Team:TU Darmstadt/Notebook/Methods/Chemically competent cells

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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 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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<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 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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<!--TYPO3SEARCH_begin--><div id="c96" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Chemically competent cells</h1></div><div><p><b>Equipment:</b></p></div><div><p>- -80°C freezer</p></div><div><p>- Incubation shaker</p></div><div><p>- Centrifuge (cooling cababilities required!)</p></div><div><p>- Photometer</p></div><div><p>- Ice water bath</p></div><div></div><div><p><b>Chemicals &amp; consumables:</b></p></div><div><p>- Ice and/or liquid nitrogen</p></div><div><p>- Falcon tubes</p></div><div><p>- dYT Medium(50 ml p.c.)</p></div><div><p>- Ice cold 100mM CaCl2</p></div><div><p>- Glycerine</p></div><div></div><div><p><b>Procedure:</b></p></div><div></div><div><p>The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.</p></div><div><p>Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.</p></div><div><p>Inoculate 200 mL LB with the preculture.</p></div><div><p>Incubate at 37°C and 150 rpm until an OD<sub>600</sub> of 0.4-0.6 is reached.</p></div><div><p>Incubate cells on ice for 15 min.</p></div><div><p>Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).</p></div><div><p>Resuspend cell pellet in 10 mL ice cold 100 mM CaCl<sub>2</sub> (Do not vortex!).</p></div><div><p>Incubate on ice for 1 hour.</p></div><div><p>Centrifuge the culture at 4°C and 3000 x g for 10 min.</p></div><div><p>Resuspend cell pellet in 10 mL ice cold 100 mM CaCl<sub>2</sub>.</p></div><div><p>Incubate on ice for 1 hour.</p></div><div><p>Centrifuge the culture at 4°C and 3000 x g for 5 min.</p></div><div><p>Resuspend cell pellet in 2mL ice cold 100 mM CaCl<sub>2</sub> and 15 % (v/v) glycerine.</p></div><div><p>Incubate on ice for 30 min.</p></div><div><p>Aliquot the cells à 100 µL.</p></div><div><p>Store at -80°C.</p></div><div></div><div><p><b>Mixtures:</b></p></div><div></div><div><p>CaCl<sub>2</sub>-Solution</p></div><div><p>- 5.55 g CaCl<sub>2</sub></p></div><div><p>- Add ddH<sub>2</sub>O to 1 L</p></div><div><p>- Sterilize by autoclaving</p></div><div></div><div><p>Cryo solution</p></div><div><p>- 0.278 g CaCl<sub>2</sub></p></div><div><p>- 10 ml glycerine</p></div><div><p>- Add ddH<sub>2</sub>O to 50 ml</p></div><div><p>- Sterilize by autoclaving</p></div><div><p><b>References:</b></p></div><div></div><div><p>Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162</p></div><div></div></div><!--TYPO3SEARCH_end-->
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<!--TYPO3SEARCH_begin--><div id="c96" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Chemically Competent Cells</h1></div><div><p><b>Equipment:</b></p></div><div><p>- -80°C freezer</p></div><div><p>- Incubation shaker</p></div><div><p>- Centrifuge (cooling cababilities required!)</p></div><div><p>- Photometer</p></div><div><p>- Ice water bath</p></div><div></div><div><p><b>Chemicals &amp; consumables:</b></p></div><div><p>- Ice and/or liquid nitrogen</p></div><div><p>- Falcon tubes</p></div><div><p>- dYT Medium(50 ml p.c.)</p></div><div><p>- Ice cold 100mM CaCl2</p></div><div><p>- Glycerine</p></div><div></div><div><p><b>Procedure:</b></p></div><div></div><div><p>The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.</p></div><div><p>- Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.</p></div><div><p>- Inoculate 200 mL LB with the preculture.</p></div><div><p>- Incubate at 37°C and 150 rpm until an OD<sub>600</sub> of 0.4-0.6 is reached.</p></div><div><p>- Incubate cells on ice for 15 min.</p></div><div><p>- Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).</p></div><div><p>- Resuspend cell pellet in 10 mL ice cold 100 mM CaCl<sub>2</sub> (Do not vortex!).</p></div><div><p>- Incubate on ice for 1 hour.</p></div><div><p>- Centrifuge the culture at 4°C and 3000 x g for 10 min.</p></div><div><p>- Resuspend cell pellet in 10 mL ice cold 100 mM CaCl<sub>2</sub>.</p></div><div><p>- Incubate on ice for 1 hour.</p></div><div><p>- Centrifuge the culture at 4°C and 3000 x g for 5 min.</p></div><div><p>- Resuspend cell pellet in 2mL ice cold 100 mM CaCl<sub>2</sub> and 15 % (v/v) glycerine.</p></div><div><p>- Incubate on ice for 30 min.</p></div><div><p>- Aliquot the cells à 100 µL.</p></div><div><p>- Store at -80°C.</p></div><div></div><div><p><b>Mixtures:</b></p></div><div></div><div><p>CaCl<sub>2</sub>-Solution</p></div><div><p>- 5.55 g CaCl<sub>2</sub></p></div><div><p>- Add ddH<sub>2</sub>O to 1 L</p></div><div><p>- Sterilize by autoclaving</p></div><div></div><div><p>Cryo solution</p></div><div><p>- 0.278 g CaCl<sub>2</sub></p></div><div><p>- 10 ml glycerine</p></div><div><p>- Add ddH<sub>2</sub>O to 50 ml</p></div><div><p>- Sterilize by autoclaving</p></div><div><p><b>References:</b></p></div><div></div><div><p>Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162</p></div><div></div></div><!--TYPO3SEARCH_end-->
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Latest revision as of 00:41, 18 October 2014

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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