Team:TU Darmstadt/protocols/Protein Expression

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<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
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<br><br><br><br>
<br><br><br><br>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font>
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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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<br>
<h2><font size="6" color="#F0F8FF" face="Arial regular">Protein Expression</font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular">Protein Expression</font></h2>
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<B> Materials<br></B></font></p>
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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 & chemicals<br></B>
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<B>Equipment<br></B>
<div align="left" style="margin-left:60px; margin-right:50px">
<div align="left" style="margin-left:60px; margin-right:50px">
<ul>
<ul>
<li class=list1>- Incubation shaker</li>
<li class=list1>- Incubation shaker</li>
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<li class=list1>- <i>E. coli</i> BL21 DE3</li>
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<li class=list1>- Photometer</li>
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<li class=list1>- DYT media</li>
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<br>
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<B>Chemicals & consumables<br></B>
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<li class=list1>- E. coli BL21 DE3</li>
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<li class=list1>- <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Media"><font size="3" color="#F0F8FF" face="Arial regular"><b>DYT Medium</b></font></a></li>
<li class=list1>- IPTG</li>
<li class=list1>- IPTG</li>
<li class=list1>- 100 ml and 3 l flasks</li>
<li class=list1>- 100 ml and 3 l flasks</li>
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<li class=list1>- Photometer</li>
 
<li class=list1>- ice</li>
<li class=list1>- ice</li>
</ul>
</ul>
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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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<li>Inoculation of 50 mL DYT medium in the 100 mL flask with <i>E. coli</i> BL21 DE3 containing <a href=http://lucerna-chem.ch/shop/558271><font size="3" color="#F0F8FF" face="Arial regular"><b> pPR-IBA2 </b></font></a>
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<B><li class=list1>Load & Run</li></B>
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<li>Inoculation of 50 mL DYT medium in the 100 mL flask with <i>E. coli</i> BL21 DE3 containing <a href=http://lucerna-chem.ch/shop/558271> pPR-IBA2 </a>
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plasmid with the sequence for the respective part to be expressed.</li>
plasmid with the sequence for the respective part to be expressed.</li>
<li>Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4</li>
<li>Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4</li>
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<li>Incubation to an OD600= 0.6 at 180 rpm and 30°C.</li>
<li>Incubation to an OD600= 0.6 at 180 rpm and 30°C.</li>
<li>Incubation for 15 minutes on ice.</li>
<li>Incubation for 15 minutes on ice.</li>
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<li>Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM).</li>
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<li>Induction of the proteinexpression with 20 mL of IPTG (stock conentration 1M).</li>
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<li>Incubation of the cell suspension over night at 180 rpm at 16°C.</li>
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<li>Incubation of the cell suspension over night at 180 rpm at 30°C.</li>
</ol>
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Latest revision as of 01:59, 5 October 2013








Lab book | Materials | Protocols

Protein Expression

Materials


Equipment

  • - Incubation shaker
  • - Photometer


Chemicals & consumables

  • - E. coli BL21 DE3
  • - DYT Medium
  • - IPTG
  • - 100 ml and 3 l flasks
  • - ice


Procedure


  1. Inoculation of 50 mL DYT medium in the 100 mL flask with E. coli BL21 DE3 containing pPR-IBA2 plasmid with the sequence for the respective part to be expressed.
  2. Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4
  3. Transferation of the starter culture into 1 L DYT medium in a 3 L flask resulting in an OD600= 0.2
  4. Incubation to an OD600= 0.6 at 180 rpm and 30°C.
  5. Incubation for 15 minutes on ice.
  6. Induction of the proteinexpression with 20 mL of IPTG (stock conentration 1M).
  7. Incubation of the cell suspension over night at 180 rpm at 30°C.

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