Maheshri:TransformationYE

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.