin and phosvitin. In this exercise, students compare the proteins in egg yolk and in hen and rooster sera by electrophoresis and identify vitellogenin, lipovitellin and phosvitin. This exercise illustrates the use of electrophoresis for protein identification and introduces concepts in molecular endocrinology.

203. Comparing Human and Bacterial AmylaseAmylase in animals, plants and bacteria can be detected and

PROT

IENS

15

cut proteins into smaller peptide fragments and the fragments derived from two or more proteins are compared. The number and size of fragments generated from a protein are determined largely by the protein’s amino acid sequence, since proteases break peptide bonds adjacent to preferred amino acid residues. In this exercise, students use peptide mapping to compare the structural relatedness of serum albumin from the human, cow, chicken and pigeon. Typical results of this exercise are shown in the figure at the bottom of the previous page.

205. Protein Evolution and the Western BlotThe Western blotting procedure is rapidly replacing conventional methods for identifying and characterizing specific proteins in complex protein mixtures. This technique is used extensively for this purpose in the research laboratory and is increasingly being used in diagnostic medicine for detecting proteins of disease agents such as the structural core proteins of the AIDS virus. Here, students will perform this technique to examine the evolutionary distance between different mammals. Guided by step-by-step instructions, they subject serum proteins from six species to gel electrophoresis and transfer the separated proteins to nitrocellulose membranes. They then use an enzyme-linked immunoassay to compare the extent to which the separated albumins in the serum samples are related to those from cow. This exercise provides an exciting lesson in molecular evolution and introduces your students to one of the most important techniques of molecular biology.

206. Affinity ChromatographyPurified proteins are often needed in the basic research laboratory and for diagnostic and therapeutic procedures. An effective technique for protein purification is affinity chromatography, which exploits a specific interaction between a protein and a complementary binding molecule. In this exercise, students isolate albumin from horse serum by affinity chromatography using a column matrix containing a reactive blue dye which binds specifically to the albumin molecule. They then use electrophoresis to analyze the isolated protein in order to verify the effectiveness of the procedure.

Price List - Standard Laboratory Program 2CAT. NO. DESCRIPTION PRICES2 The Chemical Package for 8 pairs of $243.58

students plus one student manual and one instructor manual.


INDIVIDUAL EXPERIMENTSEach of the individual experiments is supplied with the chemicals and laboratory guides needed for 16 students working in pairs. If you chose one or more of the experiments below, you should also order Electrophoresis Package 2M or Electrophoresis Package 2. Electrophoresis Package 2M provides sufficient agarose, gel stain, and electrophoresis buffers for 1 of the individual experiments in this series (four gels with 15ml of agarose per gel). Electrophoresis Package 2 provides sufficient agarose, gel stain, and electrophoresis buffers for up to 6 of the individual experiments in this series.

EP-2M Electrophoresis Package 2M $48.57EP-2 Electrophoresis Package 2 $135.65EXP-201 Molecular Weight Determination $54.86EXP-202 Identifying Sex-Speci"c Proteins $56.53EXP-203 Comparing Human and Bacterial Amylase $55.45EXP-204 Peptide Mapping Analysis $71.77EXP-205 Protein Evolution and the Western Blot $94.56EXP-206 A$nity Chromatography $87.06

Note that some of these experiments can also be performed using SDS-polyacrylamide gel electrophoresis. Please see page 16 for details.

EP-2Boiling RackAgarose (nusive)Gel Stain (Coomassie blue)Electrophoresis BufferGel BufferPush PinsExp 201Unknown Protein #1Unknown Protein #2Standard ProteinsRabbit SerumEXP 202Hen Serum ProteinsRooster Serum ProteinsEgg Yoke SampleStandard ProteinsSample BufferEXP 203Starch SolutionPotassium Iodine

Chemical Package ContentsBacterial AmylaseStandard ProteinsSample BufferGel Incubation Buffer (2 Tubes)EXP 204Bovine Serum AlbuminHuman Serum AlbuminChicken Serum AlbuminPigeon Serum AlbuminChymotrypsin Standard ProteinsSample BufferEXP 205Cow SerumHorse SerumSheep SerumGoat SerumDonkey SerumAntibodies to Cow Albumin – HRP

Chicken SerumProtein Blot Stain Dishes for Blot IncubationGelatinTris Buffer Saline (TBS)TBS + NP40ChloronaptholHydrogen PeroxideColor Development BufferStandard ProteinsNitrocelluloseBlotting paperExp 206Affi-gel BlueChromatographic ColumnsElution Buffer ConcentrateStandard ProteinsSample BufferHorse SerumColumn Buffer

This exercise requires approximately two 2-3 hour laboratory periods.

This exercise requires about two 2-3 hour laboratory periods.

PROTIENS

We now sell extra nitrocellulouse for experiment 205. See page 62 for details.

16

201P. Molecular Weight Determination A first step in characterizing a protein often involves determining its molecular weight. From this information, different proteins may be compared and the number of amino acid residues in a protein can be determined. Here, students determine the molecular weight of two unknown proteins by comparing their electrophoretic migration with the migration of standard proteins. The protein standards and unknowns have been pre-stained so that your students can follow their progress during the separation. This experiment includes instructions, prestained Unknown Protein #1,Unknown Protein #2, Standard Proteins, and Rabbit Serum. Requires three 12 well gels for 8 groups of students or two gels for 6 groups.

CAT. NO. DESCRIPTION PRICEEXP-201P Molecular Weight Determination $54.86

IND-11P. Contractile Proteins from Cow Heart In this experiment, students prepare a protein extract from cow heart. They then determine the molecular weights of major contractile proteins by comparing their migration on SDS-polyacrylamide gels to the migration of standard proteins of known size. Students also identify and determine the molecular weight of the major proteins found in milk. This exercise requires 1 three-hour or 2 two-hour lab sessions and a table top centrifuge or microcentrifuge is required. Typical results of the exercise are shown on the gel above. Includes cow heart, milk, standard proteins, sample buffer, and instructions. Requires three 12 well gels for 8 groups of students or two gels for 6 groups.

CAT. NO. DESCRIPTION PRICEIND-11P Contractile Proteins from Cow Heart $72.10

In the experiments described in this series, denatured proteins are separated according to size on high-resolution SDS polyacrylamide gels. Students perform procedures including molecular weight determinations, peptide mapping analysis, isolation and characterization of specific cellular proteins and immunological studies using the Western blot procedure. Each experiment was designed so that the protein samples of 8 groups of students are analyzed on two or four electrophoretic gels. (purchase seperately through Bio-Rad, see the bottom of page 17.) Electrophoresis equipment and an electrophoresis package containing electrophoresis buffer and gel stain can be purchased from pages 48-50 in this catalog.

Polyacrylamide Gel ElectrophoresisProtein Analysis

IND-25P. Isolation of Chromosomal Proteins The five histone molecules are the major proteins that as-sociate with DNA to form nucleosomes. In this experiment, students isolate nuclei from chicken erythrocytes and then prepare nucleosomes from the erythrocytes by using a simple procedure that employs micrococcal nuclease. They then de-termine the molecular weights of the five histone proteins by

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

comparing their migration on SDS-polyacrylamide gels to the migration of standard proteins of known size. This exercise requires a single two-three hour lab session and a micro-centrifuge is required. The exercise provides a fine introduc-tion to the nucleosome structure of chromatin and typical results are shown on the gel above. This experiment includes chicken erythrocytes, standard proteins, sample buffer, nu-clear isolation buffer, micrococcal nuclease and instructions. Requires two 12 well gels for 8 groups of students.

CAT. NO. DESCRIPTION PRICEIND-25P Isolation of Chromosomal Proteins $55.18

Electrophoresis Package for SDS Polyacrylamide GelsThe package contains Tris-Glycine-SDS electrophoresis buffer (makes 3 liters) and Coomassie Blue Gel Stain (makes 1 liter). The package provides sufficient materials for three of our polyacrylamide experiments. The gels must be ordered separately.

CAT. NO. DESCRIPTION PRICEEP-2P Electrophoresis Package for $29.41

SDS Polyacrylamide Gels

Staining is not necessary.

PROT

IENS

17

204P. Peptide Mapping Analysis SDS gel electrophoresis is used extensively to separate and identify denatured proteins. However, because this method relies on protein size alone, little information about proteins with the same molecular weight can be obtained. Peptide mapping is one of a number of techniques used to study the relatedness of similarly sized proteins. With this method, proteases are used to cut proteins into smaller peptide fragments and the fragments derived from two or more proteins are compared. The number and size of fragments generated from a protein are determined largely by the protein’s amino acid sequence, since proteases break peptide bonds adjacent to preferred amino acid residues. In this exercise, students use peptide mapping to compare the structural relatedness of serum albumin from the human, cow, chicken and pigeon. Typical results of the exercise are shown below. This experiment includes human, cow, chicken and pigeon albumins, standard proteins, sample buffer and c h y m o t r y p s i n . Requires three 12 well gels for 8 groups of students or two gels for 6 groups.

CAT. NO. DESCRIPTION PRICEEXP-204P Peptide Mapping Analysis $71.77

205P. Protein Evolution and the Western BlotThe Western blotting procedure is rapidly replacing conventional methods for identifying and characterizing specific proteins in complex protein mixtures. This technique is used extensively for this purpose in the research laboratory and is increasingly being used in diagnostic medicine for detecting proteins of disease agents. Here, students will perform this technique to examine the evolutionary distance between different mammals. Guided by step-by-step instructions, they subject serum proteins from six species to gel electrophoresis and transfer the separated proteins to nitrocellulose membranes. They then use an enzyme-linked immunoassay to compare the extent to which the separated albumins in the serum samples are related to those from cow. This exercise provides an exciting lesson in molecular evolution and introduces your students to one of the most important techniques of molecular biology. This exercise requires about two 2-3 hour laboratory sessions. The exercise was designed for 8 groups of students and includes: sera from cow, horse, sheep, goat, chicken and donkey, standard proteins, anti-cow albumin (peroxidase linked), nitrocellulose, blotting paper, protein blot stain, Tris buffer saline (TBS), TBS + NP40, color development buffer, chloronapthol, hydrogen peroxide, dishes for blot incubation, and gelatin. Requires three 12 well gels for 8 groups of students.

CAT. NO. DESCRIPTION PRICEEXP-205P Protein Evolution and the Western Blot $96.30

206P. Affinity ChromatographyPurified proteins are often needed in the basic research laboratory and for diagnostic and therapeutic procedures. An effective technique for protein purification is affinity chromatography, which exploits a specific interaction between a protein and a complementary binding molecule. In this exercise, students isolate albumin from horse serum by affinity chromatography using a column matrix containing a reactive blue dye, which binds specifically to the albumin molecule. They then use electrophoresis to analyze the isolated protein in order to verify the effectiveness of the procedure. This experiment includes Affi-gel Blue, chromatographic columns, column buffer, elution buffer, standard proteins, horse serum and instructions. Requires three 12 well gels for 8 groups of students or two gels for 6 groups.

CAT. NO. DESCRIPTION PRICEEXP-206P A$nity Chromatography $87.06

IND-27 Composition of the NuclesomeThe primary level of chromosome structure in eukaryotes occurs when the DNA molecule is wrapped around histone proteins into particles called nucleosomes. Evidence for this “beads on a string” model is derived from nuclease digestion studies. When nuclei are incubated with micrococcal nuclease, the enzyme cleaves the linker DNA between nucleosomes (the string) but not the nucleosomal core DNA (the beads). With this background information, students isolate nuclei from chicken erythrocytes and then prepare nucleosomes from the nuclei by using a simple procedure that employs micrococcal nuclease. They then analyze the resulting nucleosome DNA fragments by agarose gel electrophoresis. They also determine the molecular weights of the five histone proteins associated with the isolated nucleosomes by comparing their migration on SDS-polyacrylamide gels to the migration of prestained standard proteins of known size. Sufficient chemicals are provided so that this experiment can be performed twice by 8 groups of students. This exercise requires a microcentrifuge, EP-2P, EP-3/4 and three 12-well polyacryamide gels. This exercise provides a fine introduction to structure and composition of the nucleosome and typical results are shown on both the DNA and protein gels.

CAT. NO. DESCRIPTION PRICEIND-27 Comosition of the Nuclesome $56.36

Includes detailed instructions, chicken erythrocytes, prestained standard proteins, protein sample bu!er, DNA sample bu!er, calf thymus nucleosome standard DNA, nuclear isolation bu!er, micrococcal nuclease, nuclear stain, EDTA, small transfer pipets and large transfer pipets.

Precast Polyacrylamide gels can be ordered from Bio-Rad. (800-424-6723) Their catalog number is 1611177.

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8 9 10 11 12

PROTIENS

18

This outstanding program provides a solid foundation in the molecular biology of DNA. Your students will investigate such contemporary topics as restriction nuclease mapping, the complexity of the prokaryotic and eukaryotic genomes and the nucleosome structure of the chromosome. The studies on plasmid amplification using recombinant DNA techniques enable students to conduct the same procedures that form the basis of the biotechnology industry.

EXPERIMENTS303. Plasmid DNA StructurePlasmids are small circular DNA molecules found in most bacteria. A plasmid can exist in different structural states and these states can be distinguished by their migration on agarose gels. In this experiment, students study these structures using enzymes and electrophoresis and show that the structures are interconvertible. This exercise provides an introduction to higher-order DNA structure which is thought to be important in the control of transcription and replication in bacteria and, perhaps, in higher organisms as well.

304. Molecular CloningThis two part exercise provides state-of-the-art information and practical experience with a variety of techniques that form the foundation of the biotechnology industry. In part A, students create a strain of E.coli that is resistant to the antibiotic ampicillin by introducing a plasmid that contains an ampicillin-resistance gene. The success of the transformation is monitored by growing the bacteria on an ampicillin-containing media. This experiment provides sufficient sterile materials for sixteen platings. In Part B, students isolate the amplified plasmid from the bacteria without the use of toxic chemicals. Then they digest the isolated DNA with EcoR1 and compare the electrophoretic properties of linear and circular plasmid DNA molecules. By inserting desired DNA segments into plasmids, this procedure has enabled scientists to amplify more than 1000 specific genes including those for human interferon, insulin, and growth hormone. Part B of the exercise requires a centrifuge that can be operated at a force of at least 3,000 x g (such as a microcentrifuge or a larger floor model). The entire exercise requires three 2-3 hour laboratory periods and the materials provided are shown below.

301. The Length of DNA MoleculesElectrophoresis in agarose gels is the most common method used for determining the size of DNA molecules. In this introductory exercise, students determine the length of an unknown DNA molecule by comparing its electrophoretic mobility with six DNA molecules of known size as shown below.

MATERIALS FOR EXP 304

DNA STAINING PROCEDUREOur programs use a safe and simple two-step procedure for staining DNA in agarose gels. First, a nontoxic purple dye is incorporated into the agarose gel prior to electrophoresis. Faint purple DNA bands can be clearly seen after 30 minutes of electrophoresis. This rapid procedure enables the student to obtain preliminary results of the experiment in a short period of time. After the electrophoretic run, gels are incubated with methylene blue to complete the staining procedure.

