Team:Groningen/protocols/Ligation

From 2013.igem.org

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</ul>
</ul>
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<h3>Reaction:</h3>
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<table border="1">
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  <tr>
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    <th>Component</th>
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    <th>20&micro;l</th>
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    <th>Final concentration</th>
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  </tr>
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  <tr>
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    <td>MilliQ water</td>
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    <td>up to 20&micro;l</td>
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    <td></td>
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  </tr>
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  <tr>
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    <td>10 ligation buffer</td>
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    <td>2&micro;l</td>
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    <td>1x</td>
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  </tr>
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  <tr>
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    <td>Vector</td>
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    <td>x&micro;l</td>
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    <td>20-100ng</td>
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  </tr>
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  <tr>
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    <td>Insert</td>
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    <td>x&micro;l</td>
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    <td>3:1 molar ratio over vector</td>
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  </tr>
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  <tr>
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    <td>T4 DNA ligase 5U/&micro;l</td>
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    <td>2&micro;l</td>
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    <td>0.5U/&micro;l</td>
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  </tr>
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</table>
<h3>Reaction:</h3>
<h3>Reaction:</h3>
<table border = "1">
<table border = "1">

Revision as of 15:06, 27 July 2013

Ligation

Materials:

  • MQ water
  • 1.5ml tubes
  • Ligation buffer
  • 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

Reaction:

Component 20μl reaction Final concentration
H2O up to 20μl
10x Ligation buffer 2μl 1x
Vector 1μl 20-100μg
Insert 1μl 1:1 molar ratio
T4 DNA Ligase 5U/μl 0.2μl 1U/μl

All the reagents are added following the order listed in the table above.
After the reaction is ready mix the content of the tube and spin it down.
The tubes are incubated for at room temperature (~22°C) for 1h.

Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/fast-digestion-dna.pdf