Amberg Lab ,Upstate Medical University

http://www.upstate.edu/biochem/amberg/protocols/transformation.html

1.Grow cells to 1X10E8 or OD600 of 1.2-1.3.

2.Spin cells at 5,000rpm for 5 min, and wash pellets in an equal volume of ice cold water.

3.Wash in 1/2 volume cold water.

4.Wash in 1/25 volume ice-cold 1M. sorbitol.

5.Treat cells with 25mM. DTT for 10 minutes at room temperature.

6.Wash cells with 1M. cold sorbitol.

7.Resuspend cells in 1/200 original volume cold 1M. sorbitol.

8.Mix 50µl. of cell suspension with not more than 5µl. DNA (in low ionic strength buffer).

9.Immediately tap cell/DNA suspension to the bottom of a 0.2cm cuvette, pulse at 1.5kV, 200Æ, 25 µFaradays (pulse time Å5 millisec).

10.Immediately add 1ml. YEPD/1M. sorbitol and allow to recover at permissive temperature for one hour.

11.Spin down cells and resuspend in 1ml. 1M. sorbitol.

12.Plate on selective media.

Note that 10ml. is the minimum culture volume and that cells can be stored for a few days if they are not DTT treated.