DNA fragments of known lengths (lanes 1, 3, 5, and 7) and unknown length (lanes 2, 4, 6, 8) are separated by electrophoresis.

Lambda phage DNA was digested with EcoR1 or EcoR1 plus BamH1 prior to this electrophoretic separation.

Length of DNA EXP-301

1 2 3 4 5 6 7 8

Restriction Nuclease Mapping EXP-302

1 2 3 4 5 6 7 8

DNA

Electrophoresis equipment not required.

New Plasmid Isolating Procedure.

302. Restriction Nuclease Mapping of DNABacteriophage lambda is a DNA virus that attacks E.coli. Here, students dissect lambda DNA using the restriction endonucleases EcoR1 and BamH1 in order to identify specific sites, sequences, and structures along the phage genome. This single exercise enables students to explore a number of exciting topics in molecular biology, including the specificity of restriction endonucleases, DNA mapping strategies, complementary base-pairing of DNA, and the structure of a viral genome.

Molecular Biology of Nucleic AcidsStandard Laboratory Program 3

19

Price List - Standard Laboratory Program 3CAT. NO. DESCRIPTION PRICES3 The Chemical Package for 16 students $239.35


S3-BK 8 Student Manuals $63.14S3-SM One Student Manual plus one instructor manual. $9.61

INDIVIDUAL EXPERIMENTSEach of the individual experiments is supplied with the chemicals and laboratory guides needed for 16 students working in pairs. (Please see page 21 for additional details). If you chose one or more of the experiments below, you should also order Electrophoresis Package 3/4. Electrophoresis Package 3/4 provides sufficient agarose, gel stains and electrophoresis buffers for up to 6 of the individual experiments in this series. The package also includes the dye for rapid DNA staining during electrophoresis (See facing page). Electrophoresis Package 3/4 is also suitable for the experiments in Standard Laboratory Programs 4 and 10 and the experiments on pages 20-22.

EP-3/4 Electrophoresis Package 3/4 $67.07EXP-301 The Length of DNA Molecules $48.97EXP-302 Restriction Nuclease Mapping of DNA $62.48EXP-303 Plasmid DNA Structure $62.48EXP-304 Molecular Cloning (Part A + Part B) $99.86EXP-304A Molecular Cloning(Part A only) $71.18EXP-304B Molecular Cloning(Part B only) $62.24EXP-305 Identifying Satellite Sequences $56.37EXP-306 The Nucleosome Structure of Chromatin $71.77

305. Identifying Satellite SequencesA large fraction of the DNA of vertebrates consists of nucleotide sequences that are repeated thousands or more times in the genome. Satellite sequences are a major class of these repeated elements and these sequences are found in most eukaryotic organisms. There are about 1 million cutting sites for the restriction enzyme EcoRI in the typical vertebrate genome and about 1 million DNA fragments are generated following digestion of vertebrate DNA with this enzyme. When this DNA is separated by electrophoresis, the large number of differently-sized fragments appear as a background smear on the gel lane. However, as shown in the figure to the right, DNA fragments from the highly repeated satellites form discrete bands because they are the same size. In this exercise, students identify satellite sequences in the cow genome and determine if such sequences are present in chicken DNA.

306. The Nucleosome Structure of ChromatinThe primary level of chromosome structure in eukaryotes occurs when the DNA molecule is wrapped around histone proteins into particles called nucleosomes. Evidence for this "beads on a string" model is derived from nuclease digestion studies. When nuclei are incubated with micrococcal nuclease, the enzyme cleaves the linker DNA between nucleosomes (the string) but not the nucleosomal core DNA (the beads). When the DNA isolated from these nuclei is electrophoresed on agarose gels, the DNA exhibits a ladder pattern with the length of the DNA in each band representing a multiple of the nucleosomal unit as shown in the Figure on this page. With this background information, students first examine nucleosomal DNA from calf thymus that is given in this chemical package. They then prepare nuclei from wheat germ, incubate them with micrococcal nuclease and examine the ladder pattern in the DNA by electrophoresis. This exercise requires two 2 - 3 hour laboratory periods.

DNA from calf thymus (even numbered lanes) was digested with EcoR1 prior to this electrophoretic separation. Note the discrete satellite DNA band.

Note the ladder pattern produced when the DNA in chromatin is cleaved by micrococcal nucleases. (lanes 2-4, 6-8).

Satellite DNA EXP-305 1 2 3 4 5 6 7 8

Nucleosome DNAEXP-306 1 2 3 4 5 6 7 8

EP-3/4AgaroseGel Stain (Methylene Blue)In Gel StainElectrophoresis BufferExp 301Dye MixtureDNA MarkersUnknown DNAExp 302Phage Lambda DNABam H1Sample BufferEcoR1Restriction Nuclease BufferExp 303Plasmid DNASample BufferS1 NucleaseDye MixtureEcoR1Restriction Nuclease BufferS1 Nuclease BufferExp 304 Part AE.coliPlasmid pUC18

Chemical Package ContentsAmpicillinCalcium ChlorideNutrient BrothPerti DishesNutrient Agar + AmpicillinInoculating LoopsSterile Transfer PipetsSterile TubesExp 305Calf DNAChicken DNAEcoR1Restriction Nuclease BufferDye MixtureSample BufferExp 306Nucleosome DNA # 1Nucleosome DNA # 2Nucleosome DNA # 3Nuclear Incubation Buffer (2)Dye MixtureSample BufferWheat GermMicrococcal NucleaseCheese ClothTransfer Pipets

Exp 304 Part BE.coli -pUC18Nutrient Broth + AmpicillinElectrophoresis Sample BufferDye MixtureRestriction Nuclease BufferEcoR1IsopropanolPlasmid Isolation (found in green box)Solution I - 1 bottleSolution II - 1 bottleSolution III - 1 bottleEquilibration Buffer - 1 bottleBuffer HB - 1 bottleWash Buffer - 1 bottleElution Buffer - 1 bottleHiBind DNA Column - 24Collection Tubes - 24RNase - 1 Tube

DNA

20

The exercises in this program are designed to illustrate key principles of molecular genetics including genome organization, gene function, and DNA fingerprinting. Each exercise emphasizes an investigative approach where students draw conclusions from experimental results. Each exercise also illustrates fundamental biological concepts. The four exercises can be done independently or in sequence and are recommended for beginning and intermediate-level students.

1001. Anatomy and Evolution of the GenomeCommon plasmids are simple DNA molecules which contain a few genes and regulatory elements. Most viral genomes are more complex. For example, the genome of phage lambda contains approximately 50 genes. About 4,000 genes are present in the

E.coli genome while there is approximately 1,000 times more DNA in the genome of a mammal. This progression in genome complexity is the topic of this exercise. Here, students compare the electrophoretic patterns of restriction digests of a plasmid, phage lambda DNA, and cow DNA from thymus and kidney as shown in the figure below. The exercise serves as a good introduction for determining the size of DNA molecules and provides an appreciation for the complexity of genomes from different organisms.

1002. Analysis of a Genome Segment Phage lambda DNA and a recombinant plasmid containing a segment of the phage lambda genome are provided as the starting point for this exercise. Students digest these DNAs with EcoR1 and BamH1 and then analyze the fragments as shown below. Determination of the size of the fragments enables the student to identify the precise region in the lambda genome that is contained within the plasmid.

The New GeneticsStandard Laboratory Program 10

1003. DNA Fingerprinting (An authentic analysis)DNA fingerprinting relies on the fact that DNA can be extremely variable in sequence from one individual to another and from one organism to another. Thus, the method has been used to identify individuals in forensic and paternity cases and to study genetic lineages of closely related organisms. These applications are discussed in the text and the latter application is illustrated in this experiment. In the exercise, students are given two DNA samples which are identified in the instructor guide. One sample is from sheep and the other from cow. In part A, the DNAs are digested with restriction enzymes and the sizes of the bands composed of highly repeated sequences are determined by comparison to standard DNAs of known lengths as shown below. In part B, the nonrepeated sequences are eliminated by a simple novel procedure. This step serves to enhance the banding patterns of the repeated sequences as shown in the gel on the right. From the results of this analysis, students determine which DNA sample is from cow and which is from sheep. This exercise requiring two 2-3 hour laboratory periods is the only authentic (non-simulated) DNA fingerprinting exercise available for the teaching laboratory.

Lambda phage DNA (lanes 1, 2, 5, & 6) or a plasmid containing a segment of the phage genome (lanes 3, 4, 7, & 8) were digested with EcoR1 or EcoR1 + BamH1 prior to this electrophoretic separation.

DNA from plasmid pUC18 (lanes 1 & 5), lambda phage (lanes 2 & 6). calf thymus (lanes 3 & 7), and calf kidney (lanes 4 & 8) are separated by electrophoresis. The DNA samples for this exercise are supplied ready for electrophoresis.

Part A Part B

DNA from cow (lanes 3 & 4) and sheep (lanes 6 & 7) were digested with EcoR1 or BamH1 prior to this electrophoretic analysis. DNA markers are shown in lanes 2 & 5.

Cow and sheep DNA are subjected to a simple novel procedure in order to eliminate nonrepeated sequences. The repeated sequences were then electrophoresed on this gel.

Anatomy and Evolution of the

1 2 3 4 5 6 7 8

Analysis of a Genome Segment EXP-1002

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

See page 62 to make up your own DNA "ngerprinting story with experiment 3-20.

DNA

21

1004. Genotype to PhenotypeThis exercise was designed to provide an exciting introduction to specific gene structure and function. In the experiment, students are given two plasmids (A and B) which are identified in the instructors guide. One plasmid (A) has a functional gene for the enzyme ß-galactosidase. The ß-galactosidase gene in the other plasmid (B) is inactive because it contains a segment of foreign DNA. In the first part of the exercise, students analyze restriction digests of both plasmids in order to determine which plasmid should have a functional ß-galactosidase gene. In the second part of the exercise, the plasmids are introduced into E.coli by transformation and the color of the resulting colonies (blue or white) is then used to assess the functional status of the ß-galactosidase gene. This exercise can be completed within a single 3-hour laboratory session or two 2-hour laboratory periods.


working in pairs plus one student manual and one instructor manual (The student manual may be reproduced for educational purposes).



EP-3/4AgaroseGel Stain (Methylene Blue)In Gel StainElectrophoresis BufferEXP-1001Plasmid DNA-EcoR1 CutPhage Lambda DNA-Hind III CutCalf Thymus DNA-EcoR1 CutCalf Kidney DNA-EcoR1 CutEXP-1002Lambda DNAPlasmid-LambdaEcoR1 BamH1 Restriction Nuclease BufferSample BufferEXP-1003Unknown DNA 1Unknown DNA 2EcoR1 EndonucleaseBamH1 EndonucleaseRestriction Endonuclease Buffer (2)

INDIVIDUAL EXPERIMENTSEach of the individual experiments are supplied with the chemicals and laboratory guides needed for 16 students working in pairs. If you chose one or more of the experiments below, you should also order Electrophoresis Package 3/4. EP-3/4 provides sufficient agarose, gel stain and electrophoresis buffers for up to 6 of the individual experiments in this series. Electrophoresis Package 3/4 is also suitable for the experiments in Standard Laboratory Programs 3 and 4.

EP-3/4 Electrophoresis Package 3/4 $67.07

EXP-1001 Anatomy and Evolution of the Genome $64.91

EXP-1002 Analysis of a Genome Segment $73.37

EXP-1003 DNA Fingerprinting (An authentic analysis) $82.46

EXP-1004 Genotype to Phenotype $87.60

Sample BufferS1-NucleaseS1-Nuclease BufferDNA MarkersEXP-1004Plasmid A Plasmid B Plasmid CEcoR1 EndonucleaseEndonuclease BufferSample BufferE.coliAmpicillinDNA MarkerCaCl2Nutrient BrothNutrient Agar + AmpicillinX-galX-gal SolventInoculating LoopsSterile Transfer PipetsSterile TubesPetri Dishes

DNA



Watch a video about this popular transformation experiment at MODERNBIO.COM

WhiteColonies

BlueColonies

22

The revolution brought about by molecular biology depended heavily on nucleic acid hybridization procedures. These techniques are used extensively in the research laboratory for detecting specific nucleotide sequences in DNA and RNA and are increasingly being applied in medicine for diagnosing disease. Modern Biology, Inc. is proud to introduce these methods into your teaching laboratory. In this laboratory program, students use hybridization procedures to investigate key topics in contemporary biology including the evolution of the vertebrate genome, the complexity of the viral chromosome, and the analysis of a specific nucleotide sequence in mammalian DNA. The package comes complete with all of the biochemicals that you will need although a water bath incubator that will maintain a temperature of 60-65°C is required for these experiments. The procedures used in these exercises are safe for your students because the four hybridization probes provided with the chemical package were prepared using a nonradioactive labeling system.

EXPERIMENTS

DNA Hybridization AnalysisStandard Laboratory Program 4

401. Evolution of the Vertebrate GenomeThe history of evolution is recorded in the genomes of present-day organisms and enlightened guesses of evolutionary events can be made by comparing DNA sequences in different species. Evidence drawn from this approach has led to the construction of family trees of organisms that agree remarkably well with those obtained from more traditional procedures. In fact, on a number of occasions, comparative DNA sequence analysis has been used to clarify and expand on phylogenetic relationships that were derived from classical studies. For example, although biologists have long disagreed about the taxonomic placement of the giant panda, recent studies using DNA hybridization techniques strongly suggest that this animal is more closely related to bears than to raccoons. These important concepts are illustrated in this exercise where students use a dot-blot hybridization procedure to compare DNA sequences in salmon, turkey, chicken and cow. In the analysis, single strands of chicken or cow DNA that have been previously linked to biotin are mixed with single strands of DNA from a second species. The two types of DNA are allowed to form double-stranded hybrid molecules and the extent of hybridization is measured using a simple color-producing enzyme reaction. This exercise requires approximately two 2-3 hour laboratory periods and electrophoresis equipment is not required.

402. Application of the Southern Blot ProcedureIn l975, Edward M. Southern at the University of Edinburgh, developed a powerful technique for DNA analysis which has become known as Southern blotting. Here your students use the Southern blotting procedure to identify the major control region for transcription and replication in the lambda phage

Agarose Gel Southern Blot

Detection of the Control Region in Lambda Phage DNALeft: Restriction nuclease digests of lambda DNA were electrophoresed on an agarose gel and the gel was stained. Right: The separated DNA fragments on the gel were transferred to a nylon membrane and the DNA containing the control region was identi"ed by hybridization analysis.

genome. Following the step-by-step procedures in their manuals, students digest lambda DNA with a restriction endonuclease, electrophorese the DNA, and then transfer the separated fragments on the gel to a nylon membrane. The DNA fragment containing the control region is then identified by hybridization analysis using a biotinylated probe for the control region and the enzyme-color-producing assay. This exercise provides a wealth of practical information on one of the most powerful methods in molecular biology and illustrates an important strategy for mapping simple and complex genomes. This experiment requires about three 2-3 hour laboratory periods and typical results are shown above.

