Difference between revisions of "Team:Freiburg/Protocols/Ligation"
| Line 14: | Line 14: | ||
<div class="table sectionedit2"><table class="inline"> | <div class="table sectionedit2"><table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
| − | <th | + | <th> Amount </th><th class="col1 leftalign"> Ingredient </th> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
| − | <td | + | <td> 50 ng </td><td class="col1 leftalign"> Backbone </td> |
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
| − | <td | + | <td> 3 fold molar amount</td><td class="col1 leftalign"> Insert </td> |
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
| − | <td | + | <td> 2 µl </td><td class="col1 leftalign"> 10X T4-Ligase Buffer </td> |
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
| − | <td | + | <td> 1 µl </td><td class="col1 leftalign"> T4-Ligase </td> |
</tr> | </tr> | ||
<tr class="row5"> | <tr class="row5"> | ||
| − | <td | + | <td> ad 20 µl </td><td class="col1 leftalign"> dH<sub>2</sub>O </td> |
</tr> | </tr> | ||
</table></div> | </table></div> | ||
Revision as of 20:32, 16 September 2015
Ligation
Classical cloning as well as Gibson Assembly require the action of ligases to covalently assemble DNA fragments. Before ligation, those fragments anneal to each other due to complementary overhangs.
| Amount | Ingredient |
|---|---|
| 50 ng | Backbone |
| 3 fold molar amount | Insert |
| 2 µl | 10X T4-Ligase Buffer |
| 1 µl | T4-Ligase |
| ad 20 µl | dH2O |
Incubate for 30 min at RT
Calculating the amount of insert
required Volume of Insert = 3 x Volume(Bb) x length(In) x concentration(Bb) / [ length(Bb) x concentration(In) ]
In short: V(In) = 3 x V(Bb) x l(In) x c(Bb) / [ l(Bb) x c(In) ]
