Lab July 10 2009

Prepare buffer for Top 10 chemically competant cell procedure, http://openwetware.org/wiki/TOP10_chemically_competent_cells.

Eliminating detergent

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

CCMB80 buffer

* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) (=0.9814g) * 80 mM CaCl2.2H2O (11.8 g/L) * 20 mM MnCl2.4H2O (4.0 g/L) * 10 mM MgCl2.6H2O (2.0 g/L) * 10% glycerol (100 ml/L) * adjust pH DOWN to 6.4 with 0.1N HCl if necessary o adjusting pH up will precipitate manganese dioxide from Mn containing solutions. * sterile filter and store at 4°C * slight dark precipitate appears not to affect its function

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