PCR Amplification with AMV LongAmp™ Taq RT-PCR Kit

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.