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:
|OneTaq Hot Start 2X Master Mix
|10 µM Forward Primer
|10 µM Reverse Primer
||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.
95°C 30 seconds
94°C 15–30 seconds
45–68°C 30 seconds
68°C 1 minute per kb
68°C 5 minutes
- Analyze 5 μl of PCR products by agarose gel electrophoresis.