LiAc Transformation of Yeast Fast Version

Notes: This is a high efficiency protocol (up to 5x104 colonies/mg DNA)

1. Inoculate 50 or 100 ml of YPD with a 1:50 dillution of an overnight culture. Grow to an OD600 of .5 - 1 (1-2x107). 50ml = 5 transform, 100ml = 10.

2. Spin down at 2k for 2 min in a sterile tube at roomtemp.

3. Resuspend the cells in 10ml of TE (sterile) and spin down.

4. Resuspend in 10ml of LiAc MIX and spin down.

5. Resuspend in 500ml (50ml prep) or 1 ml (100ml prep) of LiAc MIX.

6. Aliquote 100ml of yeast into sterile ependorf tubes for each transformation.

7. Add 50ng to 5mg of plasmid DNA (if youÕre transforming with an integrating vector, the plasmid must be linearized) or about a half a minipreps worth of DNA (strains vary in there transforming ability, w303 use about 50ng).

8. Add 3-10ml (30 to 100mg of ssSalmon Sperm DNA (before each use boil for 5 min, spin and put on ice immediately) (It is critical that the salmon sperm DNA be single strained and as long as possible, see preparation protocol).

9. Add 0.7ml of PEG MIX to each tube.

10. Inc. for 30min at 30 degrees C.

11. Heat shock for 15 min at 42 degrees C, spin down (5sec micro).

12. Resuspend in 300ml of SOS (or YPER plus CaCl on gal plates), plate immediately.

LiAc MIX (store at rm temp, sterile filtered)    500ml

-H2O                                                               -400ml

-100mM LiAc                                                -50ml of 1M LiAc

-TE (tris 10mM, 1mM EDTA pH8)           -50ml of 10xTE

PEG MIX (make fresh)                                           10ml (store stocks at rm temp)

-40% PEG mw 3350                                      -8ml of 50% sterile filtered

-100mM LiAc                                                -1ml of 1M LiAc sterile filtered

-TE (tris 10mM, 1mM EDTA pH8)           -1 ml of 10xTE sterile filtered

SOS (make fresh)                                                     10ml

-1M Sorbitol                                                   -5ml of 2M sorbitol

-33% YEP                                                         3.4ml of YEP

-6.5mM CaCl                                                  -65ml of 1M CaCl

-H2O                                                                -1.6ml of H2O

RJD leaves out the sorbitol

Note: cells can be frozen at step 5 in TE + 15% glycerol and stored at -70, thaw slowly, spin down and resuspend in LiAc.