Yeast Transformation

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Sleazy Yeast Transformation

1. Create the Transformation Mix:

240ul 50% PEG3500
36ul 1M LiAc
50ul ssDNA (boil 5min and leave on ice)
34ul DNA + H20 (if plasmid 1-5ul DNA)

2. Add a match head of yeast (swirl to release)

3. Incubate overnight at 30C

4. Spin calls at 5,000g for 1min

5. Resuspend the cells in 300ul H20

6. Plate using sterile methods and incubate at 30C for 2-3days

High Efficiency Yeast Transformation

1. Dilute a Yeast Liquid Culture 1:20 in 50ml, set to shake for 4 doubling times

2. Centrifuge at 3000g for 5 min

3. Wash and Centrifuge for additional 3min

4. Resuspend in 1ml sterile H20

5. Boil a ml sample of ssDNA for 5min and set on ice

6. Transfer cells to 1.5ml tubes, centrifuge on high 30s

7. Add sterile H20 to a final volume of 1ml and resuspend

8. Pipet 100ul of samples into eppendorfs, centrifuge 30s at max

9. Remove Supernatant and add Transformation Mix (see cocktail above)

10. Resuspend incubate in water bath at 42C for 40min

11. Centrifuge 30s at max, remove supernatant

12. Resuspend in 1ml sterile H20

13. Plate 300ul, incubate 30C for 2-3days.