DNA

23

INDIVIDUAL EXPERIMENTSEach of the individual experiments is supplied with the chemicals and laboratory guides needed for 16 students working in pairs. If you chose one or more of the experiments on this page, you should also order Electrophoresis Package 3/4. Electrophoresis Package 3/4 provides sufficient agarose, gel stain and electrophoresis buffers for up to 6 of the individual experiments in this series. Note that Electrophoresis Package 3/4 is also suitable for the experiments in Standard Programs 3 and 10.

EP-3/4 Electrophoresis Package 3/4 $67.07 (for up to 6 individual experiments)

EXP-401 Evolution of the Vertebrate Genome $126.59

EXP-402 Application of the Southern Blot Procedure $149.78

EXP-403 Detecting a Speci"c Sequence in the $140.87 Mammalian Genome

IND-16. Identifying Viral DNA by Rapid Southern BlottingA limitation to the Southern blotting procedure for the teaching laboratory is that it requires multiple laboratory sessions. The procedures have been streamlined for this experiment so that they can be carried out in a single 3-hour laboratory period. In the exercise, students electrophorese three unknown DNA samples on an agarose gel. One of the samples (instructor keyed) contains biotinylated DNA from the bacterial virus lambda. Following electrophoresis, the DNA in the gel is transferred to a nylon membrane during a 15-minute blotting step and the viral DNA on the blot is then detected by using an enzyme-color-producing


Price List - Standard Laboratory Program 4S4 The Chemical Package for 16 students $238.50


S4-BK 8 Student Manuals $63.14 S4-SM 1 Student Manual and 1 Instructor Manual $9.61

Chemical Package ContentsEP-3/4AgaroseGel Stain (Methylene Blue)In Gel StainElectrophoresis BufferExp 401Cow DNAChicken DNATurkey DNASalmon DNABiotinylated Chicken DNABiotinylated Cow DNAHybridization BufferTris BufferNP40Hydrogen PeroxideGelatinChloronaptholHybridization BagsNylon MembranesBlotting PaperAvidin-PeroxidaseMethylene Blue

Paper ClipsFoam RackExp 402EcoR1 EndonucleaseHybridization BufferTris BufferNP40Hydrogen PeroxideGelatinChloronaptholHybridization BagsNylon MembranesBlotting PaperAvidin-Peroxidase4 Blotting DevicesFoam RackPaper ClipBam H1Restruction BufferElectropharesis Sample BufferBioimulated Probe

Exp 403Cow DNAChicken DNA (EcoR1 cut)Sheep DNA (BamH1 cut)EcoR1EcoR1 BufferSample BufferBiotinylated Satellite PlasmidHybridization Buffer4 Blotting DevicesDNA MarkersTris BufferNP40Hydrogen PeroxideGelatinChloronaptholHybridization BagsNylon MembranesBlotting PaperAvidin-PeroxidasePaper ClipsFoam Rack

reaction. The experiment was designed for 8 groups of students and sufficient materials are provided so that four blots can be prepared and analyzed. The exercise requires Electrophoresis Package 3/4 and a water bath maintained at 37° C.

CAT. NO. DESCRIPTION PRICEIND-16 Identifying Viral DNA by Rapid Southern Blotting $119.19

Includes instructions, DNA samples 1-3, dye mixture, blotting paper, nylon membranes, petri dishes,methylene blue, Tris-bu!er saline (TBS), TBS+NP-40, streptavidin peroxidase, Hydrogen peroxide, chloronapthol, gelatin and Tris bu!er.

403. Detecting a Specific Sequence in the Mammalian GenomeSouthern hybridization analysis can now be used for tracing defective genes in human DNA including those involved in Huntington’s disease, Duchenne Muscular Dystrophy, and Cystic Fibrosis. This application is becoming increasingly important for diagnosing these diseases in members of afflicted families and it seems likely that the method will be used to detect additional abnormal and even normal human genetic traits in the near future. In this exercise students perform Southern hybridization analysis in order to characterize a specific sequence in mammalian DNA. Students digest cow, sheep, and chicken DNA with EcoRI, electrophorese the DNA, and transfer the separated fragments to a nylon membrane. The DNA fragments containing the cow satellite is then detected by hybridization using a biotin-labeled probe made from a plasmid that contains the cow satellite sequence. This exercise requires about three 2-3 hour laboratory periods.

We now sell extra nylon membrane and blotting paper (folder).

DNA

24

Genetic Engineering(Standard Laboratory Program 5)

Recombinant DNA techniques played a central role in the recent emergence of biology into the golden age. This program enables the advanced student to approach these frontiers in biotechnology. The program provides a challenging series of seven 3-hour laboratory sessions which are intended to give students hands-on-experience and a detailed understanding of these new investigative techniques and their potentials.

In the program, students prepare a library of recombinant plasmids which contain different regions of the lambda phage genome. They then amplify and characterize the lambda DNA in selected plasmids in order to identify the phage genes that they contain. The basic strategy followed in the program is shown below and is one that has been used by investigators around the world to clone DNA molecules from literally thousands of prokaryotic and eukaryotic organisms. However, many of the procedures have been modified to make them safe and suitable for a college laboratory class. The program provides essentially all of the instructions, chemicals, sterile media, and expendable accessories that are needed to carry out DNA electrophoresis, restriction nuclease digestions, DNA ligations, bacterial transformations, bacterial selection for ampicillin resistance and ß-galactosidase production, plasmid isolation, DNA hybridization, and Southern blot procedures. A centrifuge that can be operated at a force of at least 3,000 x g (such as a microcentrifuge or a larger floor model) and a water bath incubator that will maintain a temperature of 60-65°C are required for the program. A shaking bacterial incubator is strongly recommended.

Lab Session 1Students digest phage lambda DNA with EcoR1 and ligate the resulting DNA fragments to EcoR1-digested pUC18 in order to create a series of recombinant plasmids. Some of the plasmids in the mixture will lack inserted lambda DNA while the others will have DNA inserts derived from various regions of the phage genome.

Lab Session 2The recombinant molecules are characterized by electrophoresis and then introduced into competent E.coli cells by transformation. The cells are plated onto nutrient agar containing ampicillin and X gal. The ampicillin selects for all cells that have taken up plasmid while the X gal permits students to identify colonies which contain plasmids with lambda DNA inserts. The identification is based on a powerful color selection assay. Plasmid pUC18 contains a portion of the lac Z gene which codes for ß-galactosidase and X gal is a substrate of this enzyme. When pUC18 is carried by appropriate strains of E.coli, the lac Z gene is active and the ß-galactosidase converts X gal to a blue product which gives rise to blue colonies. If foreign DNA is inserted into the EcoR1 site of pUC18, the lac Z gene is inactivated and the colonies remain white.

Lab Session 3Each student prepares overnight cultures from one blue colony which should lack lambda DNA, and three white colonies which should contain phage sequences. The plasmids are then isolated from the cultures by a unique mini-prep procedure that yields high quality plasmid DNA without the use of toxic organic solvents.

Lab Session 4Students digest the plasmids with EcoR1, BamH1, and a combination of the two enzymes and determine the size of the DNA fragments by electrophoresis. The results of this analysis provide a first step in the identification of the lambda fragments that were cloned.

Lab Session 5 - 7The DNA fragments in the gels are transferred to nylon membranes by the method of Southern using the transfer devices provided with the program. The fragments containing the lambda sequences are then detected by hybridization using a biotin-labeled probe made from phage lambda DNA. The results of this analysis and the restriction mapping studies performed during Lab Session 4 enable the student to identify the segment of the lambda genome in each plasmid.

A typical laboratory schedule for this program is given below where each lab session is approximately 3 hours.

In 2012 we changed the Plasmid Isolation procedure for this experiment.

DNA

25



S5-BK 8 Student Manuals $63.14

S5-SM 1 Student Manual and 1 Instructor Manual $9.61

In Standard Program 5, a library of recombinant plasmids is prepared by ligating DNA fragments from phage lambda to linear pUC18. The plasmids are introduced into E. coli by transformation and white colonies containing plasmids with lambda DNA are recovered. Plasmids are isolated from the E. coli and analyzed by electrophoresis, and by Southern blot hybridization procedures in order to identify the segment of the lambda genome contained in each. In the gel shown, a plasmid from a blue colony (lane 1), and plasmids from two di!erent white colonies (lanes 2 and 3), were digested with EcoR1 prior to this electrophoretic analysis. DNA standards are shown in lane 4.


Electrophoresis AgaroseGel Stain (Methylene Blue)In Gel StainElectrophoresis BufferDNA Sample Buffer (2)Dye MixtureDNA ManipulationPhage Lambda DNAPlasmid pUC18 DNAEcoR1 Endonuclease (2)Bam H1 EndonucleaseEndonuclease Buffer (2)DNA LigaseLigase BufferBacterial Transformation and GrowthE.coliCalcium ChlorideNutrient Broth (2)Nutrient Broth + AmpicillinNutrient Agar + AmpicillinX GalX Gal SolventE.coli-pUC18AmpicillinE.coli pUC18 – LambdaSterile Petri DishesSterile Transfer PipetsSterile Microtubes

Sterile Culture TubesInoculating LoopsPlasmid Isolation (found in green box)Solution I - 1 bottleSolution II - 1 bottleSolution III - 1 bottleEquilibration Buffer - 1 bottleBuffer HB - 1 bottleWash Buffer - 1 bottleElution Buffer - 1 bottleHiBind DNA Column - 24Collection Tubes - 24RNase - 1 TubeSouthern Blot AnalysisBiotinylated Lambda DNA4 Southern Transfer DevicesNylon MembraneBlotting PaperHybridization BagsHybridization BufferTris BufferNP-40ChloronaptholGelatinAvidin – PeroxidaseHydrogen PeroxideFoam RackPaper Clips

1 2 3 4

White Colonies

BlueColonies

DNA

For fast dependable service:

Call toll free: 800-733-6544

FAX: 765-523-3397

Visit: www.modernbio.com

26

A major goal of human genetics is to develop a complete map of the human genome. A high-resolution map of the genome is considered to be essential for diagnosis of genetic diseases and development of cures of these diseases by gene therapy. Studies on a specific DNA locus in a mammal usually begin with the isolation of genomic DNA from a tissue. The DNA is quantified, subjected to restriction nuclease digestion, and the fragments separated by gel electrophoresis. The DNA fragments in the gel are then transferred to a special membrane by the method of Southern and the specific DNA fragment under investigation is detected by hybridization analysis. Your students will perform each of these steps with this program series of five 3-hour laboratory sessions. The starting material for the series is calf thymus tissue. Students isolate DNA from purified thymus nuclei and then quantify it with a novel solid phase DNA assay. The DNA is digested with EcoR1, electrophoresed, and the separated fragments transferred to a nylon membrane. The DNA fragment containing the G+C-rich cow satellite is then detected by hybridization using a biotin-labeled probe made from a plasmid that contains the cow satellite sequence. Students also determine if the satellite sequence is found in sheep DNA. Microscopes, ethyl alcohol, an incubator that will maintain a temperature of 60-65°C, and a centrifuge that can be operated at a force of at least 3000 x g are needed for the program.

Tissue to Gene(Standard Laboratory Program 6)

Lab Session 1Students isolate nuclei from thymus tissue and isolate thymus DNA from the nuclei by a unique procedure that yields high quality cellular DNA without the use of toxic organic solvents.

Lab Session 2The purified DNA is quantified by a solid phase assay, digested with the restriction endonuclease EcoR1, and then electrophoresed on agarose gels. Price List - Standard Laboratory Program 6

CAT. NO. DESCRIPTION PRICES6 The Chemical Package for 16 students $231.76




Electrophoresis (ep-3/4)AgaroseGel Stain (Methylene blue)In Gel StainElectrophoresis BufferCow DNA (EcoR1 cut)Sheep DNA (BamH1 cut)DNA Sample BufferDNA PreparationCalf Thymus TissueCheese ClothNuclear Buffer (2)Sodium Dodecyl SulfateAmmonium Acetate (2)Tris-EDTA BufferDNA Standard Transfer pipetsPasteur Pipets

EcoR1 (2)Restriction Nuclease BufferSouthern Blot AnalysisFoam RackHybridization BufferNP40Hydrogen PeroxideGelatinChloronaptholHybridization BagsNylon MembranesBlotting PaperAvidin-PeroxidaseFour Transfer DevicesBiotinylated Satellite DNAPaper Clips

Lab Sessions 3-5The DNA fragments in the gels are transferred to nylon membranes using the transfer devices provided with the program. The fragment containing the satellite sequence is then detected by hybridization using a biotin-labeled probe prepared from a plasmid containing the satellite sequence.



We now sell extra nylon membrane and blotting paper (folder). This program takes 2 folders

DNA

27

Agarose Gel Southern Blot

Detection of Ribosomal RNA genes in Chloroplast DNA.

Left: Marker DNA and chloroplast DNA samples were electrophoresed on an agarose gel and the gel was stained. Right: The separated DNA fragments on the gel were transferred to a nylon membrane and the DNA containing the chloroplast ribosomal RNA genes was identi"ed by hybridization analysis.

The chloroplast genome of higher plants is relatively small and simple. This genome contains about 120 genes which code for chloroplast proteins. The organelle's genome also contains ribosomal RNA genes which code for the RNA components of the chloroplast ribosome. In this program, students identify the ribosomal RNA genes in the chloroplast genome of spinach. Students first isolate chloroplasts from fresh spinach and then prepare DNA from the isolated organelle. The DNA is digested with EcoR1, electrophoresed, and the separated fragments transferred to a nylon membrane. The DNA fragments containing the ribosomal genes are then detected by hybridization using a biotin-labeled probe made from a plasmid that contains the ribosomal gene sequences. Fresh spinach, microscopes, ethyl alcohol, a water bath incubator that will maintain a temperature of 60-65°C, and a centrifuge that can be operated at a force of at least 3000 x g are needed for the program. A microcentrifuge is also desirable, but not absolutely necessary.

Plant Molecular Biology (Standard Laboratory Program 9)

Lab Session 1Students isolate chloroplast from spinach leaves and isolate DNA from the chloroplast by a unique procedure that yields high quality DNA without the use of toxic organic solvents.

Lab Session 2The purified DNA is digested with the restriction endonuclease EcoR1, and then electrophoresed on agarose gels.

Lab Sessions 3-5The DNA fragments in the gels are transferred to nylon membranes using the transfer devices provided with the program. The fragments containing the genes for ribosomal RNA are then detected by hybridization using a biotin-labeled probe prepared from a plasmid containing the ribosomal gene sequences. Typical results are shown below.




