RT-PCR Protocol[University of Chicago]

cDNA preparation:

4-5µL RNA
3µL oligo dT primer
12µL DEPC-treated water

Incubate at 70 degrees for 10 minutes, then put on ice.

Add
8µL 5X 1st strand buffer (from Invitrogen or Gibco)
4µL 0.1M DTT
5µL dNTPs (from Ex-Taq kit)
1µL RNase inhibitor if desired

Incubate at 48 degrees for 2 minutes, then add

2µL reverse transcriptase

Incubate at 48 degrees for 60 minutes

cDNA can be frozen for later use or used immediately for PCR. You should do a no reverse transcriptase control for each sample when preparing cDNA. This will allow you to know if there is DNA contamination in your RNA samples when you do PCR.

PCR conditions:

1-5µL of cDNA
5µL 10X Ex-Taq Buffer
4µL dNTPs
2µL each primer
49µL water
0.5µL Ex-Taq

Use PCR program appropriate to the primers.