Getting full-length transcripts: PCR hell

as nospam at spam.dk
Sun Mar 6 10:35:31 EST 2005


"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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