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- | {{Biotec_Dresden/Header}}
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- | <html>
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- | <body>
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- | <div id="content_prim">
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- | <div id="materials">
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- | <h2>Materials</h2>
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- | <ul>
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- | <li>Escherichia coli DH5 alpha chemical competent cells</li>
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- | <li>1mL SOC (room temperature) for each reaction</li>
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- | <li>Ice</li>
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- | <li>Plasmid DNA</li>
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- | <li>Heating block</li>
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- | <li>1.5ml microfuge tube</li>
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- | <li>LB-agar plate with corresponding antibiotic</li>
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- | </ul>
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- | </div>
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- | <div class="visualClear"></div>
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- | <div id="procedure">
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- | <h2>Procedure</h2>
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- | <ul>
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- | <li>Chill DNA samples and tubes on ice.
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- | <li>Place LB-agar plates in <span class="markup temp">37°C</span> incubator to warm.
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- | <li>Remove chemical competent cells from <span class="markup temp">-80°C</span> freezer and thaw on ice. Alternatively, freshly prepared chemical competent cells may be used immediately.</li>
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- | <li>Dial a P2 pipetman to either <span class="markup volume">1 or 2μL</span> depending on the salt content of your DNA sample. Use <span class="markup volume">2μL</span> for samples that have been purified in some way.</li>
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- | <li>Dial a P200 pipetman to <span class="markup volume">50μL</span> or whatever volume of chemical competent cells you want to use; usually <span class="markup volume">20-50μL</span> .</li>
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- | <li>Pipet <span class="markup volume">1-2μL</span> of DNA sample and add to chemical competent cells. Swirl tip around gently in cells to mix DNA and cells. Do not pipet up and down.</li>
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- | <li>Place the mix back on <span class="markup temp">ice</span> for <span class="markup time">30 mins</span>.</li>
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- | <li>Pulse the cells with a heat shock by placing the microfuge tubes in a heating block for <span class="markup time">45 seconds</span> at <span class="markup temp">42°C</span>.</li>
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- | <li>Place sample on ice for <span class="markup time">2 mins</span> and then add SOC medium. This step should be done as quickly as possible to prevent cells from dying off.</li>
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- | <li>Chill sample on ice for <span class="markup time">2 mins</span> to permit the cells to recover.</li>
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- | <li>Transfer eppendorf tube to <span class="markup temp">37°C</span> incubator and shake to promote aeration. Incubate for <span class="markup time">1 hr</span> to permit expression of antibiotic resistance gene.</li>
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- | <li>Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic.</li>
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- | </ul>
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- | </div>
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- | <h2>Reference</h2>
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- | <p>adapted from <a href="http://openwetware.org/wiki/Knight:Electroporation">http://openwetware.org/wiki/Knight:Electroporation</a></p>
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- | </div>
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- | </body>
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- | </html>
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- | [[Category:BIOTEC Dresden/Protocol|AHL assay]]
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- | {{Biotec_Dresden/Bottom}}
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