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.
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== Johan ==
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= Johan =
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=== Autoclaved stuff for making 1000 ml competent cells ===
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== 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
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=== Step 1 in competent cell protocol ===
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== 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.
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{{Stockholm/Footer}}

Latest revision as of 10:24, 26 October 2010


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.





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/