cDNA synthesis error - (Apr/28/2011 )
I started doing cDNA synthesis, but i made an error, which i don't know if it influenced cDNA synthesis in the end..The protocol I follow goes as follows:
1. Combine 1 μg total RNA and molecular grade water to 10 μl final volume.
2. Add 1 μl OligodT.
3. Add 1 μl dNTP mix.
4. Incubate at 65°C for 5 min.
5. Place tube on ice for 2min.
6. Add 4 μl 5X Buffer, 2 μl DTT and 1μl RNAseOut.
7. Incubate at 42°C for 1 min.
8. Add 1μl SuperScript II RNase H- Reverse Transcriptase.
9. Incubate at 42°C for 60 min.
10. Incubate at 70°C for 15 min.
My error was at step 4. I accidently incubated at 42 for 1.5-2 min, then I realised and I changed it to 65 for 5 min. Does anyone knows the concequences of my mistake?
i think that is just an annealing step (poly-A to oligo-dT) so there should be no significant problem.
yes, it's the annealing step indeed, but i was not sure if my mistake would influence the cDNA synthesis efficiency somehow..Thanks for your reply