Team:TU Darmstadt/protocols/Chemically competent cells

From 2013.igem.org

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<p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular">
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<B> Short explanation<br></B>
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<B> Materials<br></B></font></p>
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The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.<br>
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<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
<br>
<br>
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<B> Equipment <br></B>
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<B>Equipment<br></B>
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-80°C freezer<br>
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<div align="left" style="margin-left:60px; margin-right:50px">
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Incubation shaker<br>
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<ul>
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Centrifuge (cooling cababilities required!)<br>
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<li class=list1>- -80°C freezer</li>
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photometer<br>
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<li class=list1>- Incubation shaker</li>
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Ice water bath<br>
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<li class=list1>- Centrifuge (cooling cababilities required!)</li>
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<li class=list1>- photometer</li>
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<li class=list1>- Ice water bath</li>
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</ul>
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</div>
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</font>
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</p>
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<br>
<br>
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<B> Chemicals & consumables <br></B>
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<B>Chemicals & consumables<br></B>
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Ice and/or liquid nitrogen<br>
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<div align="left" style="margin-left:60px; margin-right:50px">
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Falcon tubes<br>
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<ul>
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Eppis<br>
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<li class=list1>- Ice and/or liquid nitrogen</li>
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dYT Medium (50 ml p.c.)<br>
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<li class=list1>- Falcon tubes</li>
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ice cold 100mM CaCl2<br>
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<li class=list1>- dYT Medium (50 ml p.c.)</li>
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Glycerin<br>
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<li class=list1>- ice cold 100mM CaCl<sub>2</sub></li>
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<li class=list1>- Glycerin</li>
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</font>
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</p>
<br>
<br>
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<B> Procedure<br></B>
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1. Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight<br>
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<B> Procedure<br></B></font></p>
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2. Inoculate 200 mL LB with the preculture <br>
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3. Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached<br>
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The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.
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4. Incubate cells on ice for 15 min<br>
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<div align="left" style="margin-left:30px; margin-right:50px">
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5. Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice)<br>
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<ol>
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6. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!)<br>
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<li>Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.</li>
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7. Incubate on ice for 1 hour<br>
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<li>Inoculate 200 mL LB with the preculture.</li>
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8. Centrifuge the culture at 4°C and 3000 x g for 10 min<br>
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<li>Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.</li>
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9. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2<br>
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<li>Incubate cells on ice for 15 min.</li>
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10. Incubate on ice for 1 hour<br>
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<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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11. Centrifuge the culture at 4°C and 3000 x g for 5 min<br>
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<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl2 (Do not vortex!).</li>
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12. Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine<br>
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<li>Incubate on ice for 1 hour.</li>
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13. Incubate on ice for 30 min<br>
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<li>Centrifuge the culture at 4°C and 3000 x g for 10 min.</li>
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14. Aliquot the cells à 100µL<br>
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<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl2.</li>
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15. Store at -80°C<br>
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<li>Incubate on ice for 1 hour.</li>
 +
<li>Centrifuge the culture at 4°C and 3000 x g for 5 min.</li>
 +
<li>Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine.</li>
 +
<li>Incubate on ice for 30 min.</li>
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<li>Aliquot the cells à 100µ.</li>
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<li>Store at -80°C.</li>
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</ol>
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</div>
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</font></p>
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<br>
<br>
<B>Solutions<br></B>
<B>Solutions<br></B>

Revision as of 18:46, 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.


Solutions
 CaCl2
5.55 g CaCl2
Add di 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

Retrieved from "http://2013.igem.org/Team:TU_Darmstadt/protocols/Chemically_competent_cells"