Home Protocols PCR Amplification with OneTaq® RT-PCR Kit

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.
    CYCLE STEP
    		TEMP
    		TIME
    		CYCLES
    Initial Denaturation
    		95°C
    		30 seconds
    		1
    Denaturation
    Annealing
    Extension

    		94°C
    45-68°C
    68°C
    		15-30 seconds
    30 seconds
    1 minute per kb
    		25-40
    Final Extension 68°C
    		5 minutes
    		1
  4. Analyze 5 μl of PCR products by agarose gel electrophoresis.