From Powers Lab UC Davis
Previous day
Grow 5 mL culture for several hours (at least three, but longer is OK). Do a spec reading and then inoculate a small aliquot of this culture into 50 mL of YPD medium in 125 mL DeLong flask. Use formula to determine how large the aliquot should be to start the transformation protocol at a certain time the next day.
Next day
Harvest cells using 50 mL centrifuge tube, 4000 rpm Allegra tabletop centrifuge, 10'.
Resuspend cells in 25 mL sterile H2O. Centrifuge again, as above.
Resuspend cells in 1 mL 100mM LiOAc into 1.5 mL microfuge tube.
Pellet cells at max, 15 secs. Remove LiOAc with micropipet.
Resuspend cells in 400 ul 100mM LiOAc. (Will be approx. 500 ul total with cells.)
(Boil salmon sperm DNA 5', quick chill.)
Vortex cell suspension and pipet 50 ul samples into 1.5 mL tubes.
Pellet cells and remove LiOAc.
Add the following:
240 ul PEG 3350 (50% w/v)
36 ul 1 M LiOAc
10 ul salmon sperm DNA (10 mg/ml)
14 ul plasmid DNA
60 ul sterile H2O
Vortex approx. 1'.
Incubate 30 C, 30'.
Heat shock 42 C, 30'.
Microfuge at 8000 rpm, 15 secs. Remove transformation mix.
Add 200 ul sterile H2O to each tube and resuspend pellet by pipetting up and down gently.
Plate 200 ul of transformation mix
onto dropout plates.