Hi I am trying to join two linear 1.2 kb fragments with overlaps. I read a protocol which says at least 1/2 of the PCR mix has to be the equimolar concentrations of the fragment. Why is so much DNA required? Second the protocol also asked not to use Fusion polymerase. Why is it so? What should be the Tm for the PCR?
Jelena Spasic
Hey,
I did recently the overlap PCR, I used Phusion DNA Polymerase and it worked quite nice. I used 80 ng of one and 80 ng of the other template. I used less primers (0,625 microliters of each from 10 micromolar stock) in 50 microliter of PCR mixture. The conditions for PCR were:
98/30 s
10 x 98/10s
57/30s
72/40s
30x 98/10s
65/30s
72/40s
70/min
My templates were around 500-700 bp (to be able to adapt the elongation).
Cheers!
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