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.

Day 4:

• 1. 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
• 2. Centrifuge cells in 50ml tubes at 3000g for 5 minutes
• 3. Discard supernatant, resuspend in 1ml 100mM LiAc.
• 4. Transfer the mixture into 1.5ml tube.
• 5. Centrifuge at 8000rpm for 1minute and discard LiAC.
• 6. Add 50ul 100mM LiAc per transformants. (If you want to do 3 transformations, add 150ul)
• 7. Boil carrier DNA for 5 minutes and quickly chill on ice.
• 8. Centrifuge tubes and remove supernatant.
• 9. 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.