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.