Hello,
Here it is...
Innoculate 200mL selective medium (or YEPD) and grow O/N 30C 200rpm.
Take OD A600 of above culture and innoculate 500mL YEPD such that in
approximately 2 generations (4hrs) the A600=0.5-0.8. (it's important to
use YEPD in this step)
Harvest at 2500rpm 10min at room temperature (beckman)
Wash pellet once with 100mL ddH20 (it's easier to resuspend the pellet in a
small amount of liquid by swirling the tube). Spin as above.
Resuspend pellet in 50mL LiSORB and incubate at 30C for 15-30 min.
During the incubation:
boil 200uL 20mg/mL sonicated salmon sperm DNA for 10'
add 800 uL LiSORB (at room temp)
mix by pipetting up and down
cool to RT (using ice may gel mixture)
add 40 ug DNA, mix by pipetting up and down.
Spin cells (as above) and resuspend in 625 uL LiSORB.
Add 100uL of above DNA mix to 100uL cells.
To 100uL cells+100uL DNA add 900uL 40% PEG.
Incubate at 30C for 30min and heat shock 42C for 5min.
Cool in water bath at room temp.
Cells can be plated directly from PEG solution (do not try to spin cells
in PEG - if you have to concentrate them, dilute cells in about 100 mL medium,
then spin down)
PEG - PEG3350 in 100mMLiAc/TE
LiSORB - 100mMLiAc, 10mM Tris pH8, 1 mM EDTA, 1M Sorbitol
LiAc/TE - 100mMliAc, 10mM Tris pH8, 1mM EDTA
The yeast doesn't mind being frozen at -70 C in the PEG solution.
Efficiency = 5x10exp4 to 5x10exp5 colonies per ug DNA
This works well for my system. I hope someone else can get some use out
of it. I have read somewhere that the efficiency might be better if the
plates contained 1M Sorbitol, but I've never tried it.
Kath
