Check OD600 in the morning, if
the cells are overgrown then dilute them and grow to correct density-
90minutes= yeast doubling time.
EtOH ppt your digestions, and
resuspend in 30uL H20- use 10uL here and save rest for later (mark on the tube
that it is digested for transformations with NcoI enzyme).
YEAST TRANSFORMATION (7/13/01)
1) INNOCULATE 100mL YPD from a single colony or a scrape from a plate into 10mLs YPD that is then serially diluted 1:10 and 1:100 in 50-100mLs. One of these three tube will be the correct OD in the morning. (OD600= 0.5 for integrations and 0.4-0.8 for plasmids).
Background of yeast you are transforming___________________________
2) Transfer cells to 50mL Falcon Tubes, spin 3 min in IEC clinical on 4-5 setting. Start warming heat block to boiling for step 5 and start mixing the PEG mix in step 5.
3) Resuspend cells in 10mLs sterile water. Spin again.
4) Resuspend pellet in 1mL sterile 100mM LiOAc in TE.
5) Assemble the following in Eppendorf tubes:
5-10uL 10mg/mL of carrier DNA (boil and quick chill first).
1ug of plasmid or 50uL PCR reaction or 10uL of RE digest of 1ug for integrations (just
EtOH ppt the DNA after digest- “yeast like dirty things” – M. Yaffe)
100uL cells (mix up and down with pippette
0.7mL Sterile PEG mix (made fresh)
NAME OF DNA you are TRANSFORMING:__________________________________
6) Incubate at 30°C for 1 hour.
7) Add 100uL DMSO, invert; transfer to 42°C for 15 minutes
8) Spin 10 seconds in microcentrifuge. Aspirate off supernatant
9) Resuspend in 1mL SOS or YPD to wash, spin again and resuspend in 200uL SOS or YPD and wait as long as possible before plating (this is the recovery time before plating on selective media. For Kan or NEO you can plate onto YPD (recovery) and then replica plate the colonies onto selective media. In general the HIS marker they make quickly, the trp you wait slightly longer and KAN takes a lot longer.
4g PEG 3500 50mL 2M Sorbitol
1mL 1M LiOAc 34mL YEP
1mL 10X TE 650uL 1M CaCl2
up to 10mL mark witth Sterile H20. 16mL H2O
filter sterilize with 0.45uM filter
5mL 1M Tris pH 7.5
1mL 0.5M EDTA pH 8.0
44mL H2O