"
Team:TU Darmstadt/Notebook/Methods/Chemically competent cells
From 2014.igem.org
(Difference between revisions)
| (6 intermediate revisions not shown) | |||
| 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 | + | <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> |
</nav> | </nav> | ||
</div> | </div> | ||
<div id="wikicontent" class="grid_19"> | <div id="wikicontent" class="grid_19"> | ||
| - | <!--TYPO3SEARCH_begin--><div id="c96" class="csc-default"><div class="csc-header csc-header-n1"><h1 class="csc-firstHeader">Chemically | + | <!--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 & 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--> |
</div> | </div> | ||
</html> | </html> | ||
Latest revision as of 00:41, 18 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
