Summary: Auble Lab
Inoculate 100 ml YPD with 1 single (reasonably fat) yeast colony at about 5
pm. Incubate over night at 30 C with shaking.
Check OD600 next day, and also check cells under microscope for
contamination. Culture should be at about OD600=1 at 9-10 am.
When OD600 is about 1, harvest cells by centrifugation. Pellet cells 5
minutes at 5000 rpm in clinical centrifuge in each of 2 sterile 50 ml conical
Decant supernatant, wash cells in 10 ml sterile water. Pellet again as above.
Wash cells in 5 ml TE/LiOAc.
Resuspend cells in 1 ml TE/LiOAc.
Boil single-stranded carrier DNA for 5 minutes and cool on ice.
Mix 50 µl yeast cells with 1 µl plasmid mini prep and 50 µg freshly boiled
single-stranded DNA (e.g. 5 µl 10 µg/µl SS DNA) in eppendorf. Mix by
tapping with finger.
Add 300 µl TE/LiOAc/PEG solution to each tube. Vortex immediately after
solution is added to each tube.
Incubate 30 minutes at 30 C.
Heat shock 15 minutes at 42 C.