Line 234: | Line 234: | ||
<div class="panel-body"> | <div class="panel-body"> | ||
<ol> | <ol> | ||
− | <li>Prepare the | + | <li>Prepare the reagents as follow:</li> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 241: | Line 241: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>DNA </td> | + | <td>DNA template</td> |
− | <td>0. | + | <td>0.5 μg</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>10X Buffer</td> |
− | <td> | + | <td>2.5 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Enzyme</td> |
<td>2 μl</td> | <td>2 μl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Nuclease-Free Water to final colume of:</td> |
− | + | <td> 25 μl</td> | |
− | + | ||
− | + | ||
− | + | ||
− | <td> | + | |
</tr> | </tr> | ||
</table> | </table> | ||
− | <li> | + | <li>Pipette the solution up and down to ensure all reagents are mixed well.</li> |
− | <li> | + | <li>Place the reaction mixture at 37 oC incubation or dry bath for 2-4 hours. </li> |
+ | <li>Purify the DNA by PCR purification kit/gel extraction kit for downstream process. </li> | ||
</ol> | </ol> | ||
<a href=" https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012178_XbaI_10_UuL_5X1500U_UG.pdf">Click here for the manufacturer's maual</a> | <a href=" https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012178_XbaI_10_UuL_5X1500U_UG.pdf">Click here for the manufacturer's maual</a> | ||
Line 285: | Line 282: | ||
<div class="panel-body"> | <div class="panel-body"> | ||
<ol> | <ol> | ||
− | <li>Prepare the | + | <li>Prepare the reaction mixture as follow:</li> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 292: | Line 289: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>DNA | + | <td>DNA </td> |
− | <td>0. | + | <td>0.2M KCl/HCl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>DNA template</td> |
− | <td> | + | <td>10-200 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>10XT4 DNA Ligase Reaction Buffer</td> |
<td>2 μl</td> | <td>2 μl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>T4 DNA Ligase</td> |
− | <td> | + | <td>1 μl</td> |
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Water</td> | ||
+ | <td>Top up to 20 μl</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | <li> | + | <li>Allow the ligation to take place a 22-25°C for 10 minutes.</li> |
− | <li> | + | <li>Send 5 μl of the ligated sample should be used for agarose gel electrophoresis to confirm whether ligation has occurred (Optional)</li> |
− | + | ||
</ol> | </ol> | ||
+ | |||
<a href=" https://www.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202">Click here for the manufacturer's maual</a> | <a href=" https://www.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202">Click here for the manufacturer's maual</a> | ||
</p> | </p> |
Latest revision as of 14:44, 30 October 2017