Chemical Package ContentsElectrophoresis (ep-3/4)AgaroseGel Stain (Methylene blue) In Gel StainElectrophoresis Buffer Lambda DNA (Hind III cut)R-DNA Plasmid (EcoR1 cut)DNA Sample BufferDNA PreparationLarge Transfer PipetsCheese ClothBuffer (Tris-Saline)Sodium Dodecyl SulfateAmmonium AcetateEcoR1 (2)Restriction Nuclease Buffer

Southern Blot AnalysisFoam RackHybridization BufferTris BufferNP40Hydrogen PeroxideBiotinylated R-DNA PlasmidChloronaptholHybridization BagsBlotting PaperNylon MembranesAvidin-PeroxidaseFour Transfer DevicesGelatinPaper Clips

A typical laboratory schedule for this program is given where each lab session is approximately 3 hours.

Extra nylon membrane and blotting paper are available on page 62.

DNA

28

Cloning a DNA Segment from Sheep(Standard Laboratory Program 11)

Lab Session 1Students digest sheep DNA with EcoR1 and electrophorese the DNA fragments on an agarose gel in order to seperate the satellite DNA from the remaining sequences in the sheep genome.

Lab Sessions 2-3The satellite DNA band is eluted from the gel and ligated to EcoR1-digested pUC18 in order to create a recombinant plasmid that contains the satellite sequence. The recombinant plasmid is then introduced into competent E.coli cells by transformation and colonies containing foreign DNA are identified by a blue-white color selection assay.

Lab Sessions 4-5Students prepare overnight cultures from white colonies which should contain the sheep satellite sequence. Plasmids are then isolated from the cultures by a mini-prep procedure that yields high quality plasmid DNA without the use of toxic organic solvents. Students digest the plasmids with EcoR1 and determine the size of the DNA fragments by electrophoresis. The results of this gel analysis enable the student to identify the satellite sequence in the recombinant plasmid.

The sheep genome contains a highly repeated satellite sequence. In this program, students use recombinant DNA

techniques to clone this satellite DNA and then characterize the sequence by restriction enzyme mapping anaylsis.

The program provides essentially all of the instructions, chemicals, sterile media, and expendable accessories that

are needed to carry out DNA electrophoresis, restriction nuclease digestion, DNA ligations, bacterial transformations,

bacterial selection for ampicillin resistance and ß - glactosidase production and plasmid isolation. A microcentrifuge

is required for the program.

Price List - Standard Laboratory Program 11CAT. NO. DESCRIPTION PRICES11 The Chemical Package for 16 student $224.12




Electrophoresis (ep-3/4)AgaroseGel Stain (Methylene Blue)In Gel StainElectrophoresis BufferDNA Sample Buffer (2)DNA StandardsDNA ManipulationSheep DNAPlasmid pUC18 DNA (EcoR1 cut)EcoR1 Endonulcease (2)Endonuclease Buffer (2)DNA LigaseLigase BufferDNA Elutor Components1.5 ml tubes0.5 ml tubesTransfer PipetsGlass WoolPush Pins

Bacterial Transformation and GrowthE.coliCalcium ChlorideNutrient Broth (2)Nutrient Broth + AmpicillinNutrient Agar + AmpicillinX Gal and SolventAmpicillinSterile Petri DishesSterile Transfer PipetsSterile MicrotubesSterile Culture TubesInoculating LoopsPlasmid Isolation (found in green box)Solution I - 1 bottleSolution II - 1 bottleSolution III - 1 bottleEquilibration Buffer - 1 bottleBuffer HB - 1 bottleWash Buffer - 1 bottleElution Buffer - 1 bottleHiBind DNA Column - 24Collection Tubes - 24RNase

In 2012 we changed the Plasmid Isolation procedure for this experiment.

DNA


FAX: 765-523-3397 Visit: www.modernbio.com Find Equipment, Accessories, and Kits for

Teaching and Research starting on page 48.

29

IND-6. Analysis of a Mutant Hemoglobin GeneA mutation is a change in the nucleotide sequence of DNA which leads to an inherited change in an organism. Restriction endonucleases provide valuable tools for characterizing mutations at the DNA level. This principle is illustrated in the exercise where students digest a normal and a mutant gene with EcoR1 and Hae III and then analyze the DNA fragments from each by electrophoresis as shown in the figure on the right. The gene is from rabbit and codes for the ß-globin chains of hemoglobin.

DNA

IND-9. Producing a Strain of E.coli that Glows in the DarkThe emission of light by living organisms is a fascinating process. The genetic system required for luminescence in the bacterium Photobacterium (Vibiro) fischeri is the lux operon. This operon contains a gene for luciferase (the enzyme that catalyzes the light-emitting reaction) and genes for enzymes which produce the luciferins (which are the substrates for the light-emitting reaction.). In this exercise, students create a luminescent population of bacteria by introducing into E.coli a plasmid that contains this lux operon. The success of the transformation is readily apparent since the E.coli colonies that take up this plasmid glow in the dark as shown on the right. The simple procedure can be carried out during a single 1-1.5 hour laboratory

CAT. NO. DESCRIPTION PRICEIND-9 Producing a Strain of E.coli that Glows in the Dark $84.21

Includes instructions, E.coli, plasmid lux, control plasmid, calcium chloride, inoculating loops, transfer pipets, tubes, petri dishes, ampicillin,nutrient agar-ampicillin, and nutrient broth. All materials are provided sterile and ready for use.

Light

Dark

CONTROL PLASMID PLASMID LUX

The photograph in the bottom panel was taken in total darkness using only the light emitted from the bacteria that were transformed with plasmid lux.

CAT. NO. DESCRIPTION PRICEIND-6A Predigested DNA for agarose gels $66.27

Includes instructions and plasmids containing normal and mutant rabbit ß-globin genes predigested with EcoR1 and Hae III.

CAT. NO. DESCRIPTION PRICEIND-6B Nondigested DNA for agarose gels 76.54

Includes instructions and plasmids containing normal and mutant rabbit ß-globin genes, EcoR1, Hae III., restriction nuclease bu!ers and electrophoresis sample bu!er.

session. Sufficient sterile materials are provided for sixteen platings and the experiment is designed for 8 groups of students.

Additional experiments can be performed following completion of this experiment including:

1. Analysis of the effects of growth temperature and time on the glowing process. This analysis can be carried out by replating the transformed cells onto fresh nutrient agar-ampicillin plates, Nutrient agar can be purchased separately (see page 62).

2. Isolation of plasmid lux from transformed cells using the plasmid DNA isolation kit (cat. # 3-18) that is described on page 62.

PROCEDURE AND RESULTS1. Add E.coli to calcium chloride solution.2. After 15 minutes, add control plasmid and plasmid lux to two

tubes of cells and incubate both on ice for10 minutes.3. Incubate the tubes at 37°C, add nutrient broth and then spread the

bacterial suspensions onto agar plates.4. View the plates in the light and in the dark after two days at room

temperature.

1 2 3 4 5 6 7 8 DNA from the normal rabbit ß-globin gene (odd numbered lanes) and DNA from a deletion mutation of the gene, (even numbered lanes) were digested with EcoR1 (lanes 1, 2, 5, 6) or Hae III (lanes 3, 4, 7, 8) prior to this electrophoretic analysis. The mutant lacks DNA fragments which are found in the normal gene.

The results of the analysis enable the student to identify the type of mutation and the position of the mutation in the globin gene. The exercise was designed for eight groups of students and is provided in one of two forms. In IND-6A, the DNAs have been predigested with restriction enzymes and are ready for electrophoresis. In IND-6B, students digest the DNA with restriction enzymes prior to electrophoretic analysis. Electrophoresis Package 3/4 (Page 21) is needed, but not included.

DNA


This experiment can be used in a 1 hour class.

Mini Programs for DNA Analysis

Watch a video of this experiment in action at MODERNBIO.COM

30

Mini Programs for DNA Analysis

IND7. AMPLIFICATION OF A HEMOGLOBIN GENE BY THE POLYMERASE CHAIN REACTION PCR

R abbit ß- globin genes were amplified by PCR and electrophoresed on lanes 4 and 7 of this agarose gel. Lanes 2 and 5 show DNA size markers.

CAT. NO. DESCRIPTION PRICEIND-7 Ampli"cation of a Hemoglobin Gene by the $124.91

Polymerase Chain Reaction (PCR) Includes instructions , PCR bu!er, Taq DNA polymerase, DNA template, primers, nucleotides, Taq dilution bu!er, DNA size markers, electrophoresis sample bu!er, mineral oil, and PCR tubes.

The polymerase chain reaction (PCR) is one of the most powerful techniques used in molecular biology. With this method, a few ng of DNA can be amplified millions of times in a test tube in a few hours. The PCR has been used extensively in studies of gene structure and function. The method is also becoming increasingly important in DNA typing procedures such as DNA fingerprinting and in the identification and characterization of mutations that cause human diseases. This exercise is designed to illustrate the PCR in the teaching laboratory. In the experiment, students use PCR to amplify a rabbit ß-globin gene. The template for this reaction is a plasmid that contains this globin sequence. Following PCR amplification, the product of the reaction is analyzed on an agarose gel as shown on the right. The experiment was designed so that the PCR can be done manually provided that three standard water baths (45°C, 74°C, and 94°C) or three hot plates are available. The amplified globin DNA band can clearly

A ribosome is composed of a large subunit and a small subunit and each subunit contains specific RNAs that are associated with ribosomal proteins. Ribosomes play the same essential role in protein synthesis in all contemporary organisms and the genes for ribosomal RNAs in each subunit are highly conserved. The gene for the small-subunit ribosomal RNA (16 S-like) has been especially useful in evolutionary studies of distant phylogenetic relationships because it has remained quite stable during evolution of all organisms. The 16 S-like ribosomal genes can be amplified by the PCR from total DNA isolated from essentially any organism by using a single set of primers that recognize conserved regions of the gene. In this exercise, students use the PCR to amplify the gene for the 16 S-like ribosomal RNA from cow, pig, chicken and E.coli. The templates that are provided for this experiment are genomic DNAs from these organisms. In addition to these templates, students may amplify 16 S-like ribosomal genes using genomic DNAs that they have isolated from bacteria, plants or animals. These genomic DNAs can be isolated by using the genomic DNA isolation kit (Cat.# 3-19) described on page 62 of this catalog. The amplified 16 S-like ribosomal RNA genes can clearly be seen as discrete bands following staining of the agarose gels

IND10. PCR AMPLIFICATION OF A GENE FOR RIBOSOMAL RNA FROM DIFFERENT ORGANISMS

*The Polymerase Chain Reaction is covered by patents owned by Ho!man-LaRoche, Inc. The exercise described above is to be used for teaching purposes only.

CAT. NO. DESCRIPTION PRICEIND-10 PCR Ampli"cation of a Gene for Ribosomal $162.82

RNA from Di!erent Organisms Includes instructions, PCR bu!er, Taq DNA polymerase, DNA templates (cow DNA, pig DNA, chicken DNA, E.coli DNA), primers, nucleotides, Taq dilution bu!er, DNA size markers, electrophoresis sample bu!er, mineral oil, and PCR tubes.

I6 S - like ribosomal genes were ampli"ed by PCR using genomic DNA from seven different organisms. DNA size markers are shown in the "rst lane.

1 2 3 4 5 6 7 8

with methylene blue as shown on the right. The experiment was designed so that the PCR can be done manually provided that three standard water baths (45°C, 74°C, and 94°C) or three hot plates are available. The amplification reaction requires approximately 2 hours. Sufficient materials are provided so that 8 groups of students can perform the experiment with each group amplifying the ribosomal RNA gene from one template. The following materials are needed but not included: Three water baths (or an authorized DNA Thermal Cycler), electrophoresis equipment and Electrophoresis Package 3/4. (See page 21)

be seen following staining of the agarose gels with methylene blue. The amplification reaction requires approximately 1.5 hours. Sufficient materials are provided so that 8 groups of students can perform the experiment. The following materials are needed but not included: Three water baths (or an authorized DNA Thermal Cycler), electrophoresis equipment and Electrophoresis Package 3/4 (See page 21).

DNA

31

Satellite DNAs are highly repeated sequences of unknown function. The satellite DNA from the meal worm beetle is remarkable since it represents over 50% of this insects genome. In this exercise, students first isolate DNA from beetle larvae by a simple and safe procedure. They then digest the DNA with EcoR1 and examine the satellite DNA following electrophoresis as shown on the gel at the right. Sufficient materials are provided so that the experiment can be carried out twice by eight groups of students. The exercise can be completed in two, 2 to 3 hour laboratory sessions and requires ethyl alcohol, a microcentrifuge and Electrophoresis Package 3/4 (see page 21).

CAT. NO. DESCRIPTION PRICEIND-12 Characterization of the Satellite DNA from $75.52

the Meal Worm Includes instructions, meal worm beetle larvae, EcoR1(2), EcoR1 bu!er, extraction bu!er, SDS, ammonium acetate, transfer pipets, sample bu!er, and DNA Standards.

IND12. CHARACTERIZATION OF THE SATELLITE DNA FROM THE MEAL WORM

IND21. IDENTIFYING GENOMIC AND PLASMID DNA SEQUENCES IN E.COLI BY COLONY PCR

Purified DNA is most often used as a template in the PCR reaction. However, it is possible to amplify specific DNA sequences without DNA purification by starting with a single living E.coli colony. This technique is known as colony PCR and provides a powerful and reliable method for the rapid amplification and isolation of any gene in the E.coli genome or any gene on a plasmid that is carried by E.coli. In this exercise, students carry out colony PCR starting with a culture of E.coli that carries an ampicillin-resistance gene on plasmid pUC18. They first streak the cells over an nutrient agar plate in order to produce single E.coli colonies. These colonies are then used directly as templates in a PCR reaction in order to amplify a segment of the 16 S -ribosomal RNA genes which is located on the E.coli chromosome and a segment of the ampicillin-resistance gene on the plasmid. Sufficient materials are provided so that 8 groups of students can perform the experiment with each group amplifying the ribosomal RNA gene, the ampicillin-resistance gene or both genes. Typical results of the experiment are shown on the right. A DNA Thermal Cycler, electrophoresis equipment and Electrophoresis Package 3/4 (See page 23) or equivalent are needed but not included.

CAT. NO. DESCRIPTION PRICEIND-21 Identifying Genomic and Plasmid DNA

Sequences in E.coli by Colony PCR $168.39 Includes instructions, E. coli-pUC 18, nutrient agar-ampicillin, ampicillin, petri dishes, inoculating loops, ampicillin-resistance gene primers, rDNA primers, sterile pipet tips, PCR bu!er,Taq polymerase, Taq dilution bu!er, nucleotides, DNA size markers, electrophoresis sample bu!er, mineral oil, and PCR tubes.

16S-ribosomal RNA genes which are located on the E. coli chromosome and the ampicillin-resistance gene on a plasmid were ampli"ed by colony PCR using a single E. coli colony as template. From left to right, samples are DNA markers, the 16 S -ribosomal RNA gene,the ampicillin-resistance gene and both genes.

