PCR Amplification with ProtoScript^® M-MuLV Taq RT-PCR Kit

Introduction

We recommend 2–5 μl of the diluted cDNA product per 50 μl PCR reaction.

Protocol

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

   Taq 2X Master Mix	25 μl (mix well by inverting before use)
   Forward Primer (10 μM)	1 μl (final concentration 200 nm)
   Reverse Primer (10 μM)	1 μl (final concentration 200 nm)
   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 thin-wall PCR tubes on Bio-Rad iCycler or similar thermocyclers.

   INITIAL DENATURATION	95°C	1 MINUTE
   25–35 Cycles	94°C	30 seconds
   	45–68°C	10-30 seconds
   	68°C	1 minute per kb
   Final Extension	68°C	5-10 minutes

4. Analyze 5 μl of the PCR product by agarose gel electrophoresis.