Yeast transformation protocol
Day 1: streak yeast strains from freezing stock on YPD + 2% glucose plates.
Day3: Pick up single colony and inoculate into YPD + 2% glucose liquid medium. Incubate at 30 degree w/ 250 rpm shaking over night.
- 10ml of YPD + 2% glucose liquid medium is added, cells are transformed to a larger flask and cells are incubated at 30 degree, 250rpm for approximately 5 hours, until OD600=1
- Centrifuge cells in 50ml tubes at 3000g for 5 minutes
- Discard supernatant, resuspend in 1ml 100mM LiAc.
- Transfer the mixture into 1.5ml tube.
- Centrifuge at 8000rpm for 1minute and discard LiAC.
- Add 50ul 100mM LiAc per transformants. (If you want to do 3 transformations, add 150ul)
- Boil carrier DNA for 5 minutes and quickly chill on ice.
- Centrifuge tubes and remove supernatant.
- To each tube add the following: Order is very important here
10. 240ul of PEG (50%)
11. 36ul of 1M LiAc
12. 25ul single strand DNA (2ug/ml)?
13. 50ul of Water and pasmid DNA (0.1-10 ug)
14. Vertex the tubes until all samples are mixed.
15. Incubate at 30 degree for 30 minutes.
16. Heat shock at 42 degree for 20-25 minutes.
17. Centrifuge at 8000 rpm for 1 minute and remove the transformation mix by pipetting
18. Add 100ul of steriled water and mix the cell until they are resuspended
19. Pour cell mixture on selecting plates
20. Leave for 2 days at 30 degree.