1 2 3 4 5 6 7 8

In this exercise, DNA isolated from beetle larvae was digested for 0, 5, and 20 minutes (lanes 1-3, 5-7) with EcoR1 prior to this electrophoretic analysis. Note the progressive breakdown of the DNA satellite to the satellite monomer which is 142 base-pairs in length. DNA size markers are shown in lanes four and eight.

DNA

32

The budding yeast Saccharomyces cerevisiae has served as one of the most important model organism for the study of eukaryotic molecular biology. The entire yeast genome has been sequenced and the function of many yeast genes is now known. This rapid progress has relied heavily on the ability to introduce replicating plasmids into yeast. A typical replicating yeast plasmid contains an origin of replication known as an autonomously replicating sequence or ARS and a selectable marker gene that enables the yeast to grow in the absence of a nutrient such as uracil. In this exercise, students introduce such a plasmid into yeast and then select for transformants by plating the cells on a nutrient-agar medium lacking uracil. They then study the stability of the plasmid in yeast cells by growing the cells in the presence of uracil. The program lasts two, 2 to 3 laboratory sessions and requires basic training in microbiology and was designed for 8 groups of students. The following materials and equipment are needed but not included: microcentrifuge, table-top centrifuge, autoclave to prepare media, bacterial spreading rod, Bunsen burners, sterile water and microliter dispensers.

CAT. NO. DESCRIPTION PRICE IND-15 Includes instructions, four strains of E.coli $82.69

(A-D: teacher keyed), inoculating loops, nutrient agar, petri dishes, ampicillin, X-gal, X-gal solvent.

Genes introduced into E.coli by plasmid-mediated transformation can confer variation in the phenotype of the bacteria. For example, E.coli containing the ampicillin-resistance gene grows in the presence of this antibiotic while the product of the ß-galactosidase gene enables the bacteria to convert the ß-galactosidase substrate X-gal to a blue product. Similarly, E.coli containing the Lux operon produce colonies that glow in the dark. In this exercise, four different strains of E.coli are streaked onto agar plates containing media that selects for the different strains and that reveals variation in colony phenotype. Students record these variations and the results enable them to deduce the genotype of the strains as shown in the Table below. Sufficient sterile materials are provided to prepare 32 plates and the experiment was designed for 8 groups of students. The exercise requires two 1 hour laboratory sessions.

A NoB Yes White No Ampicillin ResistanceC Yes Blue No Ampicillin Resistance+GalactosidaseD Yes White Yes Ampicillin Resistance+Lux

Colony Phenotype Genotype Gene(s) ResponsibleE.coli

Strain Ampicillin on X-Gal Plates the dark

IND13. TRANSFORMATION AND ANALYSIS OF YEAST

IND15. BACTERIAL PHENOTYPE

IND28. SYNTHETIC BIOLOGY: USING BACTERIAL COMPUTERS TO SOLVE THE PANCAKE PROBLEMSynthetic biology is an exciting new field that uses engineering principles and mathematical modeling to design and construct biological devices. Synthetic biology projects include the construction of bacterial computers that can solve mathematical problems. Microbial machines, in the form of genetically engineered E. coli cells, have solved a variety of mathematical problems, which have had important applications in biology, medicine and technology. This investigation introduces students to synthetic biology principles as they use genetic recombination in bacterial computers to solve the Burnt Pancake Problem. In the project, the biological equivalent of a burnt pancake is a functional module of DNA. Similar to stacks of pancakes burnt on one side, DNA modules have directionality, require a specific order and can be flipped by genetic recombination. The bacterial computer is investigated using a modular system in which pancake stacks are assembled from flippable DNA segments. Flipping of the DNA segments "pancakes" is performed by a DNA recombination system and the flipping is monitored by antibiotic sensitivity of the bacteria and colony color. The investigation interlaces biology laboratory work with mathematical modeling, thereby allowing students to explore the rich interface between biology and mathematics. Sufficient sterile media, antibiotics, cells, plasmids and other materials are provided so that 16 students working in pairs can perform the analysis.

Panel A shows the starting con"guration of the burnt pancake bacterial computer. The double helix "gures indicate the three recombination sites for a speci"c recombinase called Hin. The two DNA segments between these sites de"ne the pancakes. Depending on the orientation of these segments, the gene for the red %uorescent protein (RFP) and tetracycline resistance gene (Tet) are either active or inactive and these activities are monitored by the student to deduce these orientations. Panel B shows the di!erential expression of the RFP in di!erent bacterial clones as a result %ipping induced by the recombinase. Panel C shows the activities carried out during the 5 laboratory sessions that comprise this exploration.

CAT. NO. DESCRIPTION PRICE IND-13 Transformation and Analysis of Yeast $190.68

Includes instructions, plasmid DNA, carrier DNA, lithium acetate, polythylene glycol, 10 x TE bu!er, dehydrated nutrient agar minus uracil, dehydrated nutrient broth minus uracil, uracil, sterile microtubes, sterile culture tubes inoculating loops, and uracil plus and uracil minus yeast cultures.

CAT. NO. DESCRIPTION PRICE IND-28 Synthetic Biology: Using Bacterial Computers

to Solve the Pancake Problem $180.00



DNA


33

Today’s cell biology is a fascinating blend of molecular biology, biochemistry, and a variety of traditional disciplines. These fields are integrated into this program in order to introduce students to selected topics in eukaryotic cell biology. In the program, students localize enzymes in plant and animal cells, perform cell fractionation procedures, and study the properties of a specific membrane receptor. Each exercise can be completed in one 3-hour or two 90-minute laboratory sessions. Microscopes, slides, microliter dispensers, small test tubes, tabletop centrifuge, and ethyl alcohol are needed but not provided.

EXPERIMENTS

Contemporary Cell BiologyStandard Laboratory Program 7

701. Enzyme CytochemistryThe concept that different enzymes are found in different tissues, cell types, and cell organelles is illustrated in this multipart exercise where students use contemporary techniques to localize specific enzymes in cells and tissues. Students are introduced to enzyme cytochemistry in an experiment on the germinating corn seed where they show that peroxidase is produced by the aleurone, a cell layer that surrounds the endosperm. They then characterize the subcellular distribution of peroxidase in giant onion epithelial cells and show that the enzyme resides in the cell wall. In the final experiment of the series, the subcellular distribution of lactate dehydrogenase in chicken erythrocytes is examined where students learn that this enzyme is confined to erythrocyte cytoplasm.

702. Analysis of a Cell-Surface ReceptorChemical signaling between cells in multicellular organisms is frequently mediated by cell-surface receptors. The receptors for neurotransmitters, protein hormones, growth factors, and plant lectins are a few of the many known examples of these important membrane components. In this exercise, students examine the cell location and properties of the receptor for the lectin concanavalin A. In the first experiment of the series, students use a concanavalin A-peroxidase complex in a microscopic assay to show that the specific receptor is found on the surface of their own cheek epithelial cells. In the second part of the exercise, students study the characteristic hemagglutination reaction that is elicited by concanavalin A and learn that the reaction is due to multiple sites for receptor binding on the concanavalin A molecule. It is known that concanavalin A binds selectively to mannose residues on the cell-surface glycoprotein receptor. This specificity is confirmed in the final experiment of the series where students show that mannose but not other sugars inhibit concanavalin A binding and the hemagglutination reaction. This innovative exercise provides a detailed picture of a cell-surface receptor and presents strategies that are used to study this important class of regulatory molecules. Typical results of selected portions of the experiment are shown on the right.

703. The Cell NucleusThe exercise is introduced with a discussion of observations that were made in the early nineteenth century. Namely, that biological stains often show specific affinity for particular subcellular components. With this information, students use differential staining procedures to identify nuclear and cytoplasmic components in cells from thymus and onion root tip. In the second part of the exercise, cell nuclei are isolated from thymus tissue and their purity assessed by staining techniques. The DNA is isolated from the nuclei by a procedure designed to provide information on the packaging of this macromolecule in the chromosome. The exercise gives students experience with both classical and contemporary techniques, and makes them aware that cell and molecular biology have long histories that can be traced to traditional disciplines.

STANDARD LABORATORY PROGRAM 7CAT. NO. DESCRIPTION PRICES7 The Chemical Package for 16 students plus one 204.50

student manual and one instructor manual.S7-BK 8 Student Manuals $63.14 S7-SM Sample Student Manual (47 pages) plus one $9.61

instructor manual.

INDIVIDUAL EXPERIMENTSEXP-701 Enzyme Cytochemistry $71.77EXP-702 Analysis of a Cell-Surface Receptor $84.12EXP-703 The Cell Nucleus $71.77


The results of EXP 702 show that mannose selectively inhibits the concanavalin A-induced hemagglutination reaction and the binding of concanavalin A to receptors on the surface of cheek epithelial cells.

EXP-701 EXP-702 EXP-703 Chicken Blood Smears Hydrogen Peroxide DNAse I Plastic Trays with Lids Tris Buffer Chloronapthol Tris Buffer Con A Methylene Blue Methylene Blue Con-A-Peroxidase Eosin Hydrogen Peroxide Mannose Onion Bulbs Chloronapthol Con-A-Buffer Calf Thymus Tetrazolium Galactose Nuclear Buffer Iodine Solution Transfer Pipets Hydrogen Transfer Pipets Peroxide Corn Seed TetrazoliumEosin Transfer Pipets Tris BufferLDH-Substrate SDS NP-40 5M NaCl

DNA

Electrophoresis equipment not required. Concanavalin A-Induced Hemagglutination

- Mannose + Mannose

Concanavalin A Binding - Mannose + Mannose

34

IND2. TISSUE PRINTINGLocating specific proteins and nucleic acid molecules in tissue sections is an important goal in cell biology. An effective and simple technique for this purpose is tissue printing which permits the localization of specific macromolecules in animal and plant tissues. Here students perform this technique to examine the tissue distribution of the enzyme peroxidase in plants. First, students section carrots, celery, and other vegetables with razor blades and transfer the proteins from the cut sections to nitrocellulose membranes by application of gentle pressure. The enzyme peroxidase is detected on the membranes using a blue color producing peroxidase substrate as shown on the left. All proteins on the membranes are then stained red with a general protein stain which highlights the selective nature of the peroxidase distribution. Students also determine the amount of peroxidase in plant extracts. This 2 hour exercise provides an exciting lesson in plant histology and introduces your student to an important technique used in contemporary cell biology. The exercise was designed for eight groups of students and includes: nitrocellulose membranes, transfer pipets, 200 x extraction buffer, peroxidase standard, chloronapthol, 4M Tris buffer, hydrogen peroxide, and 10 x protein blot stain. Assorted vegetables which can be purchased at your local supermarket and petri dishes are needed, but not provided.

Cross-sections of carrots (large circles), parsnips (small circles), and celery (horseshoe-shaped) were prepared with a razor blade. The proteins at the cut surface of the sections were transferred to nitrocellulose membranes by application of gentle pressure for 10 seconds. The nitrocellulose was then incubated with a color producing peroxidase substrate. Note that the peroxidase is found primarily in the epidermis and in some vascular (phloem) elements.

IND3. THE ELISA IMMUNOASSAY

The ELISA (enzyme-linked immunosorbant assay) is a powerful immunological method for detecting specific proteins in complex protein mixtures. The ELISA has become an important tool for the cell and molecular biologist. It is increasingly being applied in clinical medicine for detecting proteins associated with disease including antibodies produced in response to infection by the HIV virus. The method is also easy to perform and yields graphic results making it well suited for the teaching laboratory. In this exercise, students carry out the ELISA to study the specificity of antibody-antigen interactions. The basic procedures and typical results of the exercise are shown on the right. The 2-3 hour exercise was designed for sixteen students working in pairs and includes: microtitration plates, transfer pipets, chicken serum, cow serum, horse serum, rabbit serum, gelatin, tris buffer saline, tris buffer saline + NP-40, goat anti-rabbit IgG-peroxidase, TMB-peroxidase substrate, and hydrogen peroxide. Microliter dispensers are needed but not provided, and a colorimeter is desirable but not absolutely necessary.

Serum Concentration1. Varying dilutions of serum proteins from chicken (D and H), cow (C and G), horse (B and F), and rabbit (A and E) are adsorbed to the surface of wells of a microtitration plate. The antigen used for the experiment is the IgG molecule from rabbit serum.2. The immobilized serum proteins are incubated with a peroxidase-linked antibody to the rabbit antigen. During this incubation, the antibody binds preferentially to the antigen in rabbit serum.3. The bound antibody is detected by a color-producing reaction and the amount of color development is proportional to the amount of antigen present. Note maximal color development occurs with the rabbit serum proteins.

CAT. NO. DESCRIPTION PRICEIND-2 Tissue Printing $73.84

CAT. NO. DESCRIPTION PRICEIND-3 The ELISA Immunoassay $73.84

CELL

BIO

LOGY

Mini Programs in Cell Biology



35

This program was designed to actively engage students in exciting biological research projects of their own design. The projects focus on peroxidases, which form a large family of related enzymes that are ubiquitous in plants. Plant peroxidase isoenzymes can be tissue specific, developmentally regulated and display variable tissue and, high salt and disease resistance defense reactions. This induction may be related to the abilities of peroxidases to strengthen the plant cell wall and to kill microorganisms.

Students begin their projects with a hypothesis, which is a statement of an ideal that they will test in the laboratory. They then test their hypothesis by carrying out a series of experiments using the materials provided with the program and vegetables, intact plants or roots and stems. They first use the technique of tissue printing which enables them to localize the peroxidases in tissue sections. They then quantify peroxidase activity by using a DOT-blot assay and spectrophotometric procedures. Students also carry out an electrophoresis analysis in order to characterize peroxidase isoenzymes in plant extracts that they prepare. In the final section of the program, students are given detailed instructions for organizing their data for presentation in a scientific paper. They are then instructed to write a paper that conforms to the style of a scientific publication using the detailed steps that are presented in the laboratory manuals. This program for requires about three 2-3 hour laboratory sessions. Electrophoretic equipment, spectrophotometer, and plants/vegetables are needed but not provided. The materials that are provided for the program and a few of the nearly infinite number of titles of student papers are given on the right.

A FEW TITLES OF STUDENT PAPERS

isoenzyme profiles of peroxidases in the turnip.

isoenzymes in different regions of the carrot.

