Yeast Transformation High Efficiency old school





Higher efficiency – simplified transformation of yeast protocol   (LKL lab 3/2002)
Dilute from an overnight culture or yeast from a plate into YPD (OD ~0.2 ok)  a.          alternatively you can make multiple dilutions for overnight culture to attempt to achieve log phase in morning

 2. use 5-10ml YPD per transformation of yeast YPD culture to log phase (OD600 ~0.6-1.0)  at 30C

 a.       ''' This may take ~3-5 hours or more '''

 3.  Spin down   to pellet yeast, (2000RPM for 5min works in large centrifuge)

 4.  Resuspend   pellet in 0.1M LiAc (Lithium acetate)

 5. ''' Transfer to 1.5ml tubes '''

 6.  Spin down   to pellet yeast, full speed 20sec

 7.  remove supernatant   carefully  (use vacuum pump or p1000 w/big&little tips)

 8.  +   240µl 50% PEG,  ''' add slowly. ''' S crape across bottom of test tube rack to resuspend, and/or vortex/pipette

 9. ''' + 36µl 1M LiAc '''

 10.  +   5ul ssDNA salmon sperm

 a.          once every few uses ssDNA should be heated to 100C heating block for 5min and then kept on ice

 11. ''' + 70ul Water & DNA '''

 a.       For integrative transforms use >1µg of linearized DNA, maybe >4ug

<p class="MsoListParagraphCxSpMiddle" style="text-indent:-0.25in;"> 12.  42C water bath for 20min 

<p class="MsoListParagraphCxSpMiddle" style="text-indent:-0.25in;"> 13. Spin down 15s full speed

<p class="MsoListParagraphCxSpMiddle" style="text-indent:-0.25in;"> 14. Remove supernatant and discard, resuspend in 250ul sterile water

<p class="MsoListParagraphCxSpLast" style="text-indent:-0.25in;"> 15. Plate onto appropriate selection media

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