RT-PCR

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).