PCR of large fragments?

[John Alsobrook]

In <3836 at news.duke.edu> cjs at canctr.mc.duke.edu writes:

> >
> >I imagine that this has been covered in the past, but I would
> >like to know what parameters to change in PCR to acheive the
> >amplification of larger fragments (greater than 5 kb). Any suggestions
> >will be appreciated.
> >
> >Ken Garson
> 
> I too am interested in this...could you please post this to the NET or Ken if
> you receive some good personal replies, could forward or summarize them for us.
> 
> Caryl Schwartzbach
> cjs at canctr.mc.duke.edu


A short methods paper in Nucleic Acids Reserach 20(3):623, 1992, describes
conditions for generating PCR products in th 3kb - 6kb size range. The authors'
basic conclusion was that KCl is bad for big PCR; they use a KCl-free buffer
system, and claim to get several micrograms of 6kb product from 30 cycles,
but their template is a plasmid construct. I'm about to try their system
using genomic DNA as template. I'll try to remember to let you know the
outcome.

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