PCR of supercoils-observations
Dr. Duncan Clark
news at hgmp.mrc.ac.uk
Fri Aug 3 11:00:11 EST 2001
In article <AB72A0E88719D511944A00508BDF89906C1CD2 at lscmail1.na.lifetech.
com>, the eminent Hartley, Jim at BIOSCI/MRC Human Genome Mapping
Project Resource Centre 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.
Nice detective work.
The only other thing I can think of is to stick in some alkali (10mM
NaOH final to permanently alkaline denature) and then use the PCR buffer
(or one with a slightly increase Tris concn.) to neutralise. A bit like
Biotechniques Vol. 18. No 3, March 95, p376 but avoiding the ETOH
pptation. Maybe one can just resuspend a plasmid mini-prep in dilute
alkali rather than TE, leave for a few mins at room temp neutralise and
then hopefully all subsequent PCRs will work.
Your results indicate that for your test plasmid supercoiling (which is
presumably what you are disrupting at 98C) is the problem as it causes
the plasmid to not denature at a normal denaturation temp. I will try
98C for 5mins next week as I have a plasmid with an insert that is a
real pig to PCR. The 2kb insert is 70% GC and PCRs fine from xsomal but
in a column purified plasmid prep. it really plays up. I haven't yet
tried linearising and then PCRing. The prep cuts fine with RE's so I'm
guessing there isn't any inhibitor of the PCR present in the prep.
Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR:
Optimisation, Probe Technology & Future Systems
4-5 September 2001
King Alfred's College, Winchester, UK
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