PCR Amplification with AMV LongAmp™ Taq RT-PCR Kit
We recommend 2–5 μl of the diluted cDNA product for a 50 μl PCR reaction. Mix the LongAmp Taq 2X Master Mix by inverting before use.
- Mix the following components in a PCR tube on ice:
LongAmp Taq 2X Master Mix 25 μl 10 μM forward primer 1 μl 10 μM reverse primer 1 μl 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 thinwall 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 25–45 Cycles 94°C 15–30 seconds 45–68°C 30 seconds 68°C 50 seconds per kb Final Extension 68°C 5 minutes
(When using primers with annealing temperature above 60°C).
INITIAL DENATURATION 95°C 30 SECONDS 25–45 Cycles 94°C 15–30 seconds 60–68°C 60 seconds per kb Final Extension 60–68°C 5 minutes Final Soak 4–10°C
- Analyze 5 μl of PCR products by agarose gel electrophoresis.