PCR of supercoils
Dr. Duncan Clark
news at hgmp.mrc.ac.uk
Fri Jul 27 03:37:48 EST 2001
In article <AB72A0E88719D511944A00508BDF89906C1C9A at lscmail1.na.lifetech.
com>, the eminent Hartley, Jim at BIOSCI/MRC Human Genome Mapping
Project Resource Centre wrote
>I would greatly appreciate any observations or suggestions on how to obtain
>the most product from PCR of supercoiled plasmids. Any side-by-side
>comparisons of enzymes or conditions would be most helpful. Thanks.
There was quite a long thread on the problem of PCRing supercoiled
plasmids in the 7700 listserv. Maybe it is worth checking the archives.
The basic argument, if I remember correctly, was that it should not be
possible due to the inability to 'unwind' the plasmid. It was argued
that the reason people can PCR off miniprepped DNA is that there is
sufficient nicked plasmid in the preps. Various labs experienced
anything up to I think 8 Ct differences between amplifying linear and
ccc plasmid standards.
I would guess a thermostable topo. would help. I wonder if the
Methanopyrus kandleri topo would help, as patented for sequencing.
The problem with the gene pool is that there is no lifeguard.
More information about the Methods