Team:Stockholm/22 June 2010
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Andreas
Preparation of competent Top10 Escherichia coli cells
Continued from 21/6.
Based on the [http://openwetware.org/wiki/TOP10_chemically_competent_cells protocol of the Knight lab]
Materials
CCMB80 buffer
- 10 mM KOAc pH 7.0
- 80 mM CaCl2.2H2O
- 20 mM MnCl2
- 10 mM MgCl2.6H2O
- 10% glycerol
Procedures
- 250 ml LB inoculated with 2 ml from ON culture
- Grown at 28°C until an OD(600) of ≈0.3
- Cells spun down at 3000x g for 10 min in 4°C
- Cells resuspended in 80 ml ice-cold CCMB80 buffer and kept on ice for 20 min:
- Cells spun down with same settings
- Cells resuspended in 10 ml new CCMB80 and kept on ice for 20 min
- Competent cells aliquoted in 100ul aliquots and stored in -80°C.