Reverse transcriptase PCR - RTPCT (Oct/11/2005 )
I wish to know does any body has amplified 10 kb cDNA from a Viral RNA. If yes i would like to know that enzyme with high fidelity.
Thanks in advance
We're pretty succesfull in using SuperscriptIII one step with platinum taq high fidelity up to 6 kb (haven't tried longer). If your copy number is high enough, it should be possible.
You can also try transcriptor RT from Roche and then use Expand long template (also from Roche). Some people have been succesfull using that one on mRNA from about 9 kb.
Just try some enzymes, make sure your RT-step is performed at a rather low temperature (f.i. SSIII one step normally goes perfect @ 55, but for really long template I go down to 55) because viral RNA tends to be not so stable.
which primer (poly A, random??) do you use for rt pcr of long mRNA?? i never get bands of the right size, they are much shorter to be precise and i don't know the reason (primer, rt pcr protocol?)