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.