Small Scale Saccharomyces cerevisiae Transformation
1. Grow a 10 ml overnight yeast culture in YPDA at 30oC, 200rpm.
2. Pre-heat water bath to 30oC.
3. Pellet 1ml of culture in a 2ml eppendorf tube at 2000rpm for 5 minutes. 1ml of
culture is sufficient for 10 transformations.
4. Remove the supernatant and resuspend in 1ml 0.1M LiAc.
5. Pellet cells again as above.
6. Remove the supernatant and resuspend in 1ml 0.1M LiAc.
7. Incubate cells at 30oC in the water bath for 1 hour.
8. Prepare the DNA mix as follows in a 1.5ml eppendorf tube:
3ul miniprep DNA (500-1000ng)
4ul ssDNA
290ul 50% PEG 3350
(ssDNA is a 2mg/ml stock of salmon sperm carrier DNA (sigma D1626) boiled
for 10 minutes and then put on ice for 5 minutes).
9. Pre-heat DNA/PEG mix to 30oC.
10. Add 100ul cell suspension to the DNA/PEG mix and mix very gently with the pipette
tip.
11. Incubate for 50 minutes at 30oC in the water bath.
12. Heat shock cells by transferring to a 42oC water bath for 15 minutes.
13. Pellet cells at 3000rpm for 5 minutes.
14. Remove supernatant very carefully (cells will be smeared along the side of the tube
and will move easily).
15. Resuspend in 200ul sterile water.
16. Spread cell suspension on selective plates.
17. Incubate at 30oC until colonies appear.
18. Once colonies have appeared restreak 4-8 colonies onto selective media and
incubate for 1-2 days at 30oC. Plates can then be stored in the fridge. Restreak every
2-3 weeks to maintain fresh growing cells.