Team:TU Darmstadt/protocols/Chemically competent cells

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<h2><font size="6" color="#F0F8FF" face="Arial regular"> Chemically competent cells </font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular"> Chemically competent cells </font></h2>
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<li class=list1>- Ice and/or liquid nitrogen</li>
<li class=list1>- Ice and/or liquid nitrogen</li>
<li class=list1>- Falcon tubes</li>
<li class=list1>- Falcon tubes</li>
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<li class=list1>- dYT Medium (50 ml p.c.)</li>
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<li class=list1>- <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Media"> dYT Medium</a>(50 ml p.c.) </li>
<li class=list1>- ice cold 100mM CaCl<sub>2</sub></li>
<li class=list1>- ice cold 100mM CaCl<sub>2</sub></li>
<li class=list1>- Glycerin</li>
<li class=list1>- Glycerin</li>
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<li>Incubate cells on ice for 15 min.</li>
<li>Incubate cells on ice for 15 min.</li>
<li>Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).</li>
<li>Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).</li>
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<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!).</li>
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<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub> (Do not vortex!).</li>
<li>Incubate on ice for 1 hour.</li>
<li>Incubate on ice for 1 hour.</li>
<li>Centrifuge the culture at 4°C and 3000 x g for 10 min.</li>
<li>Centrifuge the culture at 4°C and 3000 x g for 10 min.</li>
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<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl2.</li>
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<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub>.</li>
<li>Incubate on ice for 1 hour.</li>
<li>Incubate on ice for 1 hour.</li>
<li>Centrifuge the culture at 4°C and 3000 x g for 5 min.</li>
<li>Centrifuge the culture at 4°C and 3000 x g for 5 min.</li>
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<li>Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine.</li>
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<li>Resuspend cell pellet in 2mL ice cold 100 mM CaCl<sub>2</sub> and 15 % (v/v) glycerine.</li>
<li>Incubate on ice for 30 min.</li>
<li>Incubate on ice for 30 min.</li>
<li>Aliquot the cells à 100µ.</li>
<li>Aliquot the cells à 100µ.</li>
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<li class=list1>- 5.55 g CaCl2</li>
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<li class=list1>- 5.55 g CaCl<sub>2</sub></li>
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<li class=list1>- add ddH2O to 1 L</li>
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<li class=list1>- add ddH<sub>2</sub>O to 1 L</li>
<li class=list1>- sterilize by autoclaving</li>
<li class=list1>- sterilize by autoclaving</li>
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<ul>
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<li class=list1>- 0.278 g CaCl2</li>
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<li class=list1>- 0.278 g CaCl<sub>2</sub></li>
<li class=list1>- 10 ml glycerin</li>
<li class=list1>- 10 ml glycerin</li>
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<li class=list1>- Add ddH2O to 50 ml</li>
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<li class=list1>- Add ddH<sub>2</sub>O to 50 ml</li>
<li class=list1>- Sterilize by autoclave</li>
<li class=list1>- Sterilize by autoclave</li>
</ul>
</ul>

Latest revision as of 00:30, 5 October 2013







Lab book | Materials | Protocols

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


Cryo solution

  • - 0.278 g CaCl2
  • - 10 ml glycerin
  • - Add ddH2O to 50 ml
  • - Sterilize by autoclave

References

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

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