Reverse transcription - (Nov/17/2005 )
I am trying to synthesize a cDNA of 10 kb with Superscript III reverse transcriptase, but I have problems to obtain it. Could it be too longer for reverse transcription? What could be the solution?
What temperature are you using, how long is your RT reaction, are you sure your RNA is high quality, intact and that you have loads of it? The subsequent PCR, is that one optimised? (do you PCR the 10 kb also, or just a smaller part?).
10 kb is very long indeed, but it should be possible. A person I know has once performed over 9 kb with transcriptor frome Roche.