DNA-Standards for quantitative PCR

Roderic Fuerst roderic at biogene.co.uk
Fri Mar 26 10:00:48 EST 1999


In article <7cqn7v$3rm$1 at mserv2.dl.ac.uk>, Thomas Pfitzner
<Thomas.Pfitzner at medizin.uni-koeln.de> writes
>I have a few questions regarding DNA-Standards for quantitative PCR. We
>have cloned our target sequence into a plasmid. What would you suggest:
>Using native or linearised Plasmid for standards ?

In my experience covalently closed plasmids without nicks are
recalcitrant in amplification when compared with linearised plasmid. The
best explanation which I have heard for this is that whilst the plasmid
duplex will be disrupted in both cases, in the case of covalently closed
plasmids the diffusion of strands apart is limited by the fact that they
are interlocked. During the subsequent cooling to annealing the
reannealing temperature of plasmid is reached before the temperature at
which primers anneal. The relatively high concentration of complementary
plasmid strands means that they reanneal before primers can anneal, thus
efficiently inhibiting PCR.

I have compared the amplification of CC plasmids with linearised
plasmids and their kinetics have been delayed by ca. 6 cycles.

The above recommendation assumes that your target is linear.

>How do you produce your linearised Standard ?

RE digest @ 250ng/ul and dilute in TE with 1ng/ul lambda as a carrier.
at this concentration of plasmid you will be able to dilute by >4 orders
of magnitude any RE buffer components, to a level where they will not
interfere with PCR.

>(We tried gel elution using QuiaExII, which seem'd to work)
>Do you recommend Carrier DNA and which type ?

Yes, for preserving template use lambda (defined sequence and easy to
get) at >=1ng/ul.

Addition of lambda to non Hot-Started reactions to 50ng/ul can also help
improve sensitivity by providing Taq and primers something to interact
with instead of each other prior to the start of cycling.

> (We used Salmon Sperm DNA
>from Stratagene which had a profound negative effect on PCR-reaction)

With such a complex genome you could have all sorts of artefactual
priming sites.

>Which method of DNA-Quantification do you prefer ? (What about your
>experience with OD at 260 nm compared to other, more sophisticated methods?)

260nm is fine - as long as all you have there is plasmid. Unfortunately
even with 230,260,280,320 measurements and the appropriate math,
polysaccharide contamination can lead to overestimation. This should not
be problem with plasmids because high quality plasmid DNA is easy to
prep using Anion Exchange.
 
>How about long-term storage of your standards ?

10mM Tris pH 8.0 
100uM EDTA

Keep your DNA at a concentration of >1ng/ul (-20oC).

Dilute fresh below this level daily, and if you have to go to <1000
copies per ul dilute as close to the time of amplification as possible.

> Does this work, or do you
>prefere to produce a new set of standards after a period of time ?

Dilute fresh below this level daily, and if you have to go to <1000
copies per ul then dilute as close to the time of amplification as
possible.


-- 
Roderic Fuerst                  E-mail: Roderic at biogene.co.uk
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