EILENE LYONS – REVISED 1/12/2010 1 FALL 2009/SPRING 2010 PREP GUIDE BIO:219 BIOTECHNOLOGY I TABLE OF CONTENTS Setup for first day of class.....................Page 2 Lab 1 Genotyping of Arabidopsis.................Page 3-6 Lab 2 DNA Concentration and Purity .......... .....Page 7-8 Lab 3 PAGE.......................................Page 9- 10 Lab 4 Purification of Eco RI(EDVOTEK Kit 302)....Page 11-12 Lab 5 Mammalian Cell Culture...................Page 13 - 14 Lab 6 Pop Bead Cloning (dry lab)....................Page 15 Lab 7 DNA Restriction for Cloning...................Page 16 Lab 8 Gene Clean....................................Page 17 Lab 9 Ligation.................................... Page 18 Lab 10 Transformation & Blue-White Selection ....Page 19-20 Lab 11 Plasmid DNA Isolation (Miniprep) ........... Page 21 Lab 12 Recombinant DNA Restriction and Mapping .....Page 22 Lab 13 Manual DNA Sequencing (EDVOTEK Kit 341)...Page 23-25 Lab 14 Gene Expression (IL-8 ELISA KIT; Qiagen RNA cleanup kit)............ ........... ........... .......Page 26 *Items marked with an asterisk do not need to be place on the cart, but should be available in the room or in the immediate prep area.
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EILENE LYONS – REVISED 1/12/2010 1
FALL 2009/SPRING 2010
PREP GUIDE
BIO:219 BIOTECHNOLOGY I
TABLE OF CONTENTS Setup for first day of class.....................Page 2
Lab 1 Genotyping of Arabidopsis.................Page 3-6
Lab 2 DNA Concentration and Purity .......... .....Page 7-8
Colored Owl or Fisher horizontal gel rigs with combs
Conical tubes, Fisher, 15 ml and 30 ml and racks
Coverage – one bottle per table
Cuvettes, cuvette racks
Electronic balances, weigh boats, spatulas and paint brush for clean up
Ethidium Bromide [10 mg/ml] stock (keep refrigerated – small refrigerator?)
Ethidium Bromide disposal station (for disposal of buffer and gels)
Glassware, including beakers, 250 ml flasks, graduated cylinders, Corning bottles
Heating block with holes for 1.5 ml tubes - 2 Kim wipes
Lab diapers
Labeling tape
Microcentrifuges – Eppendorf type – at least 2
Microcentrifuge tubes, 1.5 ml and racks
Microwave oven and hot gloves
Parafilm
Plastic wrap
Serological pipette pumps – manual and electric
Serological pipettes – all sizes
Sterile tips – all sizes
Transfer pipettes
Water bath and floating tube holders
Vortex mixers
Zip Lock bags for storing gels
ITEMS IN THE PREP AREA WHERE STUDENTS CAN ACCESS EASILY:
Ice buckets – leave in the ice room by ice machine
Power supplies
Rockers and shakers
UV spectrophotometers – 2
UV transilluminator, camera, film, face shields, spatulas
BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR
EILENE LYONS – REVISED 1/12/2010 4
LAB 1 GENOTYPING OF Arabidopsis TIMELINE
Day 1: Plant Arabidopsis seeds and prepare solutions
Day 2: DNA extraction and PCR
Day 3: Gel electrophoresis and analysis
MATERIALS for growing Arabidopsis Arabidopsis seeds (Carolina Biological Supply or [email protected] for seed ordering)
Each student needs about 25-50 clf-2 seed (from a heterozygous plant);
Modified MS agar plates, one per student
Growth chamber set on continuous light
Bleach for 30% bleach solution (students will prepare)
Sterile dH2O – one tube or bottle per group
Sterile 0.1% agarose solution (students will prepare)
Aluminum foil
MATERIALS for DNA Isolation and PCR
2 per group Ready-To-Go PCR Beads (PuReTaq Ready-To-Go™ PCR Beads from
http://www1.amershambiosciences.com 96 reactions #27-9559-01, NC9711585 from Fisher, will
last ≥ 1.5 years; cost $227.50)
Each pellet reconstituted to 25 μL contains 1.5 units Taq polymerase, 10 mM Tris-HCl
(pH 9.0), 50 mM KCl, 1.5 mM MgCl2, and 200 μM of each dNTP.
