Yeast Transformation
From Bridges Lab Protocols
Materials
Per strain, this is enough for 10 transformations per strain
- 50 mL autoclaved YPD in a 250 mL flask
- 200 mL 2xYPD
- Autoclaved empty 250 mL flask
- Herring testes DNA (Clontech Cat# 17401336)
- 50% PEG 3500, autoclaved
- 1M Lithium Acetate, autoclaved
- Sterile water
- Selective Agar Plates (see Yeast Selective Media and Agar)
Protocol
- Innoculate 50 mL YPD overnight in a 250 mL flask at 24C before going home
- The next morning check OD-600 of yeast.
- Add 10 uL cells to 1 mL of water and measure OD-600.
- Adjust for dilution factor (100X) and calculate OD.
- Dilute to a final OD of 0.5 in 50 mL of 2xYPD in an empty autoclaved 250 mL flask.
- Incubate at 24C for 2 divisions (typically ~4.5h).
- Centrifuge cells at 3000g for 5min.
- Boil hering tested DNA for 5 min at 95C then cool on ice.
- Gently resuspend in 25mL sterile water.
- Centrifuge cells at 3000g for 5min.
- Gently resuspend cells in 1mL sterile water and transfer to an eppendorf tube
- Centrifige 30s in a microfuge and aspirate supernatant
- Resuspend with 1mL sterile water ensuring to break up any clumps.
- Transfer 100 uL aliquots of cells to 1.5mL tubes, one for each transformation.
- Prepare Transformation Mixture for the appropriate number of transformations:
Reagent | Concentration | 1 | 5(6X) | 10(11X) |
---|---|---|---|---|
PEG 3500 | 50% | 240uL | 1440uL | 2640uL |
Lithium Acetate | 1M | 36uL | 216uL | 396uL |
Herring Testes DNA | 10mg/mL | 10uL | 60uL | 110uL |
Water | 34uL | 204uL | 374uL | |
Tube | eppendorf tube | 15mL Falcon Tube | 15 mL Falcon Tube |
- Add 360 uL Transformation Mixture to each tube of cells and mix by vortexing.
- Incubate at 42C for 40min.
- Centrifuge for 30s and aspirate supernatant
- Add 1mL sterile water, resuspend and mix by gently vortexing.
- Plate 50 uL on appropriate selective plate.
- Allow to grow for 3-4 days at 24C.
Based on PMID 17401336. See http://home.cc.umanitoba.ca/~gietz/method.html