1. Grow the recipient strain of yeast to mid-log (1x107).
The better the media used the better the transformation efficiency,
so use YPD when possible.
2. Pellet the cells
at 2000 rpm in 50 ml aliquots in the Sorvall bench top centrifuge
(using 50 ml Falcon® tubes).
3. Resuspend the
cells in sterile water; the volume of water used is not important
since your only washing the cells. (At this point I usually
pooled the cells in 2 Falcon® tubes).
4. Repellet the
cells as above and then resuspend the pellets in LiTE (100mM
LiAc in TE); again, the volume is not critical.
5. Pellet the cells
once more and this time resuspend the cells in 5ml of LiSORB
for every 200ml of culture (LiSORB is LiTE containing 1M Sorbitol).
6. Incubate the
cells for 1h at 30°C with shaking. (Try not to go too
long, 1h is optimal 1.5h is dangerous)
7. Pellet the cells
as above and decant.; resuspend in 250 µl of Carrier
Resuspen-sion Buffer per 200ml of culture (CRB is prepared
by diluting 20 mg/ml carrier DNA in LiSORB to a concentration
of 4 mg/ml.)
8. After removing
100µl of cells for a negative control, add 2µg
of transforming DNA for every 100µl of cells remaining.
9. Mix well, then
incubate for 30' at 30°C without shaking.
10. Add 900µl
of LiPEG (LiTE containing 40% PEG 3350) for each 100µl
of cells and mix well.
11. Divide the
cell suspension in Eppendorf™ tubes (˜1ml per tube);
this is important because it takes too long a time to heat
a large volume of PEG uniformly.
12. Incubate the
tubes at 30°C for 20 minutes.
13. Mix the contents
of each tube and then heat the tubes (in a heating block)
for 10' at 42°C.
14. Immediately
plate the cells or innoculate a second selective media with
the cell suspension. Do not try to pellet the cells through
the PEG since this will lessen the transformation frequency,
and don't wait too long to plate or grow up the transformants
since the PEG is toxic to the cells. Twenty microliters of
cell suspension can give you between 1000-2000 transformants
for a healthy yeast strain.
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