Team:TU Darmstadt/Notebook/Methods/Protein expression

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<ul class="menu"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt/" >Home</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Project" >Project</a></li><li><a href="href="https://2014.igem.org/Team:TU_Darmstadt/Results" >Results</a></li><li><a href="href="https://2014.igem.org/Team:TU_Darmstadt/PolicyandPractices" >Policy & Practices</a></li><li><a href="href="https://2014.igem.org/Team:TU_Darmstadt/Achievements" >Achievements</a></li><li class="active"><a href="href="https://2014.igem.org/Team:TU_Darmstadt/Notebook" >Notebook</a><ul class="menu2"><li class="first"><a href="href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Labjournal" >Labjournal</a></li><li><a href="href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Materials" >Materials</a></li><li class="active last"><a href="href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods" >Methods</a><ul class="menu3"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Agarose_gel_electrophoresis" >Agarose gel electrophoresis</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Dephosphorylation" >Dephosphorylation</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/DNA_ligation" >DNA ligation</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/DNA_quantification_with_NanoDrop" >DNA quantification with NanoDrop</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/PCR_purification" >PCR purification</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/PCR_with_Pfu_polymerase" >PCR with Pfu polymerase</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Plasmid_preparation" >Plasmid preparation</a></li ><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Restriction_digest" >Restriction digest</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/SDS-PAGE" >SDS-PAGE</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Bacterial_cell_culture" >Bacterial cell culture</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Cell_counting_plating" >Cell counting/plating</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Chemically_competent_cells" >Chemically competent cells</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Colony_PCR_with_Taq_polymerase" >Colony PCR with Taq polymerase</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Glycerine_stocks" >Glycerine stocks</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Heat_shock_transformation" >Heat shock transformation</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Protein expression" >Protein expression</a></li><li class="active"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/hydrochloric_acid_extraction" >37% hydrochloric acid extraction</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Dichloromethane_extraction" >Dichloromethane extraction</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Methanol_extraction" >Methanol extraction</a></li><li class="last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Ethanol_extraction" >Ethanol extraction</a></li></ul></li></ul></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Team" >Team</a></li><li class="last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Gallery" >Gallery</a></li></ul>
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<ul class="menu"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt" >Home</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Project" >Project</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results" >Results</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/PolicyandPractices" >Policy & Practices</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Achievements" >Achievements</a></li><li class="active"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook" >Notebook</a><ul class="menu2"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Labjournal" >Labjournal</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Materials" >Materials</a></li><li class="active last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods" >Methods</a><ul class="menu3"><li class="first"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Agarose_gel_electrophoresis" >Agarose gel electrophoresis</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Dephosphorylation" >Dephosphorylation</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/DNA_ligation" >DNA ligation</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/DNA_quantification_with_NanoDrop" >DNA quantification with NanoDrop</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/PCR_purification" >PCR purification</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/PCR_with_Pfu_polymerase" >PCR with Pfu polymerase</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Plasmid_preparation" >Plasmid preparation</a></li ><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Restriction_digest" >Restriction digest</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/SDS-PAGE" >SDS-PAGE</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Bacterial_cell_culture" >Bacterial cell culture</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Cell_counting_plating" >Cell counting/plating</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Chemically_competent_cells" >Chemically competent cells</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Colony_PCR_with_Taq_polymerase" >Colony PCR with Taq polymerase</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Glycerine_stocks" >Glycerine stocks</a></li><li ><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Heat_shock_transformation" >Heat shock transformation</a></li><li class="active"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Protein expression" >Protein expression</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/hydrochloric_acid_extraction" >37% hydrochloric acid extraction</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Dichloromethane_extraction" >Dichloromethane extraction</a></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Methanol_extraction" >Methanol extraction</a></li><li class="last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/Ethanol_extraction" >Ethanol extraction</a></li></ul></li></ul></li><li><a href="https://2014.igem.org/Team:TU_Darmstadt/Team" >Team</a></li><li class="last"><a href="https://2014.igem.org/Team:TU_Darmstadt/Gallery" >Gallery</a></li></ul>
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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 &amp; 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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<!--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 &amp; 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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Latest revision as of 00:48, 18 October 2014

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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.

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