Team:UNICAMP-Brazil/Protocols/Preparation of electrocompetent S. cereviseae

Revision as of 14:19, 20 October 2009 by Sula (Talk | contribs)

Prepare of eletrocompeten S. serevisiae

1- Grow yest strain in 50ml YEPD medium overnight, 250 RPM, 30ºC.

2- Inoculate 100 ml YEPD in a 500ml flask in a OD=0,1, 250 RPM, 30ºC util OD= 1,3

3- Place the culture on ice for 15 minutes to stop the growth

4- Centrifuge for 4 min at 4000g, 4ºC

5- Ressuspend cells in 20ml of cold water in 50ml tubes, complete volum to 50ml.

6- Centrifuge for 4 min at 4000g, 4ºC

7- Repeat steps 5 and 6.

8- Ressuspend cells in 10ml Sorbitol 1M

9- Centrifuge for 4 min at 4000g, 4ºC

10- Ressuspend cells in 200ul