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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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