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.
- 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
- Mix gently. Overlay with mineral oil if the thermal cycler lacks a heated lid.
- 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
- Analyze 5 μl of the PCR product by agarose gel electrophoresis.