1 | Page BIO322 Genetics Spring 2011 BIO322: Genetics Laboratory Manual Department of Biology Wilmington College of Ohio Spring 2011 Douglas J. Burks, Ph.D.
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BIO322: Genetics
Laboratory Manual
Department of Biology
Wilmington College of Ohio
Spring 2011
Douglas J. Burks, Ph.D.
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Table of Contents
Section Page
Goals and Objectives … 3
Assessment Methods … 3
Laboratory Polices … 4
Evaluation … 5
Laboratory 1: Independent Segregation … 6
Laboratory 2: Meiosis … 12
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BIO350: Topics – Molecular Pharmacology
Douglas Burks
Jim Reynolds
Department of Biology
Wilmington College of Ohio
Laboratory Manual
Goals and Objectives
We have set several goals for you this semester in the laboratory of molecular
pharmacology. These include:
1. to develop scientific questions in a way that lead to useful hypotheses,
design experiments to address the question, perform the experiments,
critically evaluate data, and analyze results of experiments;
2. to develop basic laboratory skills involved in doing modern genetics
research;
3. to enhance your ability to undertake a primary literature in developing
background and information for addressing scientific questions in the
laboratory;
4. and to strengthen written communication skills.
Assessment Methods
We will use several different methods to assess your growth and progress in the
laboratory. These include:
1. pre-lab quizzes on preparation for laboratory;
2. observation of your work at the lab bench;
3. research proposals;
4. analysis of data;
5. maintaining an accurate and current laboratory notebook;
6. and preparing research papers which include primary literature, results
and analysis of experimental results.
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Laboratory Policies
1. Laboratory personal are expected to follow the safety policies of the Biology
Department that are found in the BIO231 Laboratory Manual.
2. Students are expected to come to the laboratory prepared to undertake
research which includes doing any reading of necessary materials before
coming to lab.
3. Students may miss one laboratory period without loss of points towards their
final grade. After one absence students will be assessed -5% of the
laboratory grade per absence. Students are not excused from any
assignment for any laboratory experiment.
4. All late assignments will be assessed a penalty for each day it is late as
described in the Biology Writing Manual. The late policy states:
a. Wilmington College biology professors have adopted a unified policy
concerning late assignments. An assignment is late any time after 4:00
PM on the assignment’s due date, unless otherwise indicated. In
addition, any incomplete, substandard assignment submitted at the
deadline just to “get something in” will be considered late.
b. Submission of the final assignment after the deadline will result in a
20% deduction of points for each working day it is late. For example,
if you hand in your late assignment within one working day of the due
date, the highest score you can possibly receive is an 80%. If you
hand in your late assignment within two working days of the due date,
the highest score you can receive is a 60%. If you hand in your
assignment within three working days of the due date, the highest
score you can receive is a 40%. Late assignments will not be accepted
after three working days. (http://plato.wilmington.edu/faculty/dburks/Writing%20Manual%20August%202010.pdf)
This policy will be strictly enforced.
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Grade Evaluation for Laboratory
Percent of Laboratory Grade
Pre-lab quizzes 10%
Observation of Laboratory Work 10%
Laboratory Notebook 25%
Laboratory Reports 55%
Total 100%
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LABORATORY 1
TESTING A HYPOTHESIS
Corn Seed Color
3:1
9:3:3:1
Background:
Seed Color in corn is variable. One gene that affects color has two
alleles. For the color gene one allele is for yellow and one allele for
purple for this color gene. Purple color is dominant to yellow which is
recessive. A second trait is for seed shape. One allele for seed shape is
smooth which is dominant to a wrinkled kernel which is recessive.
In this experiment we want to test if the gene alleles for color
show independent segregation and if the two genes show independent
Name: ______________________
Date: _______________________
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assortment. A monohybrid cross for independent segregation should
give a 3:1 ratio.
Monohybrid Cross in corn
Trait Dominant
allele
Recessive
allele
Seed color
Seed Shape
Ratio
Dihybrid Cross in corn
Trait D seed
color D
seed
shape
D seed
color R
seed
shape
R seed
color D
seed
shape
R seed
color R
seed
shape
Phenotype
Ratio
To test independent segregation and assortment we have corn cob
samples that you can count the number of seeds showing various seed
types from monohybrid and dihybrid crosses. You can calculate ratios
from those samples.
Once you have those numbers how do we judge if they are the expected
ratio. It is doubtful that you will get exactly 3:1 or 9:3:3:1. How close is
close enough to say that an experimental number is the expected ratio.
The answer is to use a statistical test. The statistical test is the Chi
Square Test. The Chi Square is a statistical test used to judge the
goodness of fit of an observed distribution to a theoretical distribution.
In our case the theoretical distribution is 3:1 and 9:3:3:1. In this
statistical test we are testing the null hypothesis. The null hypothesis
is that the numbers are different due to chance.
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Chi Square is
We can look at an example in pea plants. When we do the cross
between two hybrid tall plants (Tt) we expect to see a 3 tall: 1 dwarf
plant ratio. We do the cross and plant 1000 seeds and observe 739 tall
plants and 261 dwarf plants. We expected theoretical numbers of 750
tall and 250 dwarf plants. Is this an example of independent
segregation or not. Let’s do a Chi Square Test.
