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Protocols Online
Genotyping and Ancestry
Common Media
LB
0.5% yeast extract
0.5% NaCl
1% tryptone
SOB
200 ml
1 g yeast extract
4 g tryptone
10 mMNaCl
0.2 ml of 1M KCl
0.2 ml 1M MgCl2 (1mM final)
0.2 ml 1M MgSO4 (1 mM final)
for SOC add 1/100 2M glucose
LM (1 liter)
10 g tryptone
5 g yeast extract
0.58 g NaCl (1 mM final)
2.46 g MgSO4 (1 mM final)
pH 6.8-7.0
TB
1.2% tryptone
2.4% yeast extract
0.4% glycerol
1X M9 salts
2mM MgSO4
0.1mM CaCl2
0.4% carbon source (e.g. glycerol, glucose, etc.)
In sterile H2O
5X M9 salts contains:
64 g Na2HPO4.7H2O (for 250 ml; 16 g)
15 g KH2PO4 (3.75 g)
2.5 g NaCl (0.625 g)
5 g NH4Cl (1.25 g)
dissolved in deionized water to a final volume of 1 L
YPD
1 % yeast extract
2 % peptone
2% dextrose
SD-Ura (100 ml)
2.67 g dropout base with glucose (USB #D9500)
0.2 g dropout mix minus Ura (USB #D9535)
1.5 g agar
pH 5.8 with 1M NaOH
Solutions for plasmid minipreps
Buffer P1
50 mM Tris-HCl, 10 mM EDTA, pH 8.0, 50 ug/ml RNaseA
Buffer P2
0.2 M NaOH, 1% SDS
Buffer N3
4 M guanidine HCl, 0.5 M KOAc, pH 4.2
Buffer PB (wash)
5 M guanidine HCl, 20 mM Tris-HCl, pH 6.6
38% EtOH
Buffer PE (wash)
20 mM NaCl, 2 mM Tris-HCl, pH 7.5
70% EtOH
Buffer EB (elution)
10 mM Tris-HCl, pH 8.5
E coli supplements
ampicillin 50 ug/ml from 25 mg/ml stock, filter sterilized (200 ul/ 100 ml media)
kanamycin 50 ug/ml from 50 mg/ml stock, filter sterilized (100 ul/ 100 ml media)
chloramphenicol 15 ug/ml from 34 mg/ml stock dissolved in 95% ethanol
streptomycin 50 ug/ml from 50 mg/ml stock, filter sterilized (100 ul/ 100 ml media)
x gal use 1 ul/ml media
make stock at 20 mg/ml (200 mg in 10 ml DMF)
IPTG use at 100 uM, (1 ul/ml media)
make stock (100 mM) 238 mg/10 ml ddH2O
Borax gel prep
10 X stock of borax (100 mM)
19 g of borax in 500 ml, resulting pH 9.3
Assuming Na2B4O7·10H2O, MW= 381.37
Use 1X (10 mM) for gel and running buffer (pH 9.0)
Based on BioTechniques 36:214
TSS transformation of E. coli
2X TSS
20% PEG 4000
10% DMSO
40 mM MgSO4
store at 4C
Inoculate colony of E. coli into 5 ml LB, grow overnight at 37C
Inoculate 1 ml of above into fresh flask of 50 ml SOB, shake until OD = 0.3-0.5
Place cells on ice for 10 min
Spin down to 1/10th volume (5 ml) and ice, use 1 ml original culture per sample
Mix cells 1:1 with cold 2X TSS and ice.
Add DNA to eppendorf on ice
Add 100 ul cell mixture to DNA
Incubate on ice 30-60 min
Add 0.9 ml of SOC to cells, shake at 37C for 1 hr
Plate on antibiotic media (LM + antibiotic)
adapted from PNAS 86:2172 (Chung, Niemela, Miller)
Electroporation of Saccharomyces cerevesiae
1. Inoculate 50ml YEPD with a colony and grow with shaking at 30C until early stationary (~0.6-2E8 cells/ml).
2. Harvest in a 15 ml sterile tube in clinical centrifuge spin at top speed, 4C, 2' and keep cells on ice throughout the procedure.
3. Wash cells with 40ml ICE-COLD sterile dH2O, pellet, 4C, 2'.
4. Repeat wash with 20ml sterile dH2O (ice-cold).
5. Resuspend cells in 5ml 1M Sorbitol (ice-cold) pellet, 4C, 2'.
6. Resuspend cells with 150µl 1M Sorbitol (ice-cold) - KEEP ON ICE!
7. Mix 40 µl of yeast suspension with <5 µl DNA (~5 µg) in a prechilled electroporation cuvette (0.2 cm). Tap contents to the bottom, making sure that the sample is in contact with both sides of the aluminum cuvette.
8. Give one pulse: V= 1.5kV, 25 µF, 200 Ohms. Time should be ~4-5".
9. Immediately add 1 ml 1M Sorbitol (ice-cold) and transfer with a sterile pasteur pipette to a sterile Eppendorf tube.
10. Spread onto selective plates.
Waste
All biological waste (any material coming in contact with any laboratory organism, recombinant or not, petri plate, pipette tip, etc) is placed in a plastic biohazard bag. When bag is 3/4 full it is sealed and sterilized in a 41 q pressure cooker for
45 min at 15 lbs pressure.
Alternatively, liquid media containing growth is mixed 1:1 with concentrated bleach
overnight before disposing of in sink.
Ethidium Bromide (10 mg/ml solution from Sigma)
Use as 2 ug/ml solution in 1X TAE buffer. Dispose of according to guidelines suggested by Duke University.