Primers can be ordered from http://invitrogen.com
0.4 uM minimum CLF (wildtype) primers/loading dye mix – 25 μL per Team
FORWARD 5’-TTAACCCGGACCCGCATTTGTTTCGG-3’
REVERSE 5’-AGAGAAGCTCAAACAAGCCATCGA-3’
0.4 uM minimum clf-2 (mutant) primers/loading dye mix – 25 μL per Team
FORWARD 5’-TTAACCCGGACCCGCATTTGTTTCGG-3’
Ds REVERSE 5’-GTCGGCGTGCGGCTGGCGGCG-3’
Edward’s Buffer, 2 ml per team (Students will prepare)
Cresol Red Loading Dye
Isopropanol, aliquoted – 2 ml per team
Pellet pestles – one per team
Dissecting microscopes – 1 per team
Thermal cycler
Hair dryer
TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) – 500 μl per team
MATERIALS for Electrophoresis
100 - 1000 bp ladder or pBR322 DNA-BstN I digest (inexpensive marker from NE Biolabs)
*Heating block or water bath set at 50°C
*50X TAE electrophoresis buffer (students will dilute to 1X working solution)
*D.C. power supplies – per 2 groups
*Practice loading dye
*[10 mg/ml] ethidium bromide stock solution
*Should be in room – do not place on cart
BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR
EILENE LYONS – REVISED 1/12/2010
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RECIPES:
Modified MS agar:
4.3 g MS salts (half the amount used for bacteria culture)
900 ml distilled water
Use 1.0 M NaOH or KOH to adjust to pH 5.7
BTV of 1 Liter
Then add 8 g agar and autoclave for 20 minutes at 120°C. One liter makes 40-50 10 cm
diameter plates.
Cresol Red loading dye
1% Cresol Red Dye Stock – yield 50 ml
Add 500 mg to 50 ml of ddH2O in a 50 ml tube or bottle
Shake to dissolve
Store at room temperature
Cresol Red Loading Dye – yield 50 ml of working solution
Add 17 g of sucrose to 49 ml of ddH2O in a 50 ml tube or bottle
Shake to dissolve
Add 1 ml of 1% Cresol Red Dye
Shake tube to mix. Store at 4°C.
Edward’s Buffer – yield 50 ml (solid NaCl and concentrated stocks will be used)
Mix the ingredients in a 50 ml bottle (can be stored at room temperature, indefinitely).
32.5 ml ddH2O
10 ml of 1 M Tris pH 8
2.5 ml of 5 M NaCl
2.5 ml of 0.5 M EDTA
2.5 ml of 10% SDS
50x Concentrated TAE Electrophoresis Buffer (40 mM Tris-acetate, 2 mM EDTA)
Add the following to dH2O to give a final volume of 1 liter
242 g Tris base (Tris [hydroxymethyl] aminomethane)
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA (pH 8)
Students will dilute to a 1X working solution
Primer/Loading Dye Mix
Final concentration of components:
0.25 picomoles/μL of each primer, 13.9% sucrose, and 0.0082% cresol red in Tris-low
EDTA (TLE) buffer (10 mM Tric-HCl, pH 8.0; 0.1 mM EDTA).
BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR
EILENE LYONS – REVISED 1/12/2010
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The ideal concentration is between 0.1 and 0.5 M. Primers are sold as solid DNA by
some companies and diluted in water by other companies. A certificate of analysis
packed with the primers give the mass, number of moles and if diluted, the concentration
in either μM or μg/μl or both. It can be difficult to determine how to dilute and mix the
primers for use in PCR so that the concentration is between the optimum 0.1 and 0.5 μM.
Table 1 and the sample calculation, below, may help.
