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  • Control Reactions with ProtoScript® M-MuLV Taq RT-PCR Kit

    Protocol

    1. The following control reactions can be used to examine the quality of kit components and RT-PCR products produced by the kits. The positive control reaction should give a 327 bp fragment, and no product is detectable in the -RT Reaction (Figure 3). If a PCR product is detected in the -RT control reaction, it is due to either the contamination of genomic DNA or a carry-over PCR product.
        Positive Control -RT control
      10X RT Buffer 2 μl 2 μl
      Murine RNase Inhibitor 0.5 μl 0.5 μl
      Rat Liver Total RNA (500 ng/μl) 1 μl 1 μl
      dT23 VN (50 μM) 2 μl 2 μl
      dNTP mix (2.5 mM) 4 μl 4 μl
      M-MuLV Reverse Transcriptase 1 μl -
      Nuclease-free H20 9.5 μl 10.5 μl
      Final volume 20 μl 20 μl
    2. Mix well by pipetting and incubate at 42°C for one hour.
    3. Inactivate the reverse transcriptase by heating at 80°C for 5 minutes.
    4. Dilute the cDNA by adding 30 μl H2O, and add the diluted DNA to the following PCR reaction:
      Taq 2X Master Mix 25 μl
      GAPDH Primer Set (10 μM) 1 μl
      Diluted cDNA 2 μl
      H2O 22 μl
      Total Volume 50 μl

      The following PCR cycling conditions are recommended:
      INITIAL DENATURATION 94°C 2 MINUTES
      30 Cycles 94°C 30 seconds
      55°C 15 seconds
      68°C 30 seconds
      Final Extension 68°C 5 minutes
    5. Analyze 5 μl of the reaction on a 1% agarose gel, stained with ethidium bromide.