Introduction

We recommend 2–5 μl of the diluted cDNA product for a 50 μl PCR reaction. Mix the LongAmp Taq 2X Master Mix by inverting before use.

Protocol

1. Mix the following components in a PCR tube on ice:

   LongAmp Taq 2X Master Mix	25 μl
   10 μM forward primer	1 μl
   10 μM reverse primer	1 μl
   Diluted cDNA	2–5 μl
   H2O	variable
   Total Volume	50 μl

2. Mix gently. Overlay with mineral oil if the thermal cycler lacks a heated lid.
3. The following PCR cycling conditions are recommended for 0.2 ml thinwall PCR tubes on Bio-Rad iCycler or similar thermocyclers. For other PCR tubes or cyclers, it may be necessary to modify the cycle times.

   INITIAL DENATURATION	95°C	30 SECONDS
   25–45 Cycles	94°C	15–30 seconds
   	45–68°C	30 seconds
   	68°C	50 seconds per kb
   Final Extension	68°C	5 minutes

   2-temperature PCR protocol
   (When using primers with annealing temperature above 60°C).

   INITIAL DENATURATION	95°C	30 SECONDS
   25–45 Cycles	94°C	15–30 seconds
   	60–68°C	60 seconds per kb
   Final Extension	60–68°C	5 minutes
   Final Soak	4–10°C

4. Analyze 5 μl of PCR products by agarose gel electrophoresis.