Table 1. Molar conversion for Primer Concentration* Primer length pmol/µg 20 pmol** 18-mer 168 119 ng
20-mer 152 132 ng
25-mer 121 165 ng
30-mer 101 198 ng
* From Qiagen News, Issue 5 1997
** 20 pmol of primer in a 100 µl PCR reaction gives a primer concentration of 0.2 µM
SAMPLE CALCULATION for DILUTION OF LIQUID PRIMER DNA Suppose a primer concentration is given as 40 μM and 0.32 μg/μl. You need to make up 300 μl of primer/loading dye mix. The protocol states that 22.5 μl of the primer/loading dye is used in the 30 μl reaction. The final concentration of each of the primers in the 30 μl reaction must be 0.1 – 0.5 μM. 1. The primer’s final concentration in the reaction must be 0.1 – 0.5 μM, so dilute the
primer to 0.5 μM and use this to set up the reaction. 2. Use C1 x V1 = C2 x V2 to calculate the volume of working solution to use.
C1 = 40 μM
V1 = X C2 = 0.5 μM V2 = 300 μl
(40 μM) X = (0.5 μM) (300 μl) X = 3.75 μl
Add 3.75 μl of the primer as supplied by the manufacturer to enough loading dye and the other reagents to give a total of 300 μl. When 22.5 ul of this mix is used in the reaction, the final concentration of this primer will be 0.375 μM, which is within the 0.1 – 0.5 μM final concentration limits.
(0.5 μM) (22.5 μl) = X (30 μl) X = 0.375 μM
BIOTECHNOLOGY I GENOTYPING OF ARABIDOPSIS BY PCR
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SAMPLE CALCULATION for DILUTION OF DRY PRIMER DNA Suppose a primer is 280 μg and 50 nmoles and you need to make up 300 μl of primer/loading dye mix that has a final concentration of 0.25 picomoles/μl.
1. Dilute the primer to make a 100 pmole/μl stock solution. Add 500 μl of dH20 to dissolve the DNA.
2. Dilute 1 μl in 9 μl dH20 to give a working solution of 10 pmoles/μl. 3. Use C1 x V1 = C2 x V2 to calculate the volume of working solution to use. C1 = 10 pmoles/μl
V1 = X C2 = 0.25 pmoles/μl V2 = 300 μl
(10 pmoles/μl) X = (0.25 pmoles/μl) (300 μl) X = 7.5 μl
4. Add 7.5 μl working primer solution to make the primer/loading dye mixture.
BIOTECHNOLOGY I LAB 2 DNA CONCENTRATION AND PURITY
EILENE LYONS – REVISED 1/12/2010 8
LAB 2 - DNA CONCENTRATION AND PURITY
ITEMS TO ORDER IMMEDIATELY Commercial Lambda DNA (0.3-0.5 μg/μL)
DNase
High Mass Molecular Weight Marker DNA
TIMELINE
This lab will take 1 laboratory period
MATERIALS for PART I. Prep
10X TNE Buffer (please check to make sure there is no contamination in the bottle;
buffer may need to be filtered)
MATERIALS for PART II. 0.8% Gel Casting *Powdered agarose
*50x Concentrated TAE Electrophoresis Buffer (Students will dilute to 1X)
*Horizontal gel electrophoresis boxes – one/team
*Electronic balance, weigh-boats and spatulas
*Microwave oven and hot gloves
*Electronic pipette pumps
*Thumb-operated pipette pumps
*Serological pipettes – 10 ml
*100 ml graduated cylinder - one/team
*250 ml flasks - one/team
*Dishpan in sink for dirty glassware
*Scissors – one/team
*Sharpie marking pens
MATERIALS for PARTS III & IV Electrophoresis and UV Spectrophotometry
Commercial Lambda DNA (0.3-0.5 μg/μL) Place this and other DNA on ice for lab
1/20 dilution uncut concentrated Lambda DNA to use as unknown DNA samples – 50 μL
in a 1.5 mL tube labeled as DNA A per team – do not write concentration on tubes
1/10 dilution uncut concentrated Lambda DNA mixed 1:1 with Bovine Serum Albumin
to use as DNA B – 50 μL in a 1.5 ml tube per team
Degraded DNA – 50 μL in a 1.5 mL tube labeled as DNA C per team (add DNase)
REPORT DNA SAMPLE CONCENTRATIONS TO THE INSTRUCTOR
Molecular weight DNA ladder – one 1.5 ml tube per team
*6x or 10x Gel Loading Solution – one 1.5 ml tube per team
*Practice Gel Loading Solution – one 1.5 ml tube per team
*50X TAE electrophoresis buffer (students will dilute to 1X)
*1x TNE Buffer – two 50 mL bottles
*2 Beckman UV Spectrophotometers and matching quartz cuvettes for each
*Kim wipes
*Ethidium bromide [10 mg/ml]
* Place on cart if not in the storage cabinets in the lab
BIOTECHNOLOGY I LAB 2 DNA CONCENTRATION AND PURITY
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RECIPES for Lab 2
10x TNE Buffer stock solution:
100 mM Tris base (Tris [hydroxymethyl] aminomethane)
10 mM EDTA
2.0 M NaCl
Adjust pH to 7.4 using concentrated HCl and a pH meter. Dilute to 1x working
concentration using dH2O. Filter sterilize to remove all precipitate and contaminants.
