i ve experience the same issue sometimes back. my initial problem solving approach was the same as what was suggested by B.Ramachandran. i ve even tried several brands of reverse transcriptases and found that there was a significant difference between them in thier efficacy. invitrogen kit was the most priscise. but this was not sucessfull to the level i expected. then tried to match the primer sequence to different regions of the disired mRNA. thus i played round with nearly 6 primer pairs without sucess. finally, i designed a primer set so that one of the pair complement to the utr of the mrna and it was a sucess. this indicated that there is a certain degree of mismatch between some sequences available in the data bases and the samples we are working with. in my case it was a indica rice variety where data base is consist of sequences of japonica.
hope this experience will be useful in finding a formidable solution. cheers Prasad ---------- Original Message ----------- From: Peter Ellis <[email protected]> To: [email protected] Sent: Tue, 13 Jul 2010 22:32:21 +0100 Subject: Re: 6.3Kb mRNA reverse transcription > 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 > [email protected] > http://www.bio.net/biomail/listinfo/methods ------- End of Original Message ------- _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
