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| - | <html>
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| - | <body>
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| - | <h2>Materials</h2>
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| - | <ul>
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| - | <li>Escherichia coli DH5 alpha electrocompetent cells</li>
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| - | <li>Plasmid DNA</li>
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| - | <li>Ice</li>
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| - | <li>2mm gap width Electroporation cuvette</li>
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| - | <li>Electroporater</li>
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| - | <li>1.5ml microfuge tube</li>
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| - | <li>1mL SOC (room temperature) for each reaction</li>
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| - | <li>LB-agar plate with corresponding antibiotic</li>
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| - | </ul>
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| - | <h2>Procedure</h2>
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| - | <ul>
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| - | <li>Chill electroporation cuvettes, DNA samples and tubes on ice.</li>
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| - | <li>Place LB-agar plates in 37°C incubator twarm.<li>
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| - | <li>Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice. Alternatively, freshly prepared electrocompetent cells may be used immediately.</li>
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| - | <li>If electrocompetent cells are not already in individual aliquots, then aliquot out intpre-chilled 0.6mL tubes.</li>
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| - | <li>Turn on electroporator and set voltage teither 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).</li>
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| - | <li>Dial a P2 pipetman teither 1 or 2µL depending on the salt content of your DNA sample and . Use 2µL for samples that have been purified in some way.</li>
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| - | <li>Dial a P200 pipetman t50µL or whatever volume of electrocompetent cells you want tuse. Usually 20-50µL.</li>
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| - | <li>Dial a P1000 pipetman t950µL and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything.</li>
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| - | <li>Pipet 1-2µL of DNA sample and add telectrocompetent cells. Swirl tip around gently in cells tmix DNA and cells. Dnot pipet up and down. </li>
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| - | <li>Place cells back on ice tensure they remain cold.</li>
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| - | <li>Transfer cell-DNA mixture tcuvettes using P200 pipetman. Try not thandle cuvette base tomuch sthat it stays cold.</li>
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| - | <li>Tap the cuvette on the counter gently sthat cells are at the bottom and tremove any air bubbles. </li>
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| - | <li>Wipe off excess moisture from outside of cuvette.</li>
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| - | <li>Place in chamber of electroporator.</li>
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| - | <li>Slide the chamber in sthat the cuvette sits snugly between electrodes.</li>
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| - | <li>Pulse the cells with a shock by pressing button on electroporator.</li>
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| - | <li>Remove cuvette from the chamber and immediately add SOC. This step should be done as quickly as possible tprevent cells from dying off.</li>
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| - | <li>Transfer SOC-cell mixture tchilled eppendorf tube.</li>
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| - | <li>Chill sample on ice for 2 mins tpermit the cells trecover.</li>
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| - | <li>Transfer eppendorf tube t37°C incubator and shake tpromote aeration. Incubate for 1 hr tpermit expression of antibiotic resistance gene. </li>
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| - | <li>Plate transformation ontprewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate 200µL but appropriate plating volume depends on efficiency of the transformation.</li>
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| - | <li> Incubate plate overnight at 37°C.</li>
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| - | <li> Leave remaining SOC-cell mixture on the benchtop overnight.</li>
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| - | <li> If you don't have any transformants, plate the rest of the transformation in the morning.</li>
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| - | </ul>
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| - | </body>
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| - | </html>
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| - | [[Category:BIOTEC Dresden/Protocol]]
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| - | {{Biotec_Dresden/Bottom}}
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