Method prepared by Grace Panganiban: gepangan@facstaff.wisc.edu


Yeast Transformation

1) Grow yeast to A650=0.5 (yeast doubling time is about 140 minutes at 30oC). If yeast overgrow, dilute to about A650=0.2 and let grow another 3 hrs.

2) Spin at 6000 rpm for 2 min in ufuge or at high speed for 5 min in coldroom tabletop.

3) Resuspend pellet in original volume of ice cold diw; respin.

4) Resuspend pellet in original volume of ice cold 01 M LiAc in TE; respin.

5) Resuspend pellet in 1/300 -1/100 of original volume ice cold 01 M LiAc in TE; use for transformation immediately.

6) Mix 1 ul (~100 ng) with 7 ug carrier DNA (sheared salmon sperm) in
1 ul TE and 0.2 ul 1 M LiAC.

7) Add 8 ul competent yeast

8) Add 70 ul 40% PEG, 0.1 M LiAc, TE pH 7.5; mix with pipettor.

9) Incubate for 30 min at 30oC.

10) Heat shock for 12 min at 46oC.

11) Plate on appropriate medium; grow ~ 3 days at 30oC.

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