Team:TU Darmstadt/protocols/Protein Expression

From 2013.igem.org

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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>
-
<li>Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM).</li>
+
<li>Induction of the proteinexpression with 20 mL of IPTG (stock conentration 1M).</li>
-
<li>Incubation of the cell suspension over night at 180 rpm at 16°C.</li>
+
<li>Incubation of the cell suspension over night at 180 rpm at 30°C.</li>
</ol>
</ol>

Revision as of 21:23, 4 October 2013







Protein Expression


Equipment & chemicals

  • - Incubation shaker
  • - E. coli BL21 DE3
  • - DYT media
  • - IPTG
  • - 100 ml and 3 l flasks
  • - Photometer
  • - ice



Procedure


  1. Load & Run
    2.
  2. 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.
  3. Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4
  4. Transferation of the starter culture into 1 L DYT medium in a 3 L flask resulting in an OD600= 0.2
  5. Incubation to an OD600= 0.6 at 180 rpm and 30°C.
  6. Incubation for 15 minutes on ice.
  7. Induction of the proteinexpression with 20 mL of IPTG (stock conentration 1M).
  8. Incubation of the cell suspension over night at 180 rpm at 30°C.

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