Team:TU Darmstadt/protocols/Chemically competent cells

From 2013.igem.org

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<B> Procedure<br></B></font></p>
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<B> Procedure<br></B></font></p><br>
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The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.
The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.
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<B>Mixtures<br></B></font></p><br>
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<B>CaCl2-Solution<br></B>
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<li class=list1>- 5.55 g CaCl2</li>
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<li class=list1>- add ddH2O to 1 L</li>
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<li class=list1>- sterilize by autoclaving</li>
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<B>CaCl2</B><br>
<B>CaCl2</B><br>
5.55 g CaCl2<br>
5.55 g CaCl2<br>
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Add di H2O to 1 L<br>
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Add dd H2O to 1 L<br>
Sterilize by autoclaving<br>
Sterilize by autoclaving<br>
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Revision as of 18:57, 4 October 2013





Chemically competent cells

Materials


Equipment

  • - -80°C freezer
  • - Incubation shaker
  • - Centrifuge (cooling cababilities required!)
  • - photometer
  • - Ice water bath


Chemicals & consumables

  • - Ice and/or liquid nitrogen
  • - Falcon tubes
  • - dYT Medium (50 ml p.c.)
  • - ice cold 100mM CaCl2
  • - Glycerin


Procedure


The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.

  1. Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.
  2. Inoculate 200 mL LB with the preculture.
  3. Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.
  4. Incubate cells on ice for 15 min.
  5. Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).
  6. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!).
  7. Incubate on ice for 1 hour.
  8. Centrifuge the culture at 4°C and 3000 x g for 10 min.
  9. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2.
  10. Incubate on ice for 1 hour.
  11. Centrifuge the culture at 4°C and 3000 x g for 5 min.
  12. Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine.
  13. Incubate on ice for 30 min.
  14. Aliquot the cells à 100µ.
  15. Store at -80°C.


Mixtures


CaCl2-Solution

  • - 5.55 g CaCl2
  • - add ddH2O to 1 L
  • - sterilize by autoclaving


Solutions
 CaCl2
5.55 g CaCl2
Add dd H2O to 1 L
Sterilize by autoclaving

Cryo solution
0.278 g CaCl2
10 ml glycerin
Add di H2O to 50 ml
Sterilize by autoclave

References
 Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162

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