Team:Groningen/protocols/Ligation

From 2013.igem.org

(Difference between revisions)
Line 63: Line 63:
     <td>Insert</td>
     <td>Insert</td>
     <td ALIGN=CENTER>x&micro;l</td>
     <td ALIGN=CENTER>x&micro;l</td>
-
     <td>3:1 molar ratio over vector</td>
+
     <td>3:1 molar ratio over vector&#42;</td>
   </tr>
   </tr>
Line 74: Line 74:
</table>  
</table>  
 +
<br>&#42; When restricted PCR products are ligated together a 1:1 molar ratio is used.
 +
<p>
<br>All the reagents are added following the order listed in the table above.  
<br>All the reagents are added following the order listed in the table above.  
<br>After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22&deg;C) for 20 min.
<br>After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22&deg;C) for 20 min.

Revision as of 16:58, 27 July 2013

Ligation

Materials:

  • MQ water
  • 1.5ml tubes
  • 10x T4 Ligation buffer
  • T4 Ligase
  • Insert
  • Vector

Reaction:

Component 20µl Final concentration
MilliQ water up to 20µl
10 ligation buffer 2µl 1x
Vector xµl 20-100ng
Insert xµl 3:1 molar ratio over vector*
T4 DNA ligase 5U/µl 2µl 0.5U/µl

* When restricted PCR products are ligated together a 1:1 molar ratio is used.


All the reagents are added following the order listed in the table above.
After finishing the reaction mixture, the content is mixed and incubated at room temperature (~22°C) for 20 min.
Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf