Primer dimer mystery

Gregor Bucher st2153 at zi.biologie.uni-muenchen.de
Mon Apr 3 10:02:02 EST 2000


>I wanted to have a DIG Probe (with the deg Primers), which didn't work
>when I took genomic DNA as template. I cut out the PCR band from the
>first rxn and used it as a template with the Expand Pol from my DIG
>labeling kit. I got a product AND the primer dimers were gone (mostly)!

In genomic DNA you have many places where degenerate primer could prime
(if you´r not working with the absolute optimum and even then).
Furhtermore you need more cycles to get the same amount of product because
in e.g. 5 ng of genomic template the wanted sequence is present only
rarely. And the more you amplify the larger the fraction of nonspecific
product will be. If you use a purified PCR-template every bit of DNA is
template - this makes it dificult for the primers to misprime and do their
primer dimer stuff. 

THat´s why for a amplification from a excised band you only need to tip
your pipete tip once in the template and once in your reaction (instead of
pipeting 0,1 ul or less) and 25 cycles in order to get your product. With
genomic DNA you need 5 ng or so and 30 cycles.


this is in my humble opinion the mystery

Gregor
-- 
Gregor Bucher, Zoologisches Institut der LMU München
0049/89/5902/444
st2153 at zi.biologie.uni-muenchen.de
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