Team:Groningen/protocols/Ligation

From 2013.igem.org

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<h3>Reaction:</h3>
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<table border = "1">
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<tr><td>Component</td>
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<td>20&#956;l reaction</td>
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<td>Final concentration</td>
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</tr>
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<tr><td>H<sub>2</sub>O</td>
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<td>up to 20&#956;l</td>
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<td></td>
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<tr><td>10x Ligation buffer</td>
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<td>2&#956;l</td>
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<td>1x</td>
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</tr>
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<tr><td>Vector</td>
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<td>1&#956;l</td>
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<td>20-100&#956;g</td>
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</tr>
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<tr><td>Insert</td>
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<td>1&#956;l</td>
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<td>1:1 molar ratio</td>
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</tr>
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<tr><td>T4 DNA Ligase 5U/&#956;l</td>
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<td>0.2&#956;l</td>
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<td>1U/&#956;l</td>
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</tr>
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</table>
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<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.

Revision as of 15:25, 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

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