IND22. CHARACTERIZATION OF PEROXIDASES IN PLANTS: STUDENT DESIGNED RESEARCH PROJECTS

Chemical Package ContentsPeroxidase AssaysHydrogen Peroxide (3)Chloronapthol (5)200 X Extraction Buffer (3)Color Development BufferPeroxidase Standard (3)10 X Protein Blot StainNitrocellulose (4)Transfer pipetes

ElectrophoresisAgaroseElectrophoresis Buffer Sample BufferHRP-BasicHRP-MixtureCytochrome CSerum Albumin-Hemoglobin

CAT NO. DESCRIPTION PRICEIND-22 Characterization of Peroxidases in Plants $139.21

CELL BIOLOGY

Cyanobactera, also known as blue-green algae, obtain their energy by photosynthesis using sunlight as their energy source. These organisms have been considered to be the oldest and the most important bacteria on the earth. It is believed that they were responsible for the initial oxygenation of the earth's atmosphere through photosynthesis and it is also felt they were the precursors to the chloroplasts that are found in true algae and plants. There are two classes of photosynthetic pigments in Cyanobactera. The first class contains water-soluble proteins and the major protein is called Phycocyanin, which is blue. The other classes of photosynthetic pigments that include the

carotinoids and chlorophylls are small molecular weight molecules and are insoluble in water but soluble in organic solvents such as alcohol. In this laboratory exercise, 8 groups of students isolate and characterize both groups of pigments. In part A of this exercise, students prepare a water-soluble extract from blue green algae and

IND29. ELECTROPHORETIC AND CHROMATOGRAPHIC ANALYSIS OF PHOTOSYNTHETIC PIGMENTS FROM BLUE GREEN ALGAE

Chemical Package ContentsWhat's IncludedComplete Instructions Blue –Green AlgaeDye Sample 2Dye Sample 2Glycerol SolutionThin-Layer SheetChromatographic Tubes

1.5 ml Tubes1ml Transfer Pipits

Bovine HemoglobinAccessories and Chemicals NeededElectrophoresis Equipment Automatic Pipetors (Optional)Methanol

CAT NO. DESCRIPTION PRICEIND-29 Electrophoretic and Chromatographic Analysis of $65.28

Photosynthetic Pigments from Blue- Green Algae

show that it contains the single major protein Phycocyanin by electrophoresis as shown in the gel below. They also determine the charge of this protein by comparing its electrophoretic mobility to the mobilities of dyes with known charges. In part B, they prepare an alcohol extract and analyze the smaller alcohol soluble pigments by thin layer chromatography in order to identify the chlorophylls and major carotenoid pigments. The results of this two-part study give students practical hands-on experience with isolation of components from cells as well as electrophoresis and thin layer chromatography and introduces them to one of the most important organisms on the earth.

Extra nitrocellulose packets available. See page 62 for details.

36

Mini Programs in Cell Biology

Study Questions and Data Analysis

1. Indicate the tree (A, B, C or D) shown below which is consistent with the results of your experiment and explain the reasons for your choice.

2. Does your answer given above agree with what is known about the phylogeny of these vertebrates?

3. Construct a standard curve for the gamma globulin by plotting the concentration of purified gamma globulin against relative spot intensities. Using this curve, estimate the concentration of gamma globulin in the cow serum sample.

IND17. A RAPID IMMUNOLOGICAL METHOD TO STUDY EVOLUTION

Each protein carries in its amino acid sequence information pertaining to its evolutionary history and origin, and provides clues to the evolutionary history of the organism in which it is found. Indeed, proteins existing today are in effect living fossils. This concept is illustrated in this exercise where eight groups of students examine the abilities of antibodies against cow gamma globulin to react with gamma globulins in the sera of cow, goat, sheep, horse, and chicken. The results of the experiment will enable the student to answer the questions on the right which are posed in the Student Guide provided with the exercise. Answers to these questions are given in the Instructor Guide. The method used for the experiment involves dotting small quantities of serum onto nitrocellulose and then incubating the nitrocellulose with an enzyme-linked antibody against cow gamma globulin. Following development of the nitrocellulose, purple dots appear with intensities that are proportional to the reactivity of the antibody. The entire exercise was designed for eight groups of students and can be completed within a 2-hour laboratory period. Microliter dispensers are needed but not provided and electrophoresis equipment is not required.

This miniprogram was designed to give students a basic understanding of enzyme kinetics. In the first experiment in this series, students prepare an extract from wheat germ. They then determined the initial velocity (Vo) of the reaction catalyzed by purified acid phosphatase and by the acid phosphatase activity present in the extract. From these data, they estimate the amount of the enzyme that is present in the wheat germ. In the second experiment, the student examines the effects of substrate concentration on the reaction velocity. Data are analyzed using Michaelis-Menton and Lineweaver-Burk plots and students

calculate values of Vmax and Km. The series can be completed in two 2-3 hour laboratory sessions and requires a colorimeter or spectrophotometer and a table-top centrifuge.

IND14. ENZYME KINETICS

CAT. NO. DESCRIPTION PRICEIND-14 Enzyme Kinetics $76.78

Includes instructions, wheat germ, acid phosphatase, extraction bu!er, acid phosphatase substrate, substrate dilution bu!er, nitrophenol standards, small transfer pipets and large transfer pipets.


IND-17 A Rapid Immunological Method to Study Evolution $84.86

Sheep Sheep

SheepSheep

HorseHorse

Horse

Horse

Goat Goat

GoatGoat

Cow Cow

CowCow

Chicken Chicken

Chicken

Chicken

Reptiles Reptiles

Reptiles Reptiles

A B

C D

8 sheets of nitrocelluloseblotting paperdishes for nitrocellulose incubationanti-Cow IgG (Peroxidase linked)purified cow IgGcow serumhorse serumgoat serumsheep serum

chicken serumprotein blot stainhydrogen peroxidechloronaptholgelatinTris bufferTris buffer saline (TBS)TBS+NP-40


CELL

BIO

LOGY



37

Zoology Histology Developmental Anatomy

IND19. AN INTRODUCTION TO VERTEBRATE HISTOLOGY

Students prepare and then study sections of trachea and esophagus in order to identify and characterize the major tissue types ( repithelia, connective tissue and muscle). They then study selected organs including ovary, testis, intestine, spinal cord and skin. The picture above shows a student-prepared slide illustrating the trachea and esophagus.

College students are frequently introduced to basic histology, zoology and developmental anatomy during their freshman and sophomore years. Traditionally, this is accomplished by the study of prepared microscope slides. The approach does not teach students how slides are made and students frequently become disinterested and bored because they are viewing prepared biological materials. Modern Biology Inc. now offers three miniprograms that were designed to circumvent these limitations. The starting point for the programs are precut tissue sections which are ready for staining. Thus, expensive slide–making equipment is not required. During the first 2-3 hour laboratory session, students process the sections through stains (hemotoloxin and eosin) and prepare permanent slides of the tissues. A nontoxic and biodegradable histological clearing agent is used in the procedure which eliminates hazards associated with the use of xylene or toluene-based clearing agents. During the second and third laboratory sessions, students follow the well-illustrated laboratory guides to receive a fine introduction to histology, zoology or developmental anatomy. Each of these programs is an independent teaching unit and is designed for a class of 16 students working in pairs. Sufficient materials are provided so that 8 groups can make 2 slides each. Microscopes and forceps are needed but not provided. Although the programs were designed to integrate three 2-3 hour laboratory sessions in classical biology into freshman or sophomore–college level biology classes, they can also can be used to supplement more advanced courses in histology, developmental biology and zoology.

IND18. AN INTRODUCTION TO ZOOLOGY

Molecular and cell biology can be understood and appreciated only in the light of their relations to the

whole organism. However, in recent years it has become fashionable to reduce or to eliminate altogether laboratory time allotted for organismic biology. In this program, students prepare and then study cross sections of

planaria, earthworm, lamprey (larvae), and tadpole. The laboratory guides emphasize hierarchies of animal complexity and the similarities and differences between the invertebrates and vertebrates. The picture above shows a student-prepared slide of the earth worm.

IND20. AN INTRODUCTION TO DEVELOPMENTAL

ANATOMY

Students prepare and then study sagital sections of the 72-hour chick embryo in order to obtain a clear-cut picture of their developmental anatomies. The picture above shows a student-prepared slide

CAT. NO. PRICEIND-18 An Introduction to Zoology $115.88

CAT. NO. PRICEIND-19 An Introduction to $124.19

Vertebrate Histology

CAT. NO. PRICEIND-20 An Introduction to $163.45

Developmental Anatomy

CELL BIOLOGY

Chemical Package ContentsPlanarian Slides (4)Earthworm Slides (4)Lamprey (larvae) Slides (4)Tadpole Slides(4)HematoxylinEosin YIsopropanol (2)

Clearing Agent Mounting MediumCoverslipsSlide Staining Dishes (8)Transfer Pipets (20)Complete Instructions

Chemical Package ContentsEsophagus/Trachea Slides (8)Ovary Slides (2)Testis Slides (2)Duodenum Slides (2)Spinal Cord (2)Skin (2) Hematoxylin

Eosin YIsopropanol (2)Clearing AgentMounting MediumCoverslipsSlide Staining Dishes (8)Transfer Pipets (20)Complete Instructions


72 hr Chick Slides (8)HematoxylinEosin YIsopropanol (2)Clearing Agent

Mounting MediumCoverslipsSlide Staining Dishes (8)Transfer Pipets (20)Complete Instructions

38

IND26. LOCALIZING TUBLIN BY IMMUNOHISTOCHEMISTRY

Microtubules are hollow cylinders made up of polymers of

the protein tubulin. Microtubules are major components

of cilia and flagella, which are tail like projections that

are covered by a plasma membrane and extend outwards

from the cell. Motile cilia are used for locomotion and food

gathering by some protozoa and are found in the lining of the

trachea, where their wave like motion propels mucus, dust

and other foreign matter out of the lungs.

In this exercise, students use the powerful technique of

immunohistochemistry to localize tubulin in the esophagus

and trachea. The provided tissue sections are exposed to a

tubulin monoclonal antibody, which binds to the tubulin. The

sections are then incubated with a secondary antibody, which

binds to the first antibody. The second antibody is complexed

with peroxidase, which catalyzes a color producing reaction.

Following the addition of a non-toxic peroxidase substrate,

the peroxidase converts the colorless substrate to a blue

product, thus “staining” the area containing tubulin. As can

be seen in the photographs in panels A-C at the right, the cilia

that line the trachea are stained in a highly selective manner

in keeping with the high concentrations of microtubules that

make up these structures.

Panels A-C show the localization of alpha tubulin in the esophagus and trachea. The pictures were taken at increasing magni"cation (40X-1000X). Note that the cilia (indicated by the arrows) that line the trachea are stained in a highly selective manner. Panel D shows the esophagus and trachea slides stained with hematoxylin and eosin.

IND-26 Localizing Tubulin by Immunohistochemistry $105.62(Complete Experiment)

Detailed instructions, 8 unstained trachea/esophagus slides, clearing agent, mounting medium, coverslips slide staining dishes, large transfer pipets, small transfer pipets, glass rods, tris bu!er saline, alpha tubulin antibody, peroxidase labeled second antibody, peroxidase substrate, BSA blocking solution, triton x 100, hematoxylin and eosin.

IND-26AS Tubulin Antibodies and Peroxidase Substrate $63.19 (Use your own tissue sections) This kit provides the basic chemicals and antibodies that can be

used to detect alpha tubulin by immunohistochemistry in cells or tissue sections of your choice. Includes instructions and enough reagents for the immunohistochemical detection of tubulin in 10-25 slides. Includes 1ml each of alpha tubulin antibody, peroxidase labeled second antibody, peroxidase substrate and BSA blocking solution. The IND-26AS does not include tissue sections, stains, or other solutions.

Microscopes are now available on page 56.

This exercise was designed for eight groups of students. Five

groups use the above procedure, one group carries out the

procedure without the tubulin antibody (as an important

negative control) and two groups stain their sections with

hematoxylin and eosin as seen in panel D. The analysis is

supplied complete with all of the instructions, chemicals, and

accessories needed to perform perhaps the most important

method used by the cell biologist. Sufficient antibodies,

peroxidase substrate and other reagents are provided so that

between 5-10 additional slides (provided by the instructor)

can be analyzed by the procedure. Distilled water and ethyl

alcohol or isopropyl alcohol are needed but not included.

CELL

BIO

LOGY

39

The two overview programs were prepared for the beginning college-level student and provide all of the chemicals and manuals that are needed for 16 students working in pairs to perform an integrated series of experiments on molecular biology. Each program contains three experiments and each experiment can be completed within a 2-hour laboratory session. The program manuals contain extensive background information sections and clearly outlined protocols which serve to acquaint students with molecular biology and with electrophoresis theory and techniques. These Overview Programs provide stimulating laboratory exercises and a fine introduction to molecular biology for the beginning student.

THE OVERVIEW PROGRAMS

Price List - Overview Program 1CAT. NO. DESCRIPTION PRICEO1 The Chemical Package for 16 students working in $107.12

pairs plus one student manual and one instructor manual. (The student manual may be reproduced for educational purposes).

01-BK 8 Student Manuals $48.12 O1-SM Sample Student Manual (29 Pages) plus one $6.93

instructor manual.

Price List - Overview Program 2CAT. NO. DESCRIPTION PRICEO2 The Chemical Package for 16 students working in $142.72

pairs plus one student manual and one instructor manual. (The student manual may be reproduced for educational purposes.)

Q2-BK 8 Student Manuals $48.12 O2-SM Sample Student Manual (36 pages) plus one $6.93

instructor manual.

A Molecular Approach to the Study of Genetics and Evolution

Overview Program 1 The program contains selected experiments from Standard Program 1 and stresses the relevance of molecular biology to the study of human genetics and disease. The experiments provide an introduction to electrophoresis and to protein composition, structure, and function in health and disease. The exercises in the Program are:101. Electrophoretic Separation of Proteins

*102. Genetics and Sickle Cell Anemia

*106. Protein Fingerprinting

*See page 10 for description.

An Introduction to Molecular Genetics

Overview Program 2 This outstanding program provides a solid foundation in molecular genetics and will prepare your students for advanced college-level courses. Your students will investigate such contemporary topics as DNA electrophoresis, restriction nuclease mapping, and molecular cloning procedures. Overview 2 contains the following experiments selected from Standard Program 3:*301. The Length of DNA Molecules

*302. Restriction Nuclease Mapping of DNA

*304. Molecular Cloning (Part A only)

*See page 18 for description.

This series of three experiments provides an introduction to agarose gel electrophoresis. In the first experiment, students identify unknown dye molecules by comparing their electrophoretic migration with the migration of known dyes. In the second, they identify dye molecules that bind to DNA and determine the mechanism. They then study four proteins in experiment 3 and relate differences in their charges to their migration rates in an electric field. Each protein is a different color so that its progress during the separation can easily be followed. The experiments were designed for 8 groups of students and the package includes four colorful dye mixtures, four colored proteins, DNA, agarose, electrophoresis buffer, gel loading transfer pipets and complete instructions. Each experiment can be completed within a 2-hour laboratory period. The exercises in the program are:*101. Electrophoretic Separation of Proteins *IND-24. An Introduction to Electrophoresis

*See page 10 for description. *See page 11 for description.

