Team:TU Darmstadt/Notebook/Methods/Protein expression
From 2014.igem.org
(Difference between revisions)
Line 9: | Line 9: | ||
<div id="leftNavi" class="grid_5"> | <div id="leftNavi" class="grid_5"> | ||
<nav> | <nav> | ||
- | + | <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> | |
</nav> | </nav> | ||
</div> | </div> |
Revision as of 16:34, 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.