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<li>Incubate overnight at 37°C with plates upside down.</li> | <li>Incubate overnight at 37°C with plates upside down.</li> | ||
</ol> | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="P11"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P11-collapse" aria-expanded="false" aria-controls="P11-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11">DNA gel eletrophoresis</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="P11-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P11"> | ||
+ | <div class="panel-body"> | ||
+ | |||
+ | <ol> | ||
+ | <li> Prepare your 1% gel by using 0.5g of agarose in 50 ml of TAE buffer</li> | ||
+ | <li>Add 2.5ul of Ethidium Bromide before you pour your gel into the chamber. </li> | ||
+ | <li>Mix 5ul of DNA with 1ul of loading buffer by pipetting up and down a couple of times. </li> | ||
+ | |||
+ | |||
+ | <li>Load your samples and appropriate marker into your wells.</li> | ||
+ | <li>Pipette 900 µl of room temperature SOC or LB media into the mixture.</li> | ||
+ | <li>Incubate at 37°C and 250 rpm for 60-120 minutes.</li> | ||
+ | <li>Pellet cells at 13000rpm for 1 minutes</li> | ||
+ | <li>Apply 130 volts to the chamber for 20 mins. </li> | ||
+ | <li>Re-suspend cells by light vortexing</li> | ||
+ | <li>Plate resuspended cells as above</li> | ||
+ | <li>Check your gel using a transilluminator or other UV emitting device.</li> | ||
+ | </ol> | ||
</div> | </div> | ||
</div> | </div> | ||
+ | </div> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="P12"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P12-collapse" aria-expanded="false" aria-controls="P12-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11">DNA gel purification</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="P12-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P12"> | ||
+ | <div class="panel-body"> | ||
+ | |||
+ | <ol> | ||
+ | <li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by trimming off extra agarose gel. | ||
+ | <br></li> | ||
+ | <li>Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel </li> | ||
+ | <li>Incubate the tube at 60°C until the gel completely dissolved.</li> | ||
+ | |||
+ | <li>Place on ice for 5 minutes. </li> | ||
+ | <li>Pipette 900 µl of room temperature SOC or LB media into the mixture.</li> | ||
+ | <li>Incubate at 37°C and 250 rpm for 60-120 minutes.</li> | ||
+ | <li> For DNA fragments <500 bp and >4 kb, add 1 gel volume of isopropanol to the sample and mix the reagents well. </li> | ||
+ | <li>Place a QIAquick spin column in a provided 2 ml collection tube.</li> | ||
+ | <li>To bind DNA, apply 800 μl sample to the QIAquick column, and centrifuge for 1 min. </li> | ||
+ | <li>Discard flow-through and place QIAquick column back in the same collection tube. It can be reused to reduce plastic waste.</li> | ||
+ | <li> Add 0.75 ml of Buffer PE to wash the QIAquick column, and centrifuge for 1 min..</li> | ||
+ | <li> | ||
+ | Place QIAquick column into a clean 1.5 ml micro centrifuge tube.</li> | ||
+ | <li> | ||
+ | To elute DNA, add 30-60 μl of distilled water.</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
Revision as of 08:00, 24 August 2017