PCR Amplification with OneTaq® RT-PCR Kit
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.
- 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
- 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. For other PCR tubes or cyclers, it may be necessary to modify the cycle times.
CYCLES Initial Denaturation
1 minute per kb
Final Extension 68°C
- Analyze 5 μl of PCR products by agarose gel electrophoresis.