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  • Nick Translation and Amplification of Adaptor Ligated DNA (E6260)

    Protocol

    1. Mix the following components in a sterile microfuge tube:
      Adaptor ligated DNA 30 μl
      Primer 1 (50 μM stock) 5 μl
      Primer 2 (50 μM stock) 5 μl
      LongAmp Taq 2X Master Mix 125 μl
      Sterile H2O 85 μl
      Total volume 250 μl
    2. Aliquot 125 μl into two PCR tubes.
    3. PCR cycling conditions:
      Cycle step Temp Time Cycles
      Nick Translation 72°C 20 min 1
      Initial denaturation 95°C 5 min 1
      Denaturation
      Annealing
      Extension
      95°C
      62°C
      70°C
      15 sec
      15 sec
      1 min
      10–15*
      Final extension 70°C 5 min 1
      Hold 4°C 1
      *Avoid over-amplification to optimize the number of unique molecules.