DNA concentrations for megaprimer annealing and extension

TR via methods%40net.bio.net (by taskan4 At gmail.com)
Wed Jan 31 12:46:55 EST 2007


I forgot, I did it one and I used 50 ng of each overalpping fragment
in 25 ul (for the first 5 cycles)

> I think this is called fusion PCR. You just mix the two
> overlapping fragments and do something like 5 PCR cycles
> without primers in, lets say, 25 ul. Then add another 25 ul
> of PCR mix containg the primers at 2x concentration and no
> template, and go for 25 more cycles.
>
> Have fun
>
>
> >
> > Dear experts,
> >
> > I would like to anneal two partially complementary pieces of DNA
> > obtained by pcr  (about 500 bp each with a 50bp overlap) and extend
> > them (adding dNTP and Pol). So each strand will compete for the
> > complementary strand an the other strand with the
> overlapping region.
> > Is there a concentration range where this might work best?
> >
> > Next step would be a PCR amplification with end-to-end primers.
> >
> > Your input is welcome!
> >
> > Best regards,
> >
> > WS



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