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Team:TU Darmstadt/Notebook/Methods/Chemically competent cells
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| - | <!--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--> |
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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
