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  • PCR Amplification (E6160)

    Protocol

    1. Mix the following components in a sterile PCR tube:
      RT reaction mixture: 20 μl
      LongAmp Taq® 2X Master Mix: 25 μl
      SR Primer F3: 2.5 μl
      SR Primer R3: 2.5 μl
      ------------------------------------------------------------
      Total volume: 50 μl
    2. PCR cycling conditions:.
      Cycle step Temp Time Cycles
      Initial denaturation 94°C 30 sec 1
      Denaturation
      Annealing
      Extension
      94°C
      60°C
      65°C
      10 sec
      30 sec
      15 sec
      12*
      Final extension 65°C 5 min 1
      Hold 4°C    
      *Amplification conditions may vary based on RNA input amount, tissue, and species. This protocol was optimized using 5 μg of total RNA from human brain. The number of PCR cycles can be adjusted if clear and distinct bands are not observed in the gel image. However, only run between 12 and 15 cycles.