Hi, I have never tried to reverse transcribe on such a long distance but, from my experience on regular RT, SuperscriptIII works better than SuperscriptII. In addition, based on extensive testing ~3 years ago by a colleague from my former lab, "LA Taq" from Takara was the best polymerase for the amplification of long (5-10kb) fragments.
Sébastien On Tue, Jul 13, 2010 at 5:32 PM, Peter Ellis <pj...@cam.ac.uk> wrote: > On 13/07/2010 16:16, B.Rama chandran wrote: > >> Dear all, >> I'm trying to make 6.3kb reverse transcript from mRNA. I tried >> both poly dT primer as well as gene specific primers for reverse >> transcription. But I didn't get 6.3kb product. So tried to amplify 'C' >> terminal half (3.1kb). But I didn't get that also. I have used Superscript >> II fro reverse transcription and Phusion polymerase for PCR. If anybody >> have >> any suggestion please give me. >> > > Without more information, we can't give you specific help. Here are some > possibilities. > > 1) Your RNA may be degraded - try a formaldehyde agarose gel to check the > integrity of the sample. > > 2) There may be a problem with the PCR reaction: run a control PCR on a > working template (e.g. plasmid-specific primers on plasmid DNA) to check > that your PCR enzyme / buffers / mastermix is working. > > 3) There may be a problem with the cDNA synthesis: run a control RT-PCR > using primers for a ubiquitous gene such as beta actin. > > 4) Is your gene of interest actually expressed in your mRNA sample? Can > you amplify short fragments, say ~200 bp in length? > > Peter > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
