RT-PCR Protocol 1. Place 5L each RNA sample in PCR tubes, label. Pre-mix: 1L 70 M oligoDT 4L DEPC H2O 2.

Add 5L of premix to each PCR tube. 3. Incubate at 70C for 10 minutes in PCR AB machine 4. Hit pause and add 10L of appropriate reaction. Pre-mix: RT(+) RT(-) 5L DEPC H2O 6L DEPC H2O 1L RNasn 1L RNasn 1L 10x dNTPs 1 L 10x dNTPs 2L MuMLV buffer 2L MuMLV buffer 1 L RT 5. Incubate at 25C for 15 minutes. 6. Incubate at 42C for 60 minutes. 7. Set up PCR for rp49 and l(2)dtl primers. Add to specific PCR tubes. l(2)dtl rp49 2.5L 10x PCR buffer 2.5L 10x PCR buffer 2.5L 10x dNTPs 2.5L 10x dNTPs 2.5L primer forward 2.5L primer forward 2.5L primer reverse 2.5L primer reverse 0.5L Taq 0.5L Taq 14.5L DEPC H2O 14.5L DEPC H2O 8. Set PCR machine. Cycle Temperature Time 1 94C 5 mins 29 94C 30 secs 58C 30 secs 72C 1 min 10 secs Hold at 10C