Melbourne/Ligation Protocol

From 2007.igem.org

(Difference between revisions)
(Method including controls)
(Method including controls)
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=====Method including controls=====
=====Method including controls=====
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#Add to a sterile 1.7ml Eppendorf
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Add to a sterile 1.7ml Eppendorf
*Roughly an equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified)
*Roughly an equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified)
*2ul of 10X buffer or 10ul of 2X buffer
*2ul of 10X buffer or 10ul of 2X buffer
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*milliQ water to make up to 20ul
*milliQ water to make up to 20ul
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#Incubate Overnight (if using 10X buffer) at 4 degrees, 2h or 5min (if using 2X buffer) at room temprature.
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Incubate Overnight (if using 10X buffer) at 4 degrees, 2h or 5min (if using 2X buffer) at room temprature.
=====Equipement Required=====
=====Equipement Required=====

Revision as of 09:14, 28 September 2007

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  • Applications: Ligate DNA fragments together


  • Time to complete protocol: Overnight, 2 hours, or 10 minutes
    • Lab time: 5 minutes
    • Waiting time: Overnight, 2h, 10min
  • Approximate cost of materials: $

Contents

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls
  • Ligase buffer (10X or 2X)
  • DNA for ligation
  • T4 DNA ligase
  • milliQ water
Method including controls

Add to a sterile 1.7ml Eppendorf

  • Roughly an equal quantity of DNA fragments to be ligated together (usually 5ul of both vector and insert if gel-purified)
  • 2ul of 10X buffer or 10ul of 2X buffer
  • 1ul of T4 DNA ligase
  • milliQ water to make up to 20ul

Incubate Overnight (if using 10X buffer) at 4 degrees, 2h or 5min (if using 2X buffer) at room temprature.

Equipement Required
  • 1.5mL Microfuge tubes
  • Pipettes
  • Fridge
References