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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.)


PLTE solution (make fresh day of transformation)

 800µl 50% PEG 4000

 100 µl 1M LiAcetate (LiAc)

2µl 0.5M EDTA

100µl 1M DTT
(DTT is stored at -20C,  relatively unstable so make fresh periodically)

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