Genetic complexity of living systems

LiAc yeast transformation V3

Adapted from Gottschling lab’s page

Prepare LiAc 0.1M (510 mg qsp 50 mL H2O, autoclave at 1 bar).

Prepare LiAc 1M (510 mg qsp 5 mL H2O, autoclave at 1 bar).

Prepare PEG3350 50% (25 g qsp 50 mL H2O, autoclave at 1 bar).

Prepare Boiled ssDNA (dilute at 2mg/mL, incubate in boiling water for 5 minutes and put directly on ice. Store at -20°C).

Check the stock of plasmids (or DNA) and the stock of plates with selective medium.

Day 0 :

Grow your yeast strain (1 single colony) in 4 mL YPD overnight, 30°C, 220 rpm.
 
Day 1 :
Inoculate 4 mL of YPD with 200 to 250 uL of your overnight culture, incubate 3 to 5 hours at 30°C, 220 rpm.

Set waterbath at 42°C
Centrifuge at 4600 rpm, 5 min room temp, discard supernatant.
Wash cells with 4 mL sterile water
Centrifuge at 4600 rpm, 5 min room temp, discard supernatant.
Resuspend in 1 mL sterile LiAc 0.1M, transfer to eppendorf tube.
Centrifuge 15 sec 13000 rpm.
Resuspend cells with 100 to 200 uL LiAc 0.1 M. Vortex, aliquote if necessary. Take 50 uL for each transformation.

On the 50 uL aliquot of cells, add in the following order : (LiAc 1M always after PEG 50%)

Vortex thoroughly.
Incubate at 42°C for 40 minutes.
Spin at 4600 rpm for 1 min, discard supernatant.

If no drug resistance expression is needed : resuspend (gently!) in 200 uL water and plate on selective medium. Incubate 2 days at 30°C.

If drug resistance is needed: resuspend (gently!) in 400 uL YPD, tranfer to 14 mL tube and incubate at 30°C for at least 4 hours, then spin at 4600 rpm, discard most liquid, resuspend cells with what’s left of it and plate.