Primer Degradation?

Paul Rohde P.Rohde at mailbox.uq.edu.au
Sat Oct 4 06:30:30 EST 1997


snyder.9 at osu.edu (Pam Snyder) writes: > In article <gilbride-3009971316320001 at ppp-10x1-5.bu.edu>, gilbride at bu.edu
> (Kevin Gilbride) wrote:
> 
> > I need some advise regarding the stabilty of primers. I am using a primer
> > set which consists of a 25 and 22 mer oligos with a balanced AT / CG
> > content. My main concern is a laddering of bands appearing in the dH2O
> > control lanes. It is not a contamination, my other primers are free of
> > this problem, including Beta Globin and Actin sets. My water source
> > remains constant and when I have tried other sources the problem persists.
> > I have tried storage at 4º, -20º and -80º avoiding multiple freeze thaw. 
> > The primers were synthisized by Oligo's Etc. they work initially upon
> > arrival but soon after the waters show these bands, it wouldn't be that
> > bad but they are in right in the range of which my positive bands appear.
> > Any advise or comiseration would be welcome.       
> > 
> > 
> > Thanks
> > 
> > KJ Gilbride
> > Mallory Institute of Path.
> > BUSM
> > gilbride at bu.edu


As mentioