Electro-Transformation of Yeast
Note that 10ml. is the minimum culture volume and that cells can be stored for a few days if they are not DTT treated.
- Grow cells to 1X10E8 or OD600 of 1.2-1.3.
- Spin cells at 5,000rpm for 5 min, and wash pellets in an equal volume of ice cold water.
- Wash in 1/2 volume cold water.
- Wash in 1/25 volume ice-cold 1M. sorbitol.
- Treat cells with 25mM. DTT for 10 minutes at room temperature.
- Wash cells with 1M. cold sorbitol.
- Resuspend cells in 1/200 original volume cold 1M. sorbitol.
- Mix 50µl. of cell suspension with not more than 5µl. DNA (in low ionic strength buffer).
- Immediately tap cell/DNA suspension to the bottom of a 0.2cm cuvette, pulse at 1.5kV, 200˝, 25 µFaradays (pulse time Ĺ5 millisec).
- Immediately add 1ml. YEPD/1M. sorbitol and allow to recover at permissive temperature for one hour.
- Spin down cells and resuspend in 1ml. 1M. sorbitol.
- Plate on selective media.
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