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LiAc yeast transformation V2
Adapted from Gottschling lab’s page
Day 0 : Grow your yeast strain in 4ml YPD overnight.
Day 1 :
Inoculate 4ml of YPD with 200 ul of your overnight culture, incubate 3 to 5 hours at 30°C.
Set waterbath at 42°C
Centrifuge 4000rpm 5min room temp, discard supernatant.
Wash cells with 4 ml sterile water
Centrifuge 4000rpm 5min 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 50ul for each transformation.
On the 50ul aliquot of cells, add in the following order:
Day 1 :
Inoculate 4ml of YPD with 200 ul of your overnight culture, incubate 3 to 5 hours at 30°C.
Set waterbath at 42°C
Centrifuge 4000rpm 5min room temp, discard supernatant.
Wash cells with 4 ml sterile water
Centrifuge 4000rpm 5min 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 50ul for each transformation.
On the 50ul aliquot of cells, add in the following order:
| PEG3350 50%: | 240ul |
| LiAc 1M: | 36ul |
| boiled-ssDNA (2mg/ml): | 50ul |
| your DNA solution: | 34ul |
Vortex thoroughly.
Incubate at 42°C for 40 minutes.
Spin at 6000 rpm for 1 min, discard supernatant.
If no drug resistance expression is needed: resuspend (gently!) in 200ul water and plate on selective medium.
If drug resistance is needed: resuspend (gently!) in 400ul YPD, tranfer to 14ml tube and incubate at 30°C for at least 4 hours, then spin at 10000rpm, discard most liquid, resuspend cells with what’s left of it and plate.
Buffer and Reagents:
PEG3350 50%:
Prepare 50% PEG3350 solution, mix well, filter-sterilize.
Boiled ssDNA:
Prepare salmon-sperm DNA at 2mg/ml, incubate in boiling water for 5 minutes and puce directly on ice. Store at -20°C.