Observed Expected (O-E) (O-E)2
Tall 739 750 -11 121
Dwarf 261 250 11 121
(O-E)2/expectd
Tall
121/150 .1637
(O-E)2/expectd
Tall
121/750 .860
X2 ∑(O-
E)2/expected
.970
Degrees of freedom is the number of classes – 1. df = n-1. In our case
here we have two classes (dominant and recessive) and therefore df = 2-
1 = 1.
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The p value from a p calculator is 0.327. We reject the null hypothesis
that the numbers are different by chance (they are the same).
(http://www.danielsoper.com/statcalc/calc11.aspx )
Experiment
You are to test three hypotheses in this experiment.
1. Purple vs. yellow seed (independent segregation; 3:1 ratio from
monohybrid cross).
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2. Smooth vs. wrinkled seed (independent segregation; 3:1 ratio from
monohybrid cross).
3. Smooth vs. wrinkled; purple vs. yellow (independent assortment;
9:3:3:1 ratio from dihybrid cross).
For each experiment you are to count all the seed on one cob and
calculate Chi Square and a p value.
Monohybrid Cross: Corn Kernel Coloration
Number of Purple
Seeds
Number of Yellow
seeds
Actual Number
Actual Ratio
Expected Number
Expected Ratio
Monohybrid Cross: Corn Kernel Shape
Number of Smooth
Seeds
Number of Wrinkled
seeds
Actual Number
Actual Ratio
Expected Number
Expected Ratio
Dihybrid Cross: Corn Kernel Coloration and Shape
# Purple
Smooth
# Purple
Wrinkled
# Yellow
Smooth
# Yellow
Wrinkled
Actual
Number
Actual
Ratio
Expected
Number
Expected
Ratio
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Show calculation of Chi Square for each.
Determine the p value for each
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Do the alleles show independent segregation and do the two genes show
independent assortment?
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LABORATORY 2
MEIOSIS
Background
Meiosis is the cellular division that occurs in germ cells that leads to a reductive
division and the production of gametes for sexual reproduction. Chromosomes, the units
of Mendelian segregation and assortment, are randomly divided and given to gametes in
this process. Homologous chromosomes pair and separate during the first division of
meiosis leading to the reductive division (2n � 1n) (Figure 1). It is also during
meiosis 1 that crossing-over occurs leading to new linkage patterns in genes within a
homologous pair of chromosomes. In meiosis II duplicated chromosomes separate sister
chromatids leading to 1n cells containing unduplicated chromosomes that are ready for
fertilization. Because of crossing-over the daughter cells that result from meiosis II are
not identical (Figure 2).
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The lilium anther and pollen production is a classic model system to study meiosis
by microscopy. Meiosis in plants occurs in structures called sporangia. The sporangia of
flowering plants are located in the flower. Flowers have both male and female sporangia.
The male sporangium is called the anther and the female sporangium is an ovule. Haploid
microspores are produced inside the pollen sacs by meiosis, and they usually are in
tetrads (groups of four). In lilium there are four pollen sacs (microsporangia) per anther.
In the center of each pollen sac are tetrads of microspores. A tetrad is simply a group of
four. Why are the microspores in groups of four? The tetrads are surrounded by the
tapetum, a nutritive tissue derived from the innermost layer of the pollen sac (Figure 3).
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In this laboratory you will undertake a study of meiosis in lilium anthers (Figure 4).
Protocol
Identifying meiotic stages.
1. Obtain a set of lilium meiosis slides and a compound light microscope.
a. Review how to use a microscope
b. Review using an oil immersion lens
i. Focus very carefully with the 40x objective over the stained
specimen on the slide.
1. Once focused, do not alter focus for the next three steps!
ii. Rotate turret half way so that the 40x and 100x objectives straddle
specimen.
iii. Apply a small drop of oil directly on the slide over the specimen.
iv. Rotate 100x objective into the immersion oil
v. Never get oil on any other lens.
1. Never go back to the 10x or 40x objectives after you
have applied oil to the specimen since oil can ruin the
lower power objectives
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vi. Clean up all oil when finished. 1. When you have finished for the day, wipe the 100x oil
immersion objective carefully with lens paper to remove all
oil. Wipe oil from the slide thoroughly with a Kimwipe.
Cleanse stage should any oil have spilled on it. Recap the
immersion oil container securely, replace in drawer.
2. Focus on anther with the 4X lens
3. Exam the tissue at 40X and learn landmarks of tissue
4. Exam the tissue at 100X and find and identify each stage of meiosis.
Photographing meiotic cells.
1. Obtain a set of lilium meiosis slides and a compound light microscope.
2. Focus on the lilium tissue at 4X
3. Obtain a photomicroscopy camera.
4. Log onto the computer.
5. Carefully insert camera into eyepiece and insert the USB cord into the computer.
6. Launch the microscopy software.
7. Place your USB drive into the computer
8. Photograph the tissue at 4X, 10X, and 40X
9. At 100X photograph each stage of meiosis.
10. Transfer images to your USB drive
11. Erase file on microscopy software.
Report
You are to write a description of meiosis in lilium in which you describe each stage of
meiosis using your photographs of stages as figures to illustrate your description. The
report should have a general background on the structure of the reproductive organs in
lilium and on meiosis. It should then have a detailed description of each stage of meiosis.
Reports should be typewritten, Figures should be placed within text and all figures
should have a figure legend (in a font smaller than text).
The report is due @ 1:00 PM on February 1, 2011. Late policy
applies.