Team:Marburg/Protocols/Protocol4
Competent yeast for electroporation
- inoculate 100 mL YEPD and let cells grow to OD = 1.3 - 1.5
- centrifuge (2 x 50 mL, 10 min, 4000 rpm, 4 °C)
- discard the supernatant
- resuspend cells in 8 mL ddH2O
- add 1 mL TE-Buffer, invert the tube multiple times
- add 1 mL 1 M LiAc, invert the tube multiple times
- incubate 45 min @ 100 rpm (shake tube horizontally)
- add 250 µL 1 M DTT
- incubate 15 min @ 100 rpm
- fill Falcon tubes up to 50 mL with ddH2O
- centrifuge (10 min, 5000 rpm, 4 °C)
- discard the supernatant
- resuspend pellets in 25 mL ddH2O and unite 2 samples
- centrifuge (10 min, 5000 rpm, 4 °C)
- discard the supernatant
- resuspend pellet in 5 mL cold 1 M Sorbitol
- centrifuge (10 min, 5000 rpm, 4 °C)
- discard the supernatant
- resuspend pellet in 100 µL cold 1 M Sorbitol
- store cells either on ice or @ - 20 °C. Cells can be used for a few days
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