RT Reaction

1. Before performing the RT reaction, heat 5 ug of total RNA in a 10 uL volume at 65 °C for 5-10 minutes , then quench on ice.
2. Set up the following components in a 1.5 mL Eppendorf tube:
• 10.0 uL Heat denatured RNA
• 3.0 uL 10x PCR Buffer
• 2.5 uL 10 mM dNTPs
• 6.0 uL 25 mM MgCI₂
• 1.0 uL Random primers (1.8mg/mL)
• 0.5 uL SuperScript II reverse transcriptase
• 17.0 uL water
Warning: The use of DEPC-treated water for the RT reaction is unavailable, as DEPC will inhibit the RT and PCR reactions!
3. Leave the samples at 25°C for 10 minutes then incubate at 42°C for 1 hour.
4. Denature the cDNA at 95°C and place on ice.

PCR Reaction

1. Set up the following components in a 0.5 mL PCR tube:
• 6.0 uL cDNA product
• 1.5 uL 10x PCR Buffer
• 0.2 uL Taq polymerase
• 0.5 uL primer 1 (1.0mg/mL)
• 0.5 uL primer 2 (1.0mg/mL)
• 10.3 uL water
2. Perform PCR with 30 cycles. Each cycle includes denaturation (30 seconds at 95°C); annealing (45 seconds at 60°C); and extension (60 seconds at 72°C).