Yeast Transformation 2000

Dilute a saturated overnight culture 1:40 into YPAD (1:25 if the overnight is grown in SC medium). It’s usually convenient to dilute 1.25 ml into 50 ml of YPAD in a 250 ml flask (enough for 10 transformations).

Grow 3-5 h in a 30 C shaker.

Transfer to a Falcon tube and pellet cells (5 min at 2000 K in a clinical centrifuge)

Resuspend pellet in 1 ml of 0.1 M LiOAc

Transfer to an Eppendorf tube

Quick spin to pellet cells

Resuspend pellet in 500 ul of 0.1 M LiOAc (this assumes you spun down 50 ml of cells)

For each transformation:

Aliquot 50 ul of cells in an Eppendorf tube, quick spin, and take off sup.

Then add over the pellet in this order:

250 ul of 50% PEG 3350 (use a repeater pipettor if you’ve got lots of samples)

5 ul of boiled 10 mg/ml single-stranded DNA (Herring sperm is the cheapest)

36 ul of 1 M LiOAc

2 ug of the desired DNA brought up to a volume of 50 ul (e.g. 4 ul of DNA plus 46 ul of water)

Vortex to mix

30 C water bath for 30 min

42 C water bath for 20 min

Quick spin to pellet

Take off sup

Optional outgrowth (for highest efficiency):

Resuspend pellet in 1 ml YPAD

30 C 30-60 min

quick spin

Resuspend in 200 ul of water

Plate on selective medium (unless you’re using a drug resistance marker like kanMX6—in which case you should plate on YPAD and replica plate the next day to a drug plate)