Difference between revisions of "Team:Bielefeld-CeBiTec/Protocols"
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Revision as of 23:00, 12 August 2015
Protocols
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- Thaw 50 µl electrocompetent E. coli cells on ice, dilute with icecold 50 µl glycerine (10%) if necessary
- Add 0.5-5 µl plasmid to 50 µl electrocompetent cells
- Store cells on ice for 1 minute
- Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ω
- Transfer transformation reaction to 450 µl SOC-Medium and incubate 1 h at 37 °C
- Plate on selective LB-Medium
- Incubate over night at 37 °C
- Thaw 100 µl chemo competent E. coli cells on ice
- Add 0.5-5 µl plasmid to 100 µl chemocompetent cells
- Store cells on ice for 10-30 min on ice
- Heat shock for 90 seconds at 42 °C
- Store reaction on ice for 60 seconds
- Optional: Preheat SOC medium to 37 °C
- Transfer reaction to 1 ml SOC medium and incubate at 37 °C for at least 1 hour
- Centrifuge 3 minutes at 12000 rpm and plate on selective LB medium
- Incubate at 37 °C over night