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
