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From 2010.igem.org

Contents 1 Nina 1.1 Preparation of competent DH5α Escherichia coli cells 2 Johan 2.1 Autoclaved stuff for making 1000 ml competent cells 2.2 Step 1 in competent cell protocol

Nina

Preparation of competent DH5α Escherichia coli cells

Autoclaving bottles & solutions for next day:

• Two 1000 ml empty bottles
• 500 ml 50 mM CaCl2 for 1000 ml cell culture
• 16 ml 50 mM CaCl2 + 15 % Glycerol

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Set a 5 ml LB ON culture of DH5α in 37°C, 250 rpm.

Johan

Autoclaved stuff for making 1000 ml competent cells

• Two 2L bottles, each with made 500 ml 50 mM CaCl2
• 500 ml 50 mM CaCl2
• Small bottle with 16 ml CaCl2 + 15 % Glycerol

Made from 87 % Glycerol.

c1 * v1 = c2 * v2

87 % * ? = 15 % * 16 ml

v1 = (15 % * 16 ml) / 87 % = 2,76 ml

2,76 ml 87 % glycerol was diluted with 16 - 2,76 = 13,24 ml 50 mM CaCl2

Step 1 in competent cell protocol

5 ml of LB in each of two 50 ml Falcon tubes.

Pipette tip dipped in commercial NEB5α (=DH5α) cells, then dipped in the Falcon tubes and put in 37 °C shaker.