Team:TU Darmstadt/protocols/Chemically competent cells
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+ | <font size="8" color="#F0F8FF" face="Arial regular">Protocols</font> | ||
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+ | </center> | ||
+ | |||
+ | <!-- Chemically competent cells --> | ||
+ | <center> | ||
+ | |||
+ | <br> | ||
+ | <h2><font size="6" color="#F0F8FF" face="Arial regular"> Chemically competent cells </font></h2> | ||
+ | <div id="all"> | ||
+ | <body> | ||
+ | |||
+ | |||
+ | <p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular"> | ||
+ | <B> Materials<br></B></font></p> | ||
+ | |||
+ | <p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular"> | ||
+ | <br> | ||
+ | <B>Equipment<br></B> | ||
+ | <div align="left" style="margin-left:60px; margin-right:50px"> | ||
+ | <ul> | ||
+ | <li class=list1>- -80°C freezer</li> | ||
+ | <li class=list1>- Incubation shaker</li> | ||
+ | <li class=list1>- Centrifuge (cooling cababilities required!)</li> | ||
+ | <li class=list1>- photometer</li> | ||
+ | <li class=list1>- Ice water bath</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </font> | ||
+ | </p> | ||
- | + | <p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular"> | |
- | + | <br> | |
- | + | <B>Chemicals & consumables<br></B> | |
- | + | <div align="left" style="margin-left:60px; margin-right:50px"> | |
- | + | <ul> | |
+ | <li class=list1>- Ice and/or liquid nitrogen</li> | ||
+ | <li class=list1>- Falcon tubes</li> | ||
+ | <li class=list1>- <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Media"> dYT Medium</a>(50 ml p.c.) </li> | ||
+ | <li class=list1>- ice cold 100mM CaCl<sub>2</sub></li> | ||
+ | <li class=list1>- Glycerin</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </font> | ||
+ | </p> | ||
+ | <br> | ||
+ | <p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular"> | ||
+ | <B> Procedure<br></B></font></p><br> | ||
+ | <p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"> | ||
+ | The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells. | ||
+ | <div align="left" style="margin-left:30px; margin-right:50px"> | ||
+ | <ol> | ||
+ | <li>Inoculate 2 mL of LB-Media with an E. coli colony and incubate at 37 °C overnight.</li> | ||
+ | <li>Inoculate 200 mL LB with the preculture.</li> | ||
+ | <li>Incubate at 37°C and 150 rpm until an OD600 of 0.4-0.6 is reached.</li> | ||
+ | <li>Incubate cells on ice for 15 min.</li> | ||
+ | <li>Centrifuge the culture at 4°C and 3000 x g for 10 min (the following steps are carried out on ice).</li> | ||
+ | <li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub> (Do not vortex!).</li> | ||
+ | <li>Incubate on ice for 1 hour.</li> | ||
+ | <li>Centrifuge the culture at 4°C and 3000 x g for 10 min.</li> | ||
+ | <li>Resuspend cell pellet in 10mL ice cold 100 mM CaCl<sub>2</sub>.</li> | ||
+ | <li>Incubate on ice for 1 hour.</li> | ||
+ | <li>Centrifuge the culture at 4°C and 3000 x g for 5 min.</li> | ||
+ | <li>Resuspend cell pellet in 2mL ice cold 100 mM CaCl<sub>2</sub> and 15 % (v/v) glycerine.</li> | ||
+ | <li>Incubate on ice for 30 min.</li> | ||
+ | <li>Aliquot the cells à 100µ.</li> | ||
+ | <li>Store at -80°C.</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </font></p><br> | ||
- | == | + | <p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular"> |
- | + | <B>Mixtures<br></B></font></p><br> | |
+ | <p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular"> | ||
+ | <B>CaCl2-Solution<br></B> | ||
+ | <div align="left" style="margin-left:60px; margin-right:50px"> | ||
+ | <ul> | ||
+ | <li class=list1>- 5.55 g CaCl<sub>2</sub></li> | ||
+ | <li class=list1>- add ddH<sub>2</sub>O to 1 L</li> | ||
+ | <li class=list1>- sterilize by autoclaving</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </font> | ||
+ | </p> | ||
+ | <br> | ||
+ | <p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular"> | ||
+ | <B>Cryo solution<br></B> | ||
+ | <div align="left" style="margin-left:60px; margin-right:50px"> | ||
+ | <ul> | ||
+ | <li class=list1>- 0.278 g CaCl<sub>2</sub></li> | ||
+ | <li class=list1>- 10 ml glycerin</li> | ||
+ | <li class=list1>- Add ddH<sub>2</sub>O to 50 ml</li> | ||
+ | <li class=list1>- Sterilize by autoclave</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </font> | ||
+ | </p> | ||
- | |||
- | |||
- | |||
- | === | + | <h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2> |
- | + | <font size="3" color="#F0F8FF" face="Arial regular"> | |
- | + | <ol> | |
+ | <li style="margin-left:15px; margin-right:50px; text-align:justify">Mandel, M. and Higa, A.: <i>Calcium-dependent bacteriophage DNA infection</i>. J Mol Biol, 1970, 53, 159-162</li> | ||
+ | </ol> | ||
+ | </font> |
Latest revision as of 00:30, 5 October 2013
Chemically competent cells
Materials
Equipment
Chemicals & consumables
Procedure
The transformation of E. coli with plasmid DNA via heatshock transformation requires chemically competent cells.
Mixtures
CaCl2-Solution
Cryo solution
References
- Mandel, M. and Higa, A.: Calcium-dependent bacteriophage DNA infection. J Mol Biol, 1970, 53, 159-162