Oscarliu117 (Talk | contribs) |
Oscarliu117 (Talk | contribs) |
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<a class="list-group-item list-group-item-action" href="#list-item-1">PCR</a> | <a class="list-group-item list-group-item-action" href="#list-item-1">PCR</a> | ||
<a class="list-group-item list-group-item-action" href="#list-item-2">Plasmid Construction</a> | <a class="list-group-item list-group-item-action" href="#list-item-2">Plasmid Construction</a> | ||
− | <a class="list-group-item list-group-item-action" href="#list-item-3"> | + | <a class="list-group-item list-group-item-action" href="#list-item-3">SSS</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item- | + | <a class="list-group-item list-group-item-action" href="#list-item-4">Plasmid Construction</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item- | + | <a class="list-group-item list-group-item-action" href="#list-item-5">SS</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item- | + | <a class="list-group-item list-group-item-action" href="#list-item-6">Plasmid Construction</a> |
− | <a class="list-group-item list-group-item-action" href="#list-item- | + | <a class="list-group-item list-group-item-action" href="#list-item-7">SS</a> |
+ | <a class="list-group-item list-group-item-action" href="#list-item-8">Plasmid Construction</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#list-item-9">SS</a> | ||
<a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | <a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | ||
aria-hidden="true"></i></a> | aria-hidden="true"></i></a> | ||
Line 33: | Line 35: | ||
<div class="row"> | <div class="row"> | ||
<a class="btn col-md-12" data-toggle="collapse" href="#PCR" role="button" aria-expanded="false" | <a class="btn col-md-12" data-toggle="collapse" href="#PCR" role="button" aria-expanded="false" | ||
− | aria-controls="multiCollapseExample1"> | + | aria-controls="multiCollapseExample1" style="color: white; hover: #002bb8;"> |
PCR | PCR | ||
<i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
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<ol> | <ol> | ||
<li>Gel Extraction</li> | <li>Gel Extraction</li> | ||
− | a | + | <ol type="a"> |
− | + | <li>Excised the DNA fragment from the agarose gel.</li> | |
− | + | <li>Transferred up to 300 mg of the gel slice to a 1.5 ml microcentrifuge | |
− | + | tube.</li> | |
− | + | <li>Added 500 μl of the Gel/PCR Bufffer to the sample and mixed by vortex.</li> | |
− | + | <li>Incubate at 55~60℃ for 10 minutes (or until the gel slice has | |
− | + | completely dissolved).</li> | |
+ | <li>During the incubation, mixed by vortexing the tube every 2~3 minutes.</li> | ||
+ | <li>Cooled the dissolved sample mixture to the room temperature.</li> | ||
+ | </ol> | ||
+ | </ol> | ||
</p> | </p> | ||
+ | |||
<h3>DNA Binding</h3></br> | <h3>DNA Binding</h3></br> | ||
− | <p class="pcontent">2 | + | <p class="pcontent"> |
− | + | <ol start="2"> | |
− | + | <li>Placed a PG Column in a Collection Tube. Apply the supernatant to the PG | |
− | + | Column by decanting or pipetting.</li> | |
− | + | <li>Centrifuged at 16,000 xg for 30 seconds.</li>Centrifuged at 16,000 xg for | |
− | + | 30 seconds. | |
− | + | <li>Discarded the flow-through and place the PG Column back into the same | |
− | + | collection tube.</li> | |
+ | </ol> | ||
+ | </p> | ||
+ | |||
<h3>Wash</h3></br> | <h3>Wash</h3></br> | ||
