Tian He 2008. 07. 06. Media non-selective: YPD without antibiotics Selective: Synthetic complete dropout medium (SC) (auxotroph) YPD with antibiotics.
Post on 17-Dec-2015
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Media
non-selective:
YPD without antibiotics
Selective:
Synthetic complete dropout medium (SC)(auxotroph)
YPD with antibiotics
YPD: 1000 mL
• Yeast Extract 10 g
• Peptone (Tryptone) 20 g
• Glucose 20 g
• Agar (for plates) 20 g
• Distilled H2O 1000 mL
Synthetic complete dropout medium (SC) 1000 mL
• Yeast nitrogen base without amino acids (YNB) 6.7 g
• Dropout mix (-His/-Trp/-Leu DO mix) 0.62 g
• Amino acids
• Glucose 2 g
• Agar (for plates) 2 g
• Distilled H2O 1000 mL
Cultivation
• Temperature: 30 ℃ Lower: yeasts grow slowly.
Higher: yeasts die.• Shake: 200 rpm or higher • Avoid the contamination of E.Coli.• Gloves are needed to avoid infection!
Notes:
• It takes 2 hours or more to complete a cycle.
• Yeast will age. Inoculate a new plate every month.
• The selectable marker and medium should be complementary!
Commonly used selectable marker
LEU2, URA3, TRP1, HIS3, ADE1,2
pGREG 503 504 505 506
HIS TRP LEU ADE
Question: why we use pGREG503 for homologous recombination? We cannot determine from the phenotype whether recombination occurs.
pGREG 503/4/5
Autonomously replicating sequence
Auxotroph marker
Antibiotic markerAntibiotic marker
Homologous sequence
Stuffer
Promoter Tag
Transformation
Plasmid/ssDNA/dsDNA
The competent cells of yeast cannot be stored;
it must be prepared before use!
Materials
• TE: 0.1M Tris-HCl, 0.01 M EDTA, pH 7.5 • LiAc: 1M LiAc, pH 7.5• PEG: MW 4000 (50 % w/v), stored at room temperature.
Capped securely to avoid evaporation.• Single-stranded Carrier DNA(2.0 mg/mL): Salmon sperm
DNA. Boil 1.0 mL carrier DNA for 5 minutes and quickly chill on ice water. Do not boil the carrier DNA every time. Keep a small aliquot in freezer box and boil after 3-4 freeze thaws. Keep on ice when out.
Protocol
1. Inoculate cells into 50 mL YPD and grow overnight to a density of 1-2×107/mL(nearly saturated). A suspension containing 1×106 cells/mL gives an OD600 of 0.1.
2. Dilute to 2×106 /mL in fresh YPD and re-grow into exponential phase (1×107/mL). It typically takes 3~4 hours. It is important to allow the cells to complete at least 2 divisions. Transformation efficiency remains constant for 3~4 cycles.
3. Harvest the culture in a sterile centrifuge tube at 2500 rpm for 5 minutes. Wash in sterile water twice.
4. Resuspend in 1.0 mL sterile water and transfer to 1.5 mL microfuge tube.
centrifuge for 30 s at 13.000 g and discard the supernatant.
Protocol
5. Wash cells in 1.0 mL of TE/LiAc(10×) and resuspend at 2×109 cells/mL in TE/LiAc (1×)
6. Mix 50 μL(1×108 cells) with 1 μg transforming DNA and 50 μg single-stranded carrier DNA in microfuge tubes.
7. Add 300 μL sterile plate solution (40 % PEG 4000 + 1×TE/LiAc , for 1 mL plate solution, you should add 0. 8 mL 50 % PEG 4000, 0.1 mL 10×TE, 0.1 mL 10×LiAc). Vortex to mix thoroughly.
8. Incubate at 30 in the shaker for 30 minutes. ℃
9. Heat shock in a 42 waterbath for 15 minutes(different strains have different optimal heat shock time)
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