Yeast Transformation High Effeicieny newer

 Adapted from Lewis lab publication (PMID:23483586)    1. Spin down 1.5ml cells (log phase probably higher efficiency)  2. Add 5-50 µl ss DNA (boiled 5’ 100C, then put on ice or frozen down after boiling)  3. Add DNA 2-50 µl                a. Less for plasmids (reported 10-50ng sufficient)                b. More for integrative transformations (e.g., use all 50ul of a PCR of pRS30x vector)  4. + 500 µl of PLTE (see recipe below)  5. +56 µl DMSO  6. Resuspend by raking on test tube rack, or by pipetting w/ P1000  7. 42°C for 20-30 min  8. Spin down, discard spnt  9. Resuspend in YPD  10. Incubate at 30°C for 40min  11. Spin down, discard spnt  12. Resuspend in 300+ ul water and plate to selective media (G418, -ura, -his, -leu, etc.) <p class="MsoNormal" style="margin-left:0.25in;line-height:11.55pt;background-position:initialinitial;background-repeat:initialinitial;">

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<p style="text-align:right;"> PLTE solution (make fresh day of transformation)

<p class="MsoNormal" style="margin-left:0.25in;line-height:11.55pt;text-align:right;">  800µl 50% PEG 4000

<p class="MsoNormal" style="margin-left:0.25in;line-height:11.55pt;text-align:right;">  100 µl 1M LiAcetate (LiAc)

<p class="MsoNormal" style="margin-left:0.25in;line-height:11.55pt;text-align:right;"> 2µl 0.5M EDTA

<h5 class="MsoNormal" style="margin-left:0.25in;line-height:11.55pt;text-align:right;"> 100µl 1M DTT

<h5 class="MsoNormal" style="margin-left:0.25in;line-height:11.55pt;text-align:right;"> (DTT is stored at -20C,  relatively unstable so make fresh periodically)