Fast transformation of yeast
prepare competent cells
inocculate 100 ml medium with a fresh overnight culture to OD=0,15
grow culture to OD=0,4-0,6
collect cells (2k, 15 min, RT)
wash with 25 ml LiSorb (2k, 15 min, RT)
discard supernatant, briefly spin and completely remove supernatant
suspend cells in 0,6 ml LiSorb
add 60 µl of carrier DNA (10 mg/ml salmon sperm DNA), mix
prepare aliquots of 50 µl
freeze slowly to -80°C (not with liquid nitrogen)
transformation
all steps at RT
add 0,5 to 5 µl of plasmid DNA with the cells
add 300 µl LiPEG, mix gently
incubate for 30 min at 30°C (or RT), rotate tube
add 35 µl DMSO
heatshock for 20 min at 42°C (waterbath)
spin gently (1-2 min, 1,5k) suspend cells in appropriate volume or
plate the cells directly on selective plates
incubate for two to three days at 30°C
for simple transformations 10 µl of competent cells are sufficient, use
50 µl for double transformations
buffer:
LiSorb
100 mM Li-actetat
10 mM Tris/HCl pH8
1 mM EDTA
1 M Sorbitol
LiPEG
100 mM Li-actetat
10 mM Tris/HCl pH8
1 mM EDTA
40% PEG4000
store at RT