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Team:TU Darmstadt/Notebook/Methods/Protein expression
From 2014.igem.org
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| - | <!--TYPO3SEARCH_begin--><div id="c102" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Protein expression</h1></div><div><p><b>Equipment:</b></p></div><div><p>- Incubation shaker</p></div><div><p>- Photometer</p></div><div></div><div><p><b>Chemicals & consumables:</b></p></div><div><p>- E. coli BL21 DE3</p></div><div><p>- dYT medium or LB medium</p></div><div><p>- IPTG</p></div><div><p>- 100 ml and 3 l flasks</p></div><div><p>- Ice</p></div><div></div><div><p><b>Procedure:</b></p></div><div></div><div><p>Inoculation of 50 mL LB medium in a 100 mL flask with E. coli BL21 DE3 containing the plasmid with the sequence for the respective part to be expressed.</p></div><div><p>Incubation at 180 repulsion per minute (rpm) at 30°C to an OD<sub>600</sub>= 4</p></div><div><p>Transferation of the starter culture into 1 L LB medium in a 3 L flask resulting in an OD<sub>600</sub>= 0.2</p></div><div><p>Incubation to an OD<sub>600</sub>= 0.6 at 180 rpm and 30°C.</p></div><div><p>Incubation for 15 minutes on ice.</p></div><div><p>Induction of the protein expression with 20 mL of IPTG (stock concentration 1M).</p></div><div><p>Incubation of the cell suspension overnight at 180 rpm at 30°C.</p></div></div><!--TYPO3SEARCH_end--> | + | <!--TYPO3SEARCH_begin--><div id="c102" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Protein expression</h1></div><div><p><b>Equipment:</b></p></div><div><p>- Incubation shaker</p></div><div><p>- Photometer</p></div><div></div><div><p><b>Chemicals & consumables:</b></p></div><div><p>- E. coli BL21 DE3</p></div><div><p>- dYT medium or LB medium</p></div><div><p>- IPTG</p></div><div><p>- 100 ml and 3 l flasks</p></div><div><p>- Ice</p></div><div></div><div><p><b>Procedure:</b></p></div><div></div><div><p>- Inoculation of 50 mL LB medium in a 100 mL flask with E. coli BL21 DE3 containing the plasmid with the sequence for the respective part to be expressed.</p></div><div><p>- Incubation at 180 repulsion per minute (rpm) at 30°C to an OD<sub>600</sub>= 4</p></div><div><p>- Transferation of the starter culture into 1 L LB medium in a 3 L flask resulting in an OD<sub>600</sub>= 0.2</p></div><div><p>- Incubation to an OD<sub>600</sub>= 0.6 at 180 rpm and 30°C.</p></div><div><p>- Incubation for 15 minutes on ice.</p></div><div><p>- Induction of the protein expression with 20 mL of IPTG (stock concentration 1M).</p></div><div><p>- Incubation of the cell suspension overnight at 180 rpm at 30°C.</p></div></div><!--TYPO3SEARCH_end--> |
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Revision as of 18:49, 17 October 2014
Protein expression
Equipment:
- Incubation shaker
- Photometer
Chemicals & consumables:
- E. coli BL21 DE3
- dYT medium or LB medium
- IPTG
- 100 ml and 3 l flasks
- Ice
Procedure:
- Inoculation of 50 mL LB medium in a 100 mL flask with E. coli BL21 DE3 containing the plasmid with the sequence for the respective part to be expressed.
- Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4
- Transferation of the starter culture into 1 L LB medium in a 3 L flask resulting in an OD600= 0.2
- Incubation to an OD600= 0.6 at 180 rpm and 30°C.
- Incubation for 15 minutes on ice.
- Induction of the protein expression with 20 mL of IPTG (stock concentration 1M).
- Incubation of the cell suspension overnight at 180 rpm at 30°C.
