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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.
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A. For prototrophic gene selection: *Pipette 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 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.Prototrophic gene selection
(i) Pipette 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.
(B) Eukaryotic antibiotic gene selection
(i) Pipette 1.0 ml of YPD liquid medium into the transformation tube. Vortex mix to resuspend pellet.
(ii) Incubate for 2--3 h at 30 1C to ensure good expression from the input plasmid DNA.