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

From 2009.igem.org

(Difference between revisions)
(New page: ==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, 30...)
(→DNA extraction)
Line 23: Line 23:
-
==DNA extraction==
+
==''S. cerevisiae'' Transformation==
 +
1- In a 1,5ml tube, pipete 40ul of fresh eletrocompetent cells, and addthe DNA (5-100ng in 5ul)
-
{{:Team:UNICAMP-Brazil/inc_rodape}}
+
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
 +
 
 +
 
 +
{{:Team:UNICAMP-Brazil/inc_rodape}}

Revision as of 14:46, 20 October 2009

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


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
Retrieved from "http://2009.igem.org/Team:UNICAMP-Brazil/Protocols/Preparation_of_electrocompetent_S._cereviseae"