An Introduction to ElectrophoresisOverview Program 3

CAT. NO. DESCRIPTION PRICEO3 The Chemical Package for 16 students working in $54.82

pairs plus 1 Student Manual and 1 Instructor Manual. (The student manual may be reproduced for educational purposes)

CAT. NO. DESCRIPTION PRICEO3-SM Sample Student Manual (29 pages) plus $6.12

one instructor manual.03-BK 8 Student Manuals $28.16

CELL BIOLOGY

40

A Laboratory Course in Innovative Biology (LC6)

Proteins and Pigments 1. Electrophoretic and Chromatographic Analysis of Photosynthetic Pigments from Blue-Green Algae. Cyanobactera, also known as blue-green algae, obtain their energy by photosynthesis using sunlight as their energy source. These organisms have been considered to be the oldest and the most important bacteria on the earth. It is believed that they were responsible for the initial oxygenation of the earth’s atmosphere through photosynthesis and it is also felt they were the precursors to the chloroplasts that are found in true algae and plants. There are two classes of photosynthetic pigments in Cyanobactera. The first class contains water-soluble proteins and the major protein is called Phycocyanin, which is blue. The other classes of photosynthetic pigments that include the carotinoids and chlorophylls are small molecular weight molecules and are insoluble in water but soluble in organic solvents such as alcohol. In this laboratory exercise, students isolate and characterize both groups of pigments. In part A of this exercise, students prepare a water-soluble extract from blue green algae and show that it contains the single major protein Phycocyanin by electrophoresis as shown in the gel. They also determine the charge of this protein by comparing its electrophoretic mobility to the mobilities of proteins and dyes with known charges. In part B, they prepare an alcohol extract and analyze the smaller alcohol soluble pigments by thin layer chromatography in order to identify the chlorophylls and major carotenoid pigments. The results of this two-part study give students practical hands-on experience with isolation of components from cells as well as electrophoresis and thin layer chromatography and introduces them to one of the most important organisms on the earth.

2. Speci!city of Albumin BindingThe binding of an enzyme to its substrate is only one example of the many specific molecular interactions that occur in biological systems. An analogous binding process occurs with serum albumin, which binds certain small molecular weight compounds and serves as a carrier molecule for these compounds in blood. In this exercise, students use an electrophoretic assay to examine the binding of various dyes to cow albumin. The results of this graphic analysis show that the binding of dyes to albumin is saturable, specific, compatible, and dependent on the native structure of the protein.

Enzymes and Metabolism3. Enzyme Action and KineticsThis set of experiments was designed to give students a basic understanding of enzyme kinetics. In the first experiment in this series, students prepare an

This Program is a complete laboratory course for teaching general biology or introductory cell and molecular biology. This program, like all of our others, is designed for 16 students working in pairs. This course provides essentially all of the chemicals and instructions that are needed to teach fifteen 3-hour laboratory sessions or twenty-five 1-hour laboratory sessions. The course consists of a series of experiments that are presented in a comprehensive integrated laboratory manual. In the first two sections of the course, students study topics in protein biology and biochemistry such as protein structure, function, and isolation. Experiments on enzyme kinetics and cellular metabolism are then carried out. Students perform a project of their own design in the second section of the course. The projects focus on the characterization of plant peroxidases. A number of other optional student-designed experiments are outlined in the Instructor manual of the program and basic reagents are provided in order for the student to carry out their hypothesis driven projects. Experiments on the properties and structure of DNA are presented in the next section of the course. Here, students perform experiments that deal with genome organization, and specific gene function. Techniques include DNA electrophoresis, cell fractionation, DNA isolation, restriction nuclease mapping, and basic cloning procedures. In the final section of the course students study the genetics, biochemistry and molecular biology of hemoglobin.

extract from wheat germ. They then determined the initial velocity of the reaction catalyzed by purified acid phosphatase and by the acid phosphatase activity present in the extract. From these data, they estimate the amount of the enzyme that is present in the wheat germ. In the second experiment, the student examines the effects of substrate concentration on the reaction velocity. The results enable them to determine the Vmax and Km of the enzyme catalyzed reaction.

4. E"ects of Temperature on RespirationRespiration can be viewed as a series of enzyme catalyzed reactions in which carbohydrates, proteins, and fats are broken down to carbon dioxide and water with the release of energy. During the process, hydrogen is removed from the fuel molecules and oxygen is consumed. With this background information, students measure oxygen consumption and hydrogen liberation in germinating barley and corn at different temperatures. The program provides eight calibrated respirometers for measurement of oxygen consumption and the chemicals required to perform a graphic dye reduction assay.

Cell Biology and Molecular Evolution5. A Rapid Immunological Method to Study EvolutionEach protein carries in its amino acid sequence information pertaining to its evolutionary history and origin, and provides clues to the evolutionary history of the organism in which it is found. Indeed, proteins existing today are in effect living fossils. This concept is illustrated in this exercise where students examine the abilities of antibodies against cow gamma globulin to react with gamma globulins in the sera of cow, goat, sheep, horse, and chicken. The results of the experiment will enable the student to answer the questions posed in the Student Guide provided with the exercise. Answers to these questions are given in the Instructor Guide. The method used for the experiment involves dotting small quantities of serum onto nitrocellulose and then incubating the nitrocellulose with an enzyme-linked antibody against cow gamma globulin. Following development of the nitrocellulose, purple dots appear with intensities that are proportional to the reactivity of the antibody.

6. Localizing Tubulin by Immunohistochemistry Microtubules are hollow cylinders made up of polymers of the protein tubulin. Microtubules are major components of cilia and flagella, which are tail like projections that are covered by a plasma membrane and extend outwards from the cell. Motile cilia are used for locomotion and food gathering by some protozoa and are found in the lining of the trachea, where their wave like motion propels mucus, dust and other foreign matter out of the lungs. In this exercise, students use the powerful technique of immunohistochemistry to localize tubulin in the

FeaturesSuitability – General college-level biology, advanced high school biology or introductory cell and molecular biology for college sophomores and juniors.

Topics include evolution, protein biochemistry and enzyme action, photosynthesis and cell respiration, immunology, animal and plant physiology, anatomy and histology, cell biology, molecular biology and biotechnology, bacteriology, genetics, molecular genetics and genomics, and human genetic diseases.

Lab Schedule – Fifteen 3 hour lab sessions or twenty-five 1-hour lab sessions.

Cost - $52 per student per student per fifteen 3-hour laboratory sessions, excluding costs of manuals if the students work in pairs.

Student Designed Experiments - Ideas for over 50 optional student-designed experiments are outlined in the Instructor manual of the program and basic reagents are provided in order for the student to carry out their hypothesis driven projects.

Equipment Requirements include horizontal electrophoresis equipment, microliter dispensers, a microcentrifuge and water bath. A colorimeter is recommended but not absolutely necessary for parts of two experiments.

Description of the Laboratory Exercises

COUR

SES

41

ManualPDFs

Includedon CD

esophagus and trachea. The provided tissue sections are exposed to a tubulin monoclonal antibody, which binds to the tubulin. The sections are then incubated with a secondary antibody, which binds to the first antibody. The second antibody is complexed with peroxidase, which catalyzes a color producing reaction. Following the addition of a non-toxic peroxidase substrate, the peroxidase converts the colorless substrate to a blue product, thus “staining” the area containing tubulin. As can be seen in the photographs in panels A-C at the right, the

cilia that line the trachea are stained in a highly selective manner in keeping with the high concentrations of microtubules that make up these structures. In the course of these studies, students also become familiar with the four basic tissue types and the structure of tubular organs and identify representative examples of these features in tissue sections that they prepare.

Student Designed Experiments7. Characterization of Peroxidase in Plants-Student Designed ProjectsThese experiments were designed to actively engage students in exciting biological research projects of their own design. The projects focus on peroxidases, which form a large family of related enzymes that are ubiquitous in plants. Plant peroxidase isoenzymes can be tissue specific, developmentally regulated and display variable tissue and, high salt and disease resistance defense reactions and this induction may be related to the abilities of peroxidases to strengthen the plant cell wall and to kill microorganisms. Students begin their projects with a hypothesis, which is a statement of an ideal that they will test in the laboratory. They then test their hypothesis by carrying out a series of experiments using the materials provided with the program and vegetables, intact plants or roots and stems. They first use the technique of tissue printing which enables them to localize the peroxidases in tissue sections. They then quantify peroxidase activity by using a DOT-blot assay and Spectrophotometric procedures. Students also carry out an electrophoresis analysis in order to characterize peroxidase isoenzymes in plant extracts that they prepare. In the final section of the program, students are given detailed instructions for organizing their data for presentation in a scientific paper. They are then instructed to write a paper that conforms to the style of a scientific publication using the detailed steps that are presented in the laboratory manuals.

DNA and the Cell Nucleus8. Properties of DNA and Cell Fractionation and DNA isolationA DNA molecule from a single human chromosome is about 4 cm long and the length of DNA in an individual is about 200 times the distance from the earth to the sun. Isolated DNA in a test tube is also a long, stiff molecule. When alcohol is added to a DNA solution, the DNA fibers precipitate and can be spooled onto a glass rod. This feature of DNA is illustrated in the first part of this lab period. In the second part, students isolate nuclei from calf thymus tissue. The DNA is then extracted from the nuclei by a simple procedure that uses a detergent and alcohol.

9. Anatomy and Evolution of the GenomeCommon plasmids are simple DNA molecules, which contain a few genes and regulatory elements. Most viral genomes are more complex. For example, the genome of phage lambda contains approximately 50 genes. About 4,000 genes are present in the E.coli genome while there is approximately 1,000 times more DNA in the genome of a mammal. This progression in genome complexity is the topic of this exercise. Here, students compare the electrophoretic patterns of restriction digests of a plasmid, phage lambda DNA, and cow DNA from thymus and kidney.

10. Speci!c Binding of Dyes to DNAProteins that bind to DNA control the processes of gene regulation and DNA replication. The electrophoretic mobility band shift assay is a common technique used to study such specific protein-DNA interactions. In this exercise, students use this assay to identify dye molecules that bind to DNA and attempt to determine the mechanism by which these drugs interact with the DNA molecule.

Molecular Genetics Genetics and Biochemistry of Hemoglobin

11. DNA Cloning and Genotype to PhenotypeThis exercise was designed to provide an exciting introduction to specific gene structure and function. The students are given four tubes that are labeled Plasmid A-D. which are identified in the instructor’s guide. One plasmid (A) has a functional gene for the enzyme the ß-galactosidase while tube B contains an inactive form of this gene because it contains a segment of foreign DNA. The tube labeled C contains water while tube D contains the lux operon. Bacteria that carry this plasmid glow in the dark. In the first part of the exercise, students analyze restriction digests of the plasmids in order to determine which plasmid should have a functional ß-galactosidase gene. In the second part of the exercise, the plasmids are introduced into E.coli by transformation and the color of the resulting colonies (blue or white) is then used to assess the functional status of the ß-galactosidase gene. The bacteria are also viewed in the dark for glowing in order to see which plasmid contains the lux operon. By comparing the results they identify the plasmids and relate the genotypes of the plasmids to the phenotypes conferred by the plasmids in E.coli.

12. Sickle Cell Anemia Many changes in the structure of hemoglobin have arisen by mutations. About one person in 100 carries a mutant hemoglobin gene, and these individuals have abnormal hemoglobin molecules in their blood. One of the most common abnormal hemoglobins is hemoglobin S, which causes sickle cell anemia. When the gene for hemoglobin S is inherited from both parents, all of the hemoglobin in the circulation is hemoglobin S and the individual suffers from severe anemia. An electrophoretic procedure is used to illustrate the various types of hemoglobin in the first part of this laboratory exercise. In the second part, Students use the technique of gel filtration chromatography to isolate hemoglobin and then to determine its size.

13. Analysis of a Mutant Hemoglobin Gene A mutation is a change in the nucleotide sequence of DNA, which leads to an inherited change in an organism. Restriction endonucleases provide valuable tools for characterizing mutations at the DNA level. This principle is illustrated in the exercise where students digest a normal and a mutant gene with EcoR1 and Hae III and then analyze the DNA fragments from each by electrophoresis as shown in the figure below. The gene is from rabbit and codes for the ß-globin chain-s of hemoglobin.

Price List - Laboratory Course 2CATALOG # DESCRIPTION PRICE

LC6 The Chemical Package for 16 students working in pairs $845 plus one student manual, one instructor manual and a (The student manual may be reproduced for educational purposes).

LC6SM Sample Student Manual (164 pages) plus one $37 instructor manual

LC6-BK 8 Student Manuals $148

COURSES

1 2 3 4 5 6 7 8

42

A Laboratory Course in College-Level Biology

This program is a complete laboratory course for teaching contemporary biology at the beginning college level. The program is suitable as a full-semester course that meets once per week and presents an experimental approach to the study of the biological sciences. The table of contents of the comprehensive program manual is shown on the right. The program is designed for up to 16 students working in teams of two and provides essentially all of the chemicals, accessories, and instructions that are needed to teach fifteen 60-90 minute laboratory sessions. Electrophoresis equipment is not required. To use this program, you will need microscopes, a small centrifuge, ethyl alcohol and acetic acid. The price of the course is about $29 per student per semester if the equipment listed is available in your teaching laboratory.

As an introduction to the course, students study selected topics in contemporary animal and plant biology including enzyme action, cell respiration, and evolution at the molecular level. Novel experiments are then presented on mitosis, osmoregulation and enzyme distribution which serve to help students gain an appreciation for cell structure and function. Protein biological chemistry forms the next section of the course where the technique of gel filtration chromatography is used to study the structure and function of biologically important proteins in blood. In the final section of the program, students are introduced to selected topics on the molecular biology of DNA including DNA isolation and basic DNA cloning procedures.

The experiments that comprise this course were carefully selected from Basic Programs 1-4. Program Experiment Description Basic 1 1, 2, 3 Page 6 Basic 2 1, 2, 3, 5, 6 Page 7 Basic 3 1, 2, 3 Page 8 Basic 4 1, 2, 3, 4, 5 Page 9

Price List - Laboratory Course 1CAT. NO. DESCRIPTION PRICELC1 The Chemical Package for 16 students working in pairs plus one

student manual and one instructor manual. (The student manual may be reproduced for educational purposes.) $477.16

LC1-BK 8 Student Manuals $86.35LC1-SM Sample Student Manual $21.39

(132 pages) plus one instructor manual.

FEATURESSuitability College-Level General Biology Lab Schedule One 1-2 hour lab session per week for a full

semester.Cost $29 per student per semester excluding costs

of manuals and equipment.

Table of ContentsIntroductory Remaks .............................................................................1Experimental Analysis I: Animal Biology and Evolution 1. The Mammalian Digestive System ............................................4 2. Quantifying Amylase Activity ..................................................12 3. Proteases and Factors that Influence Enzyme Activity ............21 4. Evolution of Serum Proteins ..................................................27Experimental Analysis II: Plant Physiology and Cell Biology 1. Extraction and Analysis of an Enzyme from Wheat ................33 2. Effects of Temperature on Cell Respiration ...........................46 3. Osmolarity and a Cytological Bioassay ...................................59 4. Location of an Enzyme in Plant Cells and Tissues ..................67 5. Mitotic Activity and Cell Respiration ......................................74 6. Enzyme Cytochemistry ...........................................................81Experimental Analysis III: Protein Biological Chemistry and Gel Filtration Chromatography General Backgrond ...................................................88 1. Separating Molecules by Gel Filtration ..................................92 2. The Molecular Weight of Hemoglobin ...................................99 3. Binding Specificity of Serum Albumin .................................104Experimental Analysis IV: Structure, Isolation, and Function of DNA General Background ..........................................................................108 1. Properties of DNA ................................................................114 2. Cell Fractionation and DNA Isolation ...................................119 3. Gene Function and Cloning in Bacteria ...............................126


ManualPDFs

Includedon CDCO

URSE

S

43



LC2-BK 8 student manuals. $93.62LC2-SM Sample Student Manual (148 pages) $21.39

plus one instructor manual.

