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  • LongAmp™ Taq 2X Master Mix Protocol (E6060)

    Protocol

    1. Mix the following components in a sterile microfuge tube: 
      Size Selected DNA (≤100 μl ):   variable 
      Primer 1 (50 μM stock):   10 μl 
      Primer 2 (50 μM stock):   10 μl 
      LongAmp Taq 2X Master Mix:   250 μl 
      Sterile H2O:   variable 
      -----------------------------------------------------------------
      Total volume:   500 μl
    2. Aliquot 125 μl into four 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
      2–10*
      Final Extension 70°C 5 min 1
      Hold 4°C  1

      *Avoid over-amplification to optimize the number of unique molecules.

      Cycling suggestions:
      For starting DNA quantities of: Cycles
      10 ng–100 ng 10 cycles of amplification
      100 ng–1 ug 6–8
      1 ug–2 ug 4–6
      2 ug–5 ug 2–3
    4. Purify sample on a DNA purification column, elute in 50 μl of water or EB buffer.