Nick Translation and Amplification of Adaptor Ligated DNA (E6260)

Protocol

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

   Adaptor ligated DNA	30 μl
   Primer 1 (50 μM stock)	5 μl
   Primer 2 (50 μM stock)	5 μl
   LongAmp Taq 2X Master Mix	125 μl
   Sterile H2O	85 μl
   Total volume	250 μl

2. Aliquot 125 μl into two 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	10–15*
   Final extension	70°C	5 min	1
   Hold	4°C	∞	1

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