First Strand cDNA Synthesis with ProtoScript® M-MuLV Taq RT-PCR Kit
Proper precautions should be used to avoid ribonuclease contamination. This includes the use of autoclaved tubes, baked glassware, ultra-pure solutions, sterile pipette tips and latex gloves during manipulations (3).
Thaw system components and place on ice. The 10X RT Buffer can be warmed briefly at 42°C and vortexed to dissolve any precipitate. (Note: It is important to set up a -RT control reaction (no reverse transcriptase) to insure there is no DNA contamination).
- 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 1.5 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.