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From 2010.igem.org

Contents 1 Andreas 1.1 Preparation of competent Top10 Escherichia coli cells 1.1.1 Materials 1.1.2 Procedures

Andreas

Preparation of competent Top10 Escherichia coli cells

Continued from 21/6.
Based on the 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

1. 250 ml LB inoculated with 2 ml from ON culture
   • Grown at 28°C until an OD(600) of ≈0.3
2. Cells spun down at 3000x g for 10 min in 4°C
3. Cells resuspended in 80 ml ice-cold CCMB80 buffer and kept on ice for 20 min:
4. Cells spun down with same settings
5. Cells resuspended in 10 ml new CCMB80 and kept on ice for 20 min
6. Competent cells aliquoted in 100ul aliquots and stored in -80°C.