PCR of supercoils-observations

Hartley, Jim Jim.Hartley at invitrogen.com
Fri Aug 3 08:24:16 EST 2001

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.

Jim Hartley

James L. Hartley, Ph.D.
Invitrogen, Inc.
PO Box 6482
9800 Medical Center Drive
Rockville, MD 20849-6482
Tel. 301-610-8337
Fax 301-610-8371
jhartley at lifetech.com


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