− | <p class="pcontent"> | + | <p class="pcontent"> |
− | + | <ol start="5"> | |
− | + | <li>Added 400 μl of the Buffer W1 into the PG Column.</li> | |
− | + | <li>Centrifuged at 16,000 xg for 30 seconds.</li> | |
− | + | <li>Discarded the flow-through and place the PG Column back into the same | |
− | + | collection tube.</li> | |
− | + | <li>Added 600 μl of the Buffer W2 (ethanol added) into the PG Column.</li> | |
− | + | <li>Centrifuged at 16,000 xg for 30 seconds.</li> | |
− | + | <li>Discarded the flow-through and place the PG Column back into the same | |
− | + | collection tube.</li> | |
− | + | <li>Centrifuged at 16,000 xg again for 2 minutes to remove the residual Buffer | |
+ | W2.</li> | ||
+ | </ol> | ||
+ | </p> | ||
+ | |||
<h3>Elution</h3></br> | <h3>Elution</h3></br> | ||
− | <p class="pcontent"> | + | <p class="pcontent"> |
− | + | <ol start="12"> | |
− | + | <li>To elute the DNA, placed the PG Column in a clean 1.5 ml | |
− | + | microcentrifuge | |
− | + | tube.</li> | |
− | + | <li></li>Added 50 μl of the H2O (pH is between 7.0 and 8.5) to the center of | |
+ | each PG | ||
+ | Column, let it stand for at least 2 minutes, and centrifuge at 16,000 xg for 2 | ||
+ | min.</li> | ||
+ | </ol> | ||
</p> | </p> | ||
Line 362: | Line 380: | ||
<div class="collapse multi-collapse" id="Preparation"> | <div class="collapse multi-collapse" id="Preparation"> | ||
− | <p class="pcontent"></br></br> | + | <p class="pcontent"></br></br> |
− | + | For E. coli DH5α,BL21(DE3) and W3100(DE3) competent cell | |
− | + | <ol> | |
− | + | <li>Streak out wild type E. coli on a plate (LB plate without antibiotics) | |
− | + | overnight | |
− | + | and pick one colony into 3 ml of media (LB) and grow overnight.</li> | |
− | + | <li>Transfer 0.4 ml of starter culture into 40 ml of fresh LB and grow culture at | |
− | + | 37 | |
− | + | ℃.</li> | |
− | + | <li>When the OD600 nm up to 0.35, put the cells on ice immediately.</li> | |
− | + | <li>Spin the cells at 4℃for 10 minutes at 4000 rpm.</li> | |
− | + | <li>Suspend the pellet on ice carefully with 16 ml chilly Transformation Buffer | |
− | + | 1(TFB1).</li> | |
− | + | <li>Leave nicely suspended bugs on ice for 10 minutes.</li> | |
− | + | <li>Spin the cells at 4℃ for 10 min. at 4000 rpm.</li> | |
− | + | <li>Suspend the pellet on ice with 1.6 ml of Transformation Buffer 2 (TFB2).</li> | |
− | + | <li>Leave on immediately on ice for 30 minutes.</li> | |
+ | <li>Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with | ||
+ | liquid nitrogen.</li> | ||
+ | <li>Store the frozen cells in the -80℃ freezer.</li> | ||
+ | </ol> | ||
+ | </p> | ||
</div> | </div> | ||
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<div class="collapse multi-collapse" id="Plasmid_Extraction"> | <div class="collapse multi-collapse" id="Plasmid_Extraction"> | ||
− | <p class="pcontent"></br></br>1. Transfer 1.4 ml of well-grown bacterial culture to | + | <p class="pcontent"></br></br> |
+ | 1. Transfer 1.4 ml of well-grown bacterial culture to | ||
a centrifuge tube.</br></br> | a centrifuge tube.</br></br> | ||
2. Centrifuge the tube at 16,000 xg for 1 minute to pellet the cells and | 2. Centrifuge the tube at 16,000 xg for 1 minute to pellet the cells and | ||
Line 512: | Line 536: | ||
<div class="collapse multi-collapse" id="DNS"> | <div class="collapse multi-collapse" id="DNS"> | ||
− | + | ||
</div> | </div> | ||
Revision as of 11:40, 29 September 2018