Electroporation (Yeast)

Tuesday, November 18, 2003

Description
Electroporation (Yeast)

Procedure
1. Inoculate 50ml YEPD with a colony and grow with shaking at 30 deg C until early stationary (~0.6-2E8 cells/ml).

2. Harvest in a 50ml sterile conical tube in GPR centrifuge spin at 3000rpm, 4 deg C, 5' and keep cells on ice throughout the procedure.

3. Wash cells with 40ml ICE-COLD sterile dH2O, pellet at 2.5krpm, 4 deg C, 5'.

4. Repeat wash with 20ml sterile dH2O (ice-cold).

5. Resuspend cells in 5ml 1M Sorbitol (ice-cold) pellet at 2krpm, 4 deg C, 5'.

6. Resuspend cells with 150l 1M Sorbitol (ice-cold) - KEEP ON ICE!

7. Mix 40l of yeast suspension with <5l DNA (~5g) in a prechilled electroporation cuvette (0.2 cm). Tap contents to the bottom, making sure that the sample is in contact with both sides of the aluminum cuvette.

8. Give one pulse: V= 1.5kV, 25F, 200 Ohms. Time should be ~4-5".

9. Immediately add 1ml 1M Sorbitol (ice-cold) and transfer with a sterile pasteur pipette to a sterile Eppendorf tube.

10. Spread onto selective plates

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Electroporation (Yeast)