Modified from Gietz (2002) by Greg Foley and then Rishi Jajoo
- Filter-sterilized, 1M lithium acetate.
- Filter-sterilized, 100mM lithium acetate.
- Polyethylene glycol (MW = 3015-3685) 50% w/v. (10 g in 20 ml total volume)
- Antibiotic YPD agar plates.
- 2 ng/ ul Salmon Sperm
- 5:00 PM: Inoculate the yeast strain into 5 ml of YPD and incubate in shaker overnight.
- Place 50 ml of YPD in shaker. (This is enough for 10 Reactions)
- Pipette 500 ul of overnight culture into 50 ml of pre-warmed YPD.
- Incubate in shaker for 4 hours.
- Boil a 1.0 ml sample of salmon sperm for 5 min and place on ice while harvesting the cells.
- Harvest the cells by centrifugation at 3000 g for 5 min in a 50 ml Falcon tube.
- Re-suspend cells in 25 ml of sterile water.
- Centrifuge at 3000 g for 5 min.
- Re-suspend in 1 ml of 100 mM lithium acetate.
- Transfer the cell suspension to a 1.5 ml microcentrifuge tube.
- Centrifuge for 30 seconds at max speed.
- Resuspend in enough 100 mM lithium acetate to reach 500 ul total volume.
- Make one, 50 ul aliquot for each transformation.
- Centrifuge the aliquots at top speed for 30 seconds and pipette off the supernatant.
- Add the following reagents to each transformation in the order given:
- Polyethylene glycol 3500 50% w/v: 240 µl
- Lithium acetate 1.0 M: 36 µl
- Boiled salmon sperm (
- Plasmid DNA plus Water (use 0.1 – 10
- Incubate for 30 minutes at 30 degrees.
- Incubate at 42 degrees for 20 minutes.
- Centrifuge at top speed for 30 sec and pipette off the supernatant.
- Pipette 1.0 ml of YPD into each tube;
- Let sit 10 minutes to soften the pellet.
- Stir the pellet gently with a pipette tip and vortex for 1-2 seconds until the cells are re-suspended.
- Burn a hole through the top of the epitube with a hot 20 gauge needle to prevent fermentation induced blowouts.
- Incubate at 30°C overnight.
- Plate 200ul aliquots onto selective plates.
Incubate the plates at 30°C for 3 to 4 days.