Transcript Cultivation and Transformation of Yeasts

Tian He
2008. 07. 06
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)
• Dropout mix (-His/-Trp/-Leu DO mix)
6.7 g
0.62 g
• Amino acids
• Glucose
2g
• Agar (for plates)
2g
• 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
HIS
504
TRP
505
LEU
506
ADE
Question: why we use pGREG503 for homologous
recombination? We cannot determine from the phenotype
whether recombination occurs.
pGREG 503/4/5
Promoter
Homologous sequence
Tag
Antibiotic marker
Antibiotic marker
Stuffer
Auxotroph marker
Autonomously replicating sequence
Yeast strain: AH109
GAL 4 induced promoters: GAL1, GAL2, MEL1
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 12×107/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 singlestranded 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)
Protocol
10.
Centrifuge at 13.000 g for 30 s. Remove the supernatant carefully.
11.
Resuspend the cell pellet 1.0 mL of 1×TE/sterile water. Stir the pellet
with a micropipette tip and vortex.
12.
Dilute appropriately and plate on selective medium.