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