DNA concentrations for megaprimer annealing and extension

[Ednot]

On Jan 31, 7:44 am, "WS" <novalidaddr... At nurfuerspam.de> wrote:
> 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

Hello

Sometime ago ,i fused 4 fragments of 90 -100 mers, i tried a gradient
in the annealing (55 60 65 °C) on a I-cycler.
I had good results with 35 cycles and a Kod Polymerase.
It´s better if you have good GC content in the overlapping region.

Good Luck.