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  • 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.