- Make the RNA/primer/dNTP mix by combining the following components in a sterile RNase-free microfuge tube:
Total RNA 1–10 μl (1 ng–1 μg) Primer dT23VN 2 μl dNTP Mix 4 μl nuclease-free H20 variable Total Volume 16 μl
- Heat for 5 minutes at 70°C. Spin briefly and promptly chill on ice.
- Add the following components to the 16 μl RNA/primer/dNTP solution and mix well by pipetting up and down:
-RT control 10X RT Buffer 2 μl 2 μl Murine RNase Inhibitor 0.5 μl 0.5 μl M-MuLV Reverse Transcriptase 1 μl - Nuclease-free H20 0.5 μl 2.5 μl Final volume 20 μl 20 μl
- Incubate the 20 μl cDNA synthesis reaction at 42°C for one hour. If random primers are used, an incubation step at 25°C for 5 minutes is recommended prior to the 42°C incubation.
- Inactivate the enzyme at 80°C for 5 minutes.
- Bring the reaction volume to 50 μl with water. The cDNA product should be stored at -20°C
PCR Amplification (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.