Team:Groningen/protocols/Ligation

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<h1>Ligation Reaction</h1>
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<h5>Materials:</h5>
<h5>Materials:</h5>

Revision as of 11:25, 28 July 2013

Ligation Reaction

Materials:
  • MQ water
  • 1.5ml tubes
  • 10x T4 Ligation buffer
  • T4 Ligase
  • Insert
  • Vector
Reaction mixture:
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