Maheshri:TransformationYE
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Yeast Electroporation
- Grow a 500 mL culture to an OD600 of 0.5-1.0.
- Spin down and resuspend in 80 mL sterile water.
- Add 10 mL of 10X TE (pH 7.5) and 10 mL 1 M LiAc (pH 7.5).
- Shake at 30°C, 100 RPM, for 30 minutes.
- Add 2.5 mL fresh 1M DTT.
- Shake an additional 15 minutes.
- Bring volume to 500 mL with cold water.
- Spin, resuspend in 250 mL cold water.
- Spin, resuspend in 25 mL cold 1 M sorbitol.
- Spin, resuspend in 0.5 mL 1 M sorbitol.
- Add transforming DNA to 50 μL of cells and place in cuvette.
- Electroporate: 1 KV, 25 mF, 200 ohms.
- 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.