Maheshri:TransformationYE

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Yeast Electroporation

  1. Grow a 500 mL culture to an OD600 of 0.5-1.0.
  2. Spin down and resuspend in 80 mL sterile water.
  3. Add 10 mL of 10X TE (pH 7.5) and 10 mL 1 M LiAc (pH 7.5).
  4. Shake at 30°C, 100 RPM, for 30 minutes.
  5. Add 2.5 mL fresh 1M DTT.
  6. Shake an additional 15 minutes.
  7. Bring volume to 500 mL with cold water.
  8. Spin, resuspend in 250 mL cold water.
  9. Spin, resuspend in 25 mL cold 1 M sorbitol.
  10. Spin, resuspend in 0.5 mL 1 M sorbitol.
  11. Add transforming DNA to 50 μL of cells and place in cuvette.
  12. Electroporate: 1 KV, 25 mF, 200 ohms.
  13. Add 1 mL 1 M sorbitol and plate aliquots of transformation.

Hints:

  • There is no need to add carrier DNA.
  • Transforming DNA should be in a low salt buffer or else there will be fireworks.
  • Although the Red Book places 100 ng as the maximum amount of DNA to be used, up to 6 mg have been used by this author with great results.
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