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


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


    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.
      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.