...
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.
...