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  • PCR Amplification with OneTaq® RT-PCR Kit

    Introduction

    We recommend 2–5 μl of the diluted cDNA product for a 25 μl PCR reaction. Mix the OneTaq Hot Start 2X Master Mix by inverting before use.

    Protocol

    1. Mix the following components in a PCR tube on ice:
      Reagent Volume
      OneTaq Hot Start 2X Master Mix 12.5 µl
      10 µM Forward Primer 0.5 µl
      10 µM Reverse Primer 0.5 µl
      Diluted cDNA 2–5 µl
      H2O to a total volume of 25 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 thin-wall 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

      25–40 Cycles:
      94°C   15–30 seconds
      45–68°C   30 seconds
      68°C   1 minute per kb

      Final Extension:
      68°C   5 minutes

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