50x Concentrated TAE Electrophoresis Buffer (40 mM Tris-acetate, 2 mM EDTA)
Add the following to dH2O to give a final volume of 1 liter
242 g Tris base
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA (pH 8)
BIOTECHNOLOGY I LAB 3 PAGE
EILENE LYONS – REVISED 1/12/2010 10
LAB 3 POLYACRYLAMIDE GEL ELECTROPHORESIS OF PROTEINS
TIMELINE
DAY 1: Prep for the lab, extract plant proteins, run SDS PAGE, stain and destain
DAY 2: Graph results using MS Excel and analyze
MATERIALS:
Per class
Bio-Safe Coomassie Blue stain (destains with dH2O)
*Practice gel loading solution
10X Tris-glycine-SDS buffer stock
*Vortex mixers
*Heating block set at 95°C
*1000 mL graduated cylinder
*dH2O
*1 L Corning orange capped bottles
*Plastic wrap
White light box
*Polaroid camera and film
*A supply of transfer pipettes
*Labeling tape
*1.5 ml microfuge tubes
Per every 2 teams
Flat metal spatula for separating gels
Dual vertical mini gel rig with clamps – please put on cart
Power supply – please put on cart
Per team
Kaleidoscope prestained protein molecular weight markers – aliquot 25 μl per team (Bio-
Rad # 161-0324) NOTE: heat briefly at 37°C to dissolve any precipitated SDS before aliquoting.
Flowering plants, one/team
2x Protein loading dye/buffer (≥ 100 ul)
Scalpel with sharp blade
Precast 15% polyacrylamide gel for SDS PAGE
8 disposable pellet pestles
2 ml 1X Laemmli buffer
*Should be in room – do not place on cart
BIOTECHNOLOGY I LAB 3 PAGE
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RECIPES
10x Tris-Glycine-SDS Buffer (500 ml should be allowed for each double gel rig; 1x =
25 mM Tris; 192 mM glycine, 0.1 % SDS )
3.04 g Tris base
14.41 g glycine
1.0 g SDS
dH2O to give 100 ml final volume
Dilute 100ml in 900ml dH2O for 1x working concentration
10 x Laemmli Buffer
0.25 M Tris,
1.92 M Glycine
1 % SDS in aqueous solution
Dilute to 1x working concentration
2x Protein loading dye (10 ml)
1.2 ml 1M Tris HCl pH 8**
4 ml 10% SDS
2 ml 100% glycerol
1 mg bromophenol blue
0.1 ml -mercaptoethanol
2.7 ml dH2O to give 10 ml final volume
**Adjust to pH 8 with HCl for prepoured graduated gels; if using discontinuous self poured
gels, adjust pH to 6.8.
BIOTECHNOLOGY I LAB 4 PURIFICATION OF ECO RI BY ION EXCHANGE
EILENE LYONS – REVISED 1/12/2010 12
LAB 4
PURIFICATION OF ECOR I by ION EXCHANGE CHROMATOGRAPHY