PCR of supercoils-observations
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Fri Aug 3 19:31:16 EST 2001
Jim.Hartley at invitrogen.com ("Hartley, Jim") wrote:
>I recently posted a question about improving the efficiency of PCR of a
>plasmid template. The only treatment that really worked well was to preheat
>the DNA in TE, 98 C for 5 min, then cool and add the rest of the components
>and go straight to cycling. The same heating in amplification buffer was
>not effective. I estimate the yield was about 1.8 fold per cycle, 400 fold
>in ten cycles. This yield was approximately the same as the same plasmid
>linearized. Pretreatment with topoisomerase (in amplification buffer) did
>not help. Several different enzyme cocktails did approximately the same
>(YieldAce and Pfu Turbo from Stratagene, ThermalAce from Invitrogen). Added
>magnesium did not help. Several amounts of the enhancer PCRx did not help.
>We'll do a little more work on timing and cooling, if we see anything
>striking I'll post.
It's an interesting observation and something that makes sense (supercoiles
being topologically not a good substrate), but estimates liker 1.8X/cycle,
400X/10 cycles simply do not make any sense because after first couple of
cycles your template is effectively not a supercoil at all.
IMHO, if you are to make something resembling science about this issue,
you'd have to present real-time amplification graphs together with
their dependence on initial template concentration and some important
implications of it. (Say, if this difference, with regard to a final product
yield, only exists between 1/10000th and 1/1000th part of miniprep -
then I doubt anyone cares).
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