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  • PCR Amplification of Adaptor Ligated cDNA Library (E6114)

    Protocol

    1. Mix the following components in a sterile microfuge tube: 

      DNA (3–25 ng):   variable 
      Primer 1 (50 μM stock):   10 μl 
      Primer 2 (50 μM stock):   10 μl
      LongAmp Taq:   2X 
      Master Mix:   250 μl 
      Nuclease-Free Water:   variable 
      ---------------------------------------------------------------------------
      total volume:   500 μl
    2. Aliquot 125 μl into four PCR tubes.
    3. 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
    4. Purify DNA sample on one column.