Difference between revisions of "Yeast Transformation"

Revision as of 17:56, 21 December 2009

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

1. Innoculate 50 mL YPD overnight in a 250 mL flask at 24C before going home
2. The next morning check OD-600 of yeast.
   1. Add 10 uL cells to 1 mL of water and measure OD-600.
   2. Adjust for dilution factor (100X) and calculate OD.
   3. Dilute to a final OD of 0.5 in 50 mL of 2xYPD in an empty autoclaved 250 mL flask.
3. Incubate at 24C for 2 divisions (typically ~4.5h).
4. Centrifuge cells at 3000g for 5min.
5. Boil hering tested DNA for 5 min at 95C then cool on ice.
6. Gently resuspend in 25mL sterile water.
7. Centrifuge cells at 3000g for 5min.
8. Gently resuspend cells in 1mL sterile water and transfer to an eppendorf tube
9. Centrifige 30s in a microfuge and aspirate supernatant
10. Resuspend with 1mL sterile water ensuring to break up any clumps.
11. Transfer 100 uL aliquots of cells to 1.5mL tubes, one for each transformation.
12. Prepare Transformation Mixture for the appropriate number of transformations:

1. Add 360 uL Transformation Mixture to each tube of cells and mix by vortexing.
2. Incubate at 42C for 40min.
3. Centrifuge for 30s and aspirate supernatant
4. Add 1mL sterile water, resuspend and mix by gently vortexing.
5. Plate 50 uL on appropriate selective plate.
6. Allow to grow for 3-4 days at 24C.

Based on PMID 17401336. See http://home.cc.umanitoba.ca/~gietz/method.html