Getting full-length transcripts: PCR hell

Emilie C.Emilie at gmail.com
Mon Mar 7 04:30:52 EST 2005


I gained sequence information by performing RACE PCR, so I expect that
the primer are designed to exon sequence only [or am I wrong?]. For
the ORF, looking at my RACE contig and related protein, it shouldn't
exceed 3.5 Kb (hopefully!).

I also tried to include co-solvent in my PCR reaction [poly mate from
Bioline, and likes] without any success.
I'll give betaine and DMSO a go.


"as" <nospam at spam.dk> wrote in message news:<crFWd.347$IS7.120 at news.get2net.dk>...
> "Emilie" <C.Emilie at gmail.com> skrev i en meddelelse 
> news:e1fd95c3.0503050939.49e452d2 at posting.google.com...
> > Hello,
> >
> > I am working on fungal genes. They are long transcript, one is 2.6kb,
> > the other 3.5 Kb, both with a GC% around 67.
> > I am doing RT with Thermoscript, tried a range of temperature. I tried
> > multiple polymerases [with or without proofreading activity], with
> > different Mgcl2 gradient, DNTPs concentration, template
> > concentration...
> > I tried various PCR programs, including touchdown, nested PCR etc...
> > I also looked at primer design, tried to improve that.
> > I am only successfull at reamplifying small portions of my genes, but
> > not the whole thing. My controls are ok, as well.
> > Does it sounds familiar to you? If you have any advice, please let me
> > know, because I am running out of ideas!
> >
> > Thanks!
> > -------
> > Emilie
> > Department of Biological Sciences, Warwick University
> > c.emilie at gmail.com
> 
> Maybe the genes are wrongly annotated, so that your primers are in an 
> intron, or maybe the ORF is much bigger than you expect?
> I have been using Phusion from Finnzymes (where other polymerases have 
> failed) on difficult templates (no affiliation)
> Regards AS



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