mRNA RT with ProtoScript® M-MuLV Taq RT-PCR Kit (NEB)

Published on Author Giorgio Gilestro
  1. 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
  2. Heat for 5 minutes at 70°C. Spin briefly and promptly chill on ice.
  3. 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
  4. 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.
  5. Inactivate the enzyme at 80°C for 5 minutes.
  6. 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.)

  1. 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
  2. Mix gently. Overlay with mineral oil if the thermal cycler lacks a heated lid.
  3. 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
  4. Analyze 5 μl of the PCR product by agarose gel electrophoresis.