DNA concentrations for megaprimer annealing and extension

TR via methods%40net.bio.net (by taskan4 At gmail.com)
Wed Jan 31 11:47:53 EST 2007


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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