This Program is a complete laboratory course for teaching cell biology or introductory molecular biology at the college level. The program is designed for 16 students working in pairs and provides essentially all of the chemicals and instructions that are needed to teach sixteen 2-3 hour laboratory sessions. The course consists of 14 experiments carefully selected from Standard Programs 1, 2, 3, and 7 that are presented in a comprehensive integrated laboratory manual. The Table of Contents of the manual and the catalog numbers of the experiments provided with the program are shown below. To use this program, you will need an Accessory Kit, the appropriate electrophoresis equipment, microscopes, and a water bath. A small centrifuge is required for 2 experiments. The price of the course is about $51 per student per semester if if the equipment listed is available in your teaching laboratory.

In this laboratory course, your students will be introduced to the molecular biology of the eukaryotic cell. In the first section of the course, students study topics in protein biology and biochemistry such as protein structure, function, isolation, molecular evolution, and the detection and molecular basis of human disease. Techniques used for these experiments include electrophoresis (both native and denaturing), affinity chromatography, peptide mapping, and the Western blot procedure. In the second section of the course, students localize enzymes in plant and animal cells, perform cell fractionation procedures, and study the properties of a specific cell-surface receptor. Experiments on the properties and structure of DNA are presented in the final section of the course. Techniques include DNA electrophoresis and restriction nuclease mapping.

The experiments that comprise this course were selected from four Programs:

Program Experiment Description Standard 1 101, 102, 106 Page 10 Standard 2 201, 204, 205, 206 Page 14 Standard 3 301, 302, 303, 306 Page 18 Standard 7 701, 702, 703 Page 33

A Laboratory Course in Cell Biology

FEATURESSuitability Eukaryotic Cell Biology or Introductory

Molecular Biology for sophomores and juniors.Lab Schedule One 2-3 hour lab session per week for a full



Table of ContentsPart 1. The ProteinsI. Protein Composition and Structure: A Review of the Basics ..............1II. Electrophoresis-General Information and Analysis of Native Proteins Theoretical Aspects ...........................................................................7 Practical Aspects and Procedures. ..................................................11 Experimental Analysis I 1. Electrophoretic Separation Proteins ......................................17 2. Genetics and Sickle Cell Anemia ............................................20 3. LDH Isoenzymes ....................................................................21II. Electrophoretic Analysis of Denatured Proteins General Information ........................................................................32 Experimental Analysis II 4. Molecular Weight Determination ..........................................36 5. Peptide Mapping Analysis ......................................................40 6. Protein Evolution and the Western Blo ..................................44 7. Affinity Chromatography ........................................................53Part 2. The Eucaryotic Cell General Information ........................................................................58 Experimental Analysis III 8. Enzyme Cytochemistry ...........................................................59 9. Analysis of a Membrane Receptor .........................................59 10.The Cell Nucleus ....................................................................86Part 3. NucleicAcids .............................................................................. I. Nucleic Acid Structure and Function: A Review of the Basics .............107II. Electrophoretic Analysis of DNA General Information ......................................................................118 Experimental Analysis IV 11. The Length of DNA Molecules .............................................122 12. Restriction Nuclease Mapping of DNA .................................126 13. Plasmid DNA Structure ........................................................132 14. The Nucleosome Structure of Chromatin ............................136

COURSES

ManualPDFs

Includedon CD

44



LC3-BK 8 student manuals. $107.00LC3-SM Sample Student Manual (118 pages) plus $21.39

one instructor manual.

This Program is a complete laboratory course for teaching molecular biology at the advanced college level. The program is designed for up to 16 students working in pairs and provides essentially all of the chemicals and instructions that are needed to teach seventeen 2-3 hour laboratory sessions. The course consists of experiments carefully selected from Standard Programs 3, 4, and 5 that are presented in a comprehensive integrated laboratory manual. The Table of Contents of the manual and the catalog numbers of the experiments provided with the program are shown below. To use this program, you will need an Accessory Kit, the appropriate electrophoresis equipment, a water bath for DNA hybridization, and a centrifuge that can be operated at a force of at least 3,000 x g. A bacterial shaker is also strongly recommended. The price of the course is about $39 per student, per semester if the equipment listed is available in your teaching laboratory.

This laboratory course is intended to give students a solid foundation in the molecular biology of DNA and to provide hands-on-experience with the genetic techniques that form the basis of the biotechnology industry. In the first section of the course, students use electrophoresis and restriction nuclease mapping to explore important features of prokaryotic and eukaryotic genomes. DNA hybridization techniques are used in the second segment of the course where students study molecular evolution at the DNA level and examine a specific sequence in the eukaryotic genome. In the final section, students prepare and characterize a library of recombinant plasmids where they receive an appreciation and a detailed understanding of a variety of state-of-the-art DNA cloning techniques.

The experiments that comprise this course were selected from three Programs:

Program Experiments Descriptions Standard 3 301, 302, 303, 306 Page 18 Standard 4 401, 403 Page 22 Standard 5 Entire Program Page 24

A Laboratory Course in Molecular Biology

FEATURESSuitability Molecular Biology or Molecular Genetics for

juniors and seniors.Lab Schedule One 3 hour lab session per week for a full


of manuals and equipment

Table of ContentsI. Nucleic Acid Structure and Function: A Review of the Basics ..........................1II. Electrophoretic Analysis of DNA General Information .....................................................................................12 Experimental Analysis I 1. The Length of DNA Molecules ............................................................15 2. Restriction Nuclease Mapping of DNA ...............................................20 3. Plasmid DNA Structure ......................................................................25 4. The Nucleosome Structure of Chromatin ...........................................30III. DNA Hybridization Analysis ............................................................................... General Information .....................................................................................48 Experimental Analysis II ................................................................................... 5. Evolution of the Vertebrate Genome ..................................................61 6. Identifying a Specific Sequence in the Mammalian Genome ..............71IV. Genetic Engineering General Information .....................................................................................86 Experimental Analysis III 7. Construction of a Recombinant DNA Library .....................................91 8. Transformation of E.coli ....................................................................94 9. Isolation of Plasmid DNA .................................................................102 10. Mapping of Plasmid DNA .................................................................113 11. Analysis of Plasmid DNA by Southern Blot Hybridization .................116

ManualPDFs

Includedon CDCO

URSE

S

45

A Comprehensive Laboratory Course in Molecular Biology

This Program is a complete laboratory course for teaching molecular biology or biochemistry at the advanced college level. The program is designed for up to 16 students working in pairs and provides essentially all of the chemicals and instructions that are needed to teach twenty eight 2-3 hour laboratory sessions. The course consists of experiments carefully selected from the Standard Programs that are presented in a comprehensive integrated laboratory manual. The Table of Contents of the manual and the catalog numbers of the experiments provided with the program are shown below. To use this program, you will need an Accessory Kit, the appropriate electrophoresis equipment, microscopes, a water bath for DNA hybridization, and a centrifuge that can be operated at a force of at least 3000 x g. A bacterial shaker is also strongly recommended. The price of the course is about $77 per student per semester if the equipment listed is available in your teaching laboratory.

In this laboratory course, your students will be introduced to many of the concepts and techniques that have revolutionized the biological sciences during the past two decades. In the first section of the course, students study topics in protein biology and biochemistry such as protein structure, function, isolation, location in cells, molecular evolution, and the detection and molecular basis of human disease. Techniques used for these experiments include electrophoresis (both native and denaturing), affinity chromatography, peptide mapping, enzyme cytochemistry and the Western blot procedure. In the second section of the course, students perform experiments with DNA structure and function. These exercises stress the organization and complexity of the prokaryotic and eukaryotic genomes, gene function and regulation, and the structure of the eukaryotic chromosome. Techniques include restriction nuclease mapping, Southern blot hybridization procedures, and an array of state-of-the-art DNA cloning techniques.

The experiments that comprise this course were selected from six programs.

Program Experiments Description Standard 1 101, 102, 106 Page 10 Standard 2 201, 204, 205, 206 Page 14 Standard 3 301, 302, 303, 306 Page 18 Standard 4 401, 403 Page 22 Standard 5 Entire Program Page 24 Standard 7 701, 702, 703 Page 33

Price List-Laboratory Course 4CAT. NO DESCRIPTION PRICELC4 The Chemical Package for 16 studentsworking in pairs plus one




FEATURESSuitability Molecular Biology or Biochemistry for juniors

and seniors.Lab Schedule Two 3-hour lab sessions per week for a full-



Table of Contents Part 1. The ProteinsI. Protein Composition and Structure: A Review of the Basics .............................................1II. Electrophoresis-General Information and Analysis of Native Proteins Theoretical Aspects ..........................................................................................................7 Practical Aspects and Procedures ..................................................................................11 Experimental Analysis I 1. Electrophoretic Separation of Proteins .............................................................17 2. Genetics and Sickle Cell Anemia........................................................................20 3. LDH Isoenzymes ................................................................................................26III. Electrophoretic Analysis of Denatured Proteins General Information ......................................................................................................32 Experimental Analysis II 4. Molecular Weight Determination ......................................................................36 5. Peptide Mapping Analysis ..................................................................................40 6. Protein Evolution and the Western Blot .............................................................44 7. Affinity Chromatography ....................................................................................53Part 2. The Eucaryotic Cell General Information ......................................................................................................58 Experimental Analysis III 8. Enzyme Cytochemistry .......................................................................................59 9. Analysis of a Membrane Receptor. ....................................................................74 0. The Cell Nucleus ...............................................................................................86Part 3. Nucleic AcidsI. Nucleic Acid Structure and Function: A Review of the Basics .......................................107II. Electrophoretic Analysis of DNA General Information ....................................................................................................118 Experimental Analysis IV 11. The Length of DNA Molecules .........................................................................122 12. Restriction Nuclease Mapping of DNA .............................................................126 13. Plasmid DNA Structure ....................................................................................132 14. The Nucleosome Structure of Chromatin ........................................................136III. DNA Hybridization Analysis ................................................................................................ General Information ....................................................................................................148 Experimental Analysis V ..................................................................................................... 15. Evolution of the Vertebrate Genome ................................................................161 16. Identifying a Specific Sequence in the Mammalian Genome ...........................171 Part 4. Genetic Engineering............................................................................................... General Information ....................................................................................................186 Experimental Analysis VI 17. Construction of a Recombinant DNA Library ...................................................191 18. Transformation of E.coli .................................................................................202 19. Isolation of Plasmid DNA ................................................................................207 20. Mapping of Plasmid DNA ................................................................................213 21. Analysis of Plasmid DNA by Southern Blot Hybridization ................................216

COURSES

ManualPDFs

Includedon CD

46

A Laboratory Course in Cell and Molecular Biology

This Program is a complete laboratory course for teaching cell and molecular biology at the college level. The program is designed for 16 students working in pairs and provides essentially all of the chemicals and instructions that are needed to teach sixteen 3 hour laboratory sessions. The course consists of 16 experiments carefully selected from 7 Laboratory Programs that are presented in a comprehensive integrated laboratory manuals. The Table of Contents of the manual and the catalog numbers of the experiments provided with the program are shown below. To use this program, you will need an Accessory Kit, the appropriate electrophoresis equipment, microscopes, a water bath, and a small centrifuge such as tabletop model or microcentrifuge. The price is about $52 per student per semester if the equipment listed is available in your teaching laboratory. In this laboratory course, college freshman and sophomore-level students will be introduced to the molecular biology of the eukaryotic cell. In the first two sections of the course, students study topics in protein biology and biochemistry such as protein structure, function, molecular evolution, and the detection and molecular basis of human disease. Techniques used for these experiments include electrophoresis and the Western blot procedure. In the third section of the course, students localize enzymes in plant and animal cells and study the properties of a specific cell-surface receptor. Students perform a project of their own design in the fourth section of the course. The projects focus on the characterization of plant peroxidases. For the projects, assorted vegetables or plants are needed but not included. Experiments on the properties and structure of DNA are presented in the final section of the course. Here, students perform experiments that deal with genome organization, chromatin structure, and specific gene function. Techniques include DNA electrophoresis, cell fractionation, DNA isolation, restriction nuclease mapping, and basic cloning procedures.The experiments that comprise this course were selected from 7 Programs. Program Experiment Description Basic 1 1, 2 Page 6 Basic 4 2 Page 9 Standard 1 101, 102 Page 10 Standard 3 306 Page 18 Standard 7 701, 702 Page 33 Standard 8 801, 803, 804 Page 12 Standard 10 1001, 1002, 1004 Page 20 Others IND-2, IND-5 Page 34





FEATURESSuitability Eukaryotic Cell and Molecular Biology for

freshmen and sophomores.Lab Schedule One 3-hour lab session per week for a full



Table of ContentsExperimental Analysis I: Protein Structure and Evolution Protein Composition and Structure: A Review of the Basics ............................1 Electrophoresis-General Information and Analysis of Native Proteins Theoretical Aspects .........................................................................................7 Practical Aspects and Procedures .................................................................11 1. Electrophoretic Separation of Proteins ..............................................17 2. Genetics and Sickle Cell Anemia ........................................................20 3. Serum Proteins and the Western Press-Blot ......................................26Experimental Analysis II: Protein Function 4. Specificity of Albumin Binding ...........................................................38 5. Extraction and Analysis of an Enzyme from Wheat ..............................47 6. Tissue-Specific Isoenzymes in the Cow ................................................59 Experimental Analysis III: Location of Proteins in Cells 7. Enzyme Cytochemistry .........................................................................66 8. Analysis of a Cell Surface Receptor ......................................................81Experimental Analysis IV: Characterization of Peroxidase in Plants (Student Designed Projects)Description ........................................................................................................93 9. Tissue Printing .....................................................................................94 10. Peroxidase Isoenzymes ...................................................................106 Experimental Analysis V: DNA and the Cell Nucleus Nucleic Acid Structure and Function: A Review of the Basics ......................117 Electrophoretic Analysis of DNA 11. Anatomy and Evolution of the Genome ............................................130 12. Properties of DNA ............................................................................141 13. Analysis of a Genomic Segment .......................................................146 14. Cell Fractionation and DNA Isolation ...............................................154 15. Genotype to Phenotype ....................................................................161 16. The Nucleosome Structure of Chromatin ........................................173

ManualPDFs

Includedon CDCO

URSE

S

g Modern · PDF fileZoology Histology Developmental AnatomyIndividual Experiments ... Plant Molecular Biology ... New Experiments from Modern Biology Inc.! IND-29. Electrophoretic

Documents