Team:TU Darmstadt/protocols/Chemically competent cells

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</a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/materials">
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols">
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<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font>
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<!-- Chemically competent cells -->
<!-- Chemically competent cells -->
<center>
<center>
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<br>
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<br>
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<br>
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<br>
<br>
<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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<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">
-
Incubation shaker<br>
+
<ul>
-
Centrifuge (cooling cababilities required!)<br>
+
<li class=list1>- -80°C freezer</li>
-
photometer<br>
+
<li class=list1>- Incubation shaker</li>
-
Ice water bath<br>
+
<li class=list1>- Centrifuge (cooling cababilities required!)</li>
 +
<li class=list1>- photometer</li>
 +
<li class=list1>- Ice water bath</li>
 +
</ul>
 +
</div>
 +
</font>
 +
</p>
 +
 
 +
<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> 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>
-
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>- <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Media"> dYT Medium</a>(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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</ul>
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</div>
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</font>
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</p>
<br>
<br>
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<B> Procedure<br></B>
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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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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><br>
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2. Inoculate 200 mL LB with the preculture <br>
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<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular">
-
3. Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached<br>
+
The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.
-
4. Incubate cells on ice for 15 min<br>
+
<div align="left" style="margin-left:30px; margin-right:50px">
-
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>
-
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>
-
7. Incubate on ice for 1 hour<br>
+
<li>Inoculate 200 mL LB with the preculture.</li>
-
8. Centrifuge the culture at 4°C and 3000 x g for 10 min<br>
+
<li>Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.</li>
-
9. Resuspend cell pellet in 10mL ice cold 100 mM CaCl2<br>
+
<li>Incubate cells on ice for 15 min.</li>
-
10. Incubate on ice for 1 hour<br>
+
<li>Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).</li>
-
11. Centrifuge the culture at 4°C and 3000 x g for 5 min<br>
+
<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub> (Do not vortex!).</li>
-
12. Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine<br>
+
<li>Incubate on ice for 1 hour.</li>
-
13. Incubate on ice for 30 min<br>
+
<li>Centrifuge the culture at 4°C and 3000 x g for 10 min.</li>
-
14. Aliquot the cells à 100µL<br>
+
<li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub>.</li>
-
15. Store at -80°C<br>
+
<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 CaCl<sub>2</sub> and 15 % (v/v) glycerine.</li>
 +
<li>Incubate on ice for 30 min.</li>
 +
<li>Aliquot the cells à 100µ.</li>
 +
<li>Store at -80°C.</li>
 +
</ol>
 +
</div>
 +
</font></p><br>
 +
 
 +
<p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular">
 +
<B>Mixtures<br></B></font></p><br>
 +
<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
 +
<B>CaCl2-Solution<br></B>
 +
<div align="left" style="margin-left:60px; margin-right:50px">
 +
<ul>
 +
<li class=list1>- 5.55 g CaCl<sub>2</sub></li>
 +
<li class=list1>- add ddH<sub>2</sub>O to 1 L</li>
 +
<li class=list1>- sterilize by autoclaving</li>
 +
</ul>
 +
</div>
 +
</font>
 +
</p>
<br>
<br>
 +
<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
 +
<B>Cryo solution<br></B>
 +
<div align="left" style="margin-left:60px; margin-right:50px">
 +
<ul>
 +
<li class=list1>- 0.278 g CaCl<sub>2</sub></li>
 +
<li class=list1>- 10 ml glycerin</li>
 +
<li class=list1>- Add ddH<sub>2</sub>O to 50 ml</li>
 +
<li class=list1>- Sterilize by autoclave</li>
 +
</ul>
 +
</div>
 +
</font>
 +
</p>
 +
 +
 +
<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2>
 +
<font size="3" color="#F0F8FF" face="Arial regular">
 +
<ol>
 +
<li style="margin-left:15px; margin-right:50px; text-align:justify">Mandel, M. and Higa, A.: <i>Calcium-dependent bacteriophage DNA infection</i>. J Mol Biol, 1970, 53, 159-162</li>
 +
</ol>
 +
</font>

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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