PCR Amplification (E6120)

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 F1: 2.5 μl
    SR Primer R1: 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.