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.