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Team:TU Darmstadt/Notebook/Methods/Chemically competent cells
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href="https://2014.igem.org/Team:TU_Darmstadt/Notebook/Methods/SDS-PAGE" >SDS-PAGE</a></li><li | + | <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="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 class="active"><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><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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Revision as of 01:20, 16 October 2014
Chemically competent cells
Equipment:
- -80°C freezer
- Incubation shaker
- Centrifuge (cooling cababilities required!)
- Photometer
- Ice water bath
Chemicals & consumables:
- Ice and/or liquid nitrogen
- Falcon tubes
- dYT Medium(50 ml p.c.)
- Ice cold 100mM CaCl2
- Glycerine
Procedure:
The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.
Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.
Inoculate 200 mL LB with the preculture.
Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.
Incubate cells on ice for 15 min.
Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).
Resuspend cell pellet in 10 mL ice cold 100 mM CaCl2 (Do not vortex!).
Incubate on ice for 1 hour.
Centrifuge the culture at 4°C and 3000 x g for 10 min.
Resuspend cell pellet in 10 mL ice cold 100 mM CaCl2.
Incubate on ice for 1 hour.
Centrifuge the culture at 4°C and 3000 x g for 5 min.
Resuspend cell pellet in 2mL ice cold 100 mM CaCl2 and 15 % (v/v) glycerine.
Incubate on ice for 30 min.
Aliquot the cells à 100 µL.
Store at -80°C.
Mixtures:
CaCl2-Solution
- 5.55 g CaCl2
- Add ddH2O to 1 L
- Sterilize by autoclaving
Cryo solution
- 0.278 g CaCl2
- 10 ml glycerine
- Add ddH2O to 50 ml
- Sterilize by autoclaving
References:
Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162
