Line 37: | Line 37: | ||
</div> | </div> | ||
<div class="modal-body"> | <div class="modal-body"> | ||
− | <ol | + | <ol> |
− | <li | + | <li>Gently mix the following reaction by pipetting and centrifuge briefly.</li> |
− | + | <table class="centertable"> | |
− | + | <tr> | |
− | + | <th></th> | |
− | + | <th>20 μl system</th> | |
− | + | <th>50 μl system</th> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Template</td> | |
− | + | <td>12~20 ng</td> | |
− | + | <td>30~50 ng</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Forward primer</td> | |
− | + | <td>1.0 μl</td> | |
− | + | <td>2.5 μl</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Reverse primer</td> | |
− | + | <td>1.0 μl</td> | |
− | + | <td>2.5 μl</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>dNTP</td> | |
− | + | <td>1.6 μl</td> | |
− | + | <td>4.0 μl</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>10x Buffer</td> | |
− | + | <td>2.0 μl</td> | |
− | + | <td>5.0 μl</td> | |
− | + | </tr> | |
− | + | </table> | |
− | + | <p>Program of KOD DNA polymerase</p> | |
− | + | <table class="centertable"> | |
− | + | <tr> | |
− | + | <th>Temperature</th> | |
− | + | <th>Time</th> | |
− | + | <th>Repeat</th> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>94 ℃</td> | |
− | + | <td>3 min.</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>94 ℃</br>(Denaturation)</td> | |
− | + | <td>40 sec</td> | |
− | + | <td rowspan="3">25~30 cycles</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>57.5 ℃</br>(Annealing)</td> | |
− | + | <td>30 sec</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72 ℃</br>(Extension)</td> | |
− | + | <td>Depend on sequence size</br>(2 kbp/min. for Taq)</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72 ℃</td> | |
− | + | <td>5 min.</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>4 ℃</td> | |
− | + | <td>∞</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | </table> | |
− | + | ||
− | + | ||
<li>Confirm the size of the digested product by gel electrophoresis.</li> | <li>Confirm the size of the digested product by gel electrophoresis.</li> | ||
<li>Gel purification of the target size.</li> | <li>Gel purification of the target size.</li> |
Revision as of 16:53, 2 October 2018