Yeast Transformation

This protocol was submitted by Matt Kaeberlein.

This protocol works well for transforming plasmids and PCR disruption cassettes.

Required Reagants:

• Stock Solutions
• 1M Lithium Acetate (LiAc)
• 10X TE
• 50% PEG
• 30C Incubator
• 37C Incubator
• 5mg/mL salmon sperm DNA (ssDNA)
• Yeast
• and transforming DNA

Solutions (Make fresh each time):

• TE + 100mM LiAc (Dilute stock 1M LiAc and 10X TE in water 1:1:8).
• PLATE (1X TE, 100 mM LiAc 40% PEG)

Protocol:

1. Grow yeast to be transformed overnight in YPD.
2. Dilute back overnight culture 1:100 in fresh YPD and culture at 30C for 4-6 hours.
3. Spin down 15mL of cells.
4. Wash cells 2X in 0.5mL TE + 100 mM LiAc
5. Resuspend cells in 200uL TE + LiAc
6. Add transforming DNA.
7. Add 20uL freshly boiled ssDNA.
8. Add 1mL PLATE solution. Mix by inversion several times.
9. Incubate at 30C for 30 min.
10. Incubate at 37C for 15 min. Mix by inversion every 5 min.
11. Spin down cells and plate on selective media.

For transforming DNA I use 1 uL of mini-prep DNA for a plasmid and 30-50uL of PCR product for a PCR disruption.

In practice, I usually scale up and spin down 45 mL of cells in a 50 mL tube. I do the 2X washes then split the cells into 3 individual eppendorf tubes for transformation.