...
2. Add 5 mL of overnight cultures into 50 mL of 2xYPAD in a 250 mL bottle. Record the OD600 of the solution (remember to calculate the OD600(corrected) = OD600 of cells - OD600 of 2xYPAD blank). This will likely be around OD600(corrected) ~ 0.1. Often 2xYPAD has an OD600 of about 0.2.
3. Grow at 30C 300 rpm until OD600(corrected) ~ 0.4-0.5. This will equate to about 2 cell divisions, which is necessary for good transformation efficiency. This can take 6-10 hours.
...
11. Place the tubes in a 42C water bath for 45 minutes.
12. Centrifuge the tubes at 13000g for 30 seconds and remove the supernatant.
A. For prototrophic gene selection: Pipette 200 uL - 1.0 ml of sterile water into the transformation tube. Stir the pellet with a sterile micropipette tip to break the cell pellet and then vortex mix to uniformly resuspend pellet. I usually resuspend in 200 uL.
B. For eukaryotic antibiotic gene selection: Pipette 200 uL - 1.0 ml of YPD liquid medium into the transformation tube. Vortex mix to resuspend pellet. Incubate for 2--3 h at 30C to ensure good expression from the input plasmid DNA.
13. Plate 200 uL of the cell suspension on the appropriate plates. Incubate plates at 30C for 2-3 days.