Team:Brown-Stanford/Lab/Protocol/T4Ligation

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T4 ligation

(Taken from [http://openwetware.org/wiki/DNA_Ligation openwetware.org]; this protocol was edited for amplification of [http://partsregistry.org/Part:BBa_K656013 Part BBa_K656013])

Reagents for 35µL reaction

  • 2.9 µL nuclease free water
  • 3.5 µL 10x T4 ligation buffer
  • 24 µL insert (BBa_K656013, 10 ng/µL)
  • 2.85 µL vector (pSCB3, 14.6ng/µL)
  • 1.75 µL T4 ligase

Treatment

  1. Add all reagents in order
  2. Allow to incubate for 2 hours at room temperature
  3. Denature ligase at 65˚C, store at 4˚ or -20˚
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