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.