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.