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Team:Stockholm/7 June 2010
From 2010.igem.org
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Set a 5 ml LB ON culture of DH5α in 37°C, 250 rpm. | 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 | * Two 2L bottles, each with made 500 ml 50 mM CaCl2 | ||
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2,76 ml 87 % glycerol was diluted with 16 - 2,76 = 13,24 ml 50 mM CaCl2 | 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. | 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. | Pipette tip dipped in commercial NEB5α (=DH5α) cells, then dipped in the Falcon tubes and put in 37 °C shaker. | ||
Revision as of 23:10, 25 October 2010
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Contents |
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
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.
