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

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Revision as of 14:53, 20 October 2009

Prepare of eletrocompeten S. cerevisiae

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

S. cerevisiae Transformation

1- In a 1,5ml tube, pipete 40ul of fresh eletrocompetent cells, and addthe DNA (5-100ng in 5ul)

2- mix gently and place on ice for 5 minutes

3- transfer the contents of the tube to a cold electroporation bucket.

4- Proceed electroporation

5- Add immediately 1ml of cold sorbitol (1M)

6- Plate in seletive media (YNB Ura- )

Culture Media

YEPD 10g yeast extract, 20g peptone, 20g glucose, Final volume: 1l

YNB Ura- 6,7g YNB, 30g glucose, 30g agar, 10ml tryptophan (100x), 10ml histidine (100x), 30ml leucine (100x), 30ml Drop out

  Drop out:
  0,5g/l   Adenine 
  1,2g/l   L- aspartic acid 
  1,2g/l   L- glutamic acid
  0,24g/l  L- arginine
  0,36g/l  L- lysine
  0,24g/l  L- methionine
  0,6g/l   L- phenylalanine
  4,5g/l   L- serine
  2,4g/l   L- treotonine
  0,18g/l  L- tyrosine
  1,8g/l   L- valine

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-==Prepare of eletrocompeten ''S. serevisiae''==
+==Prepare of eletrocompeten ''S. cerevisiae''==
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 - Ressuspend cells in 200ul
 ==''S. cerevisiae'' Transformation==