elisabet.einarsdottir at ucmm.umu.se
Thu Jul 25 03:00:33 EST 2002
Thank you for the tips Emir!
I have tried several different DNA templates, and in various amounts
(ranging from a lot, to a few ng). The DNA should be very pure, prepared
using phenol/chloroform. I have tried 1.0, 1.5 and 2.0mM magnecium
concentrations, I have tried gradient PCR (from 44 to 68, the AT is
calculated to be ca 56), and have tried nested PCRs. The sequence to be
amplified is not so long, only around 400bp.
But I will check the GC content of the gene, and try varying the PCR
programs. If that still does not work, I'll look into the kit you
"Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote in message
news:MuA%8.11$X4.2494 at news.uchicago.edu...
> Your "ample template DNA" sounds suspicious. Did you try varying template
DNA concentration? You don't need a lot. What else except checking the
sequence and redesigning primers did you try? Magnesium concentration, other
"tricks"? How long is the sequence? Did you try amplifying a portion of the
gene (another set of primers for internal sequence)? Did you try varying a
program? Is the gene much different in GC content? Etc, etc, etc. I think
Epicentre (.com - no affiliation) sells a FailSafe PCR kit that includes
> pre-mixed cocktails that allow to screen for best PCR conditions towards
getting a desired product.
> Good luck
> "Elisabet Einarsdottir" <elisabet.einarsdottir at ucmm.umu.se> wrote in
> news:ahm5dv$inn$1 at hudsucker.umdac.umu.se...
> > Hello there!
> > This is probably a long shot, but I am wondering if there is anyone out
> > there who has been working with the MODY genes (1-5)? And if so, if they
> > have any experience in PCRing up MODY4 genomic regions. We have tried to
> > simple PCR products from the MODY4 exons (nothing fancy, just normal PCR
> > with ample template DNA), but nothing works.
> > Have no problems with the other MODY genes, have tried different DNA
> > templates, have rechecked the genomic and primer sequence, and have
> > several primerpairs, so I am running out of ideas!
> > Any ideas would be very much appreciated,
> > Elisabet Einarsdottir, PhD student
> > elisabet.einarsdottir at ucmm.umu.se
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