UCL London/Protocol/Ligation

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: '''''Materials:'''''
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* Plasmid DNA: 15 µL
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* Restriction Enzymes (depend on the requirements of the parts)
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** EcoRI: 1 µL
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** PstI: 1 µL
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** SpeI: 1 µL
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** XbaI: 1 µL
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* 10× NE buffer: 1.5 µL/1 µL (lid)
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* BSA: 1 µL (lid)
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* Quick Ligase
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* 2× Quick Ligation Buffer
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: '''''Method:'''''
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# Digest the DNA as in [[UCL_London/Protocol/Restriction_Enzyme_Digestion|Restriction Enzyme Digestion Protocol]].
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# Heat inactive the restriction enzymes.
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# Run a diagnostic gel before the ligation.
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# After the gel is confirmed, add 1 µL quick ligase, 10 µL ligation buffer, 3 µL backbone and 6 µL inserts (or other volumes depend on the concentration of DNA) into an eppendorf.
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# Leave for 5 minutes (or up to 30 minutes) at room temperature (25°C).
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# Transform the ligated DNA onto competent cells as described in [[UCL_London/Protocol/Transformation| Transformation]], method 2 step 4 to 10.
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Latest revision as of 11:09, 18 October 2009

Materials:
  • Plasmid DNA: 15 µL
  • Restriction Enzymes (depend on the requirements of the parts)
    • EcoRI: 1 µL
    • PstI: 1 µL
    • SpeI: 1 µL
    • XbaI: 1 µL
  • 10× NE buffer: 1.5 µL/1 µL (lid)
  • BSA: 1 µL (lid)
  • Quick Ligase
  • 2× Quick Ligation Buffer


Method:
  1. Digest the DNA as in Restriction Enzyme Digestion Protocol.
  2. Heat inactive the restriction enzymes.
  3. Run a diagnostic gel before the ligation.
  4. After the gel is confirmed, add 1 µL quick ligase, 10 µL ligation buffer, 3 µL backbone and 6 µL inserts (or other volumes depend on the concentration of DNA) into an eppendorf.
  5. Leave for 5 minutes (or up to 30 minutes) at room temperature (25°C).
  6. Transform the ligated DNA onto competent cells as described in Transformation, method 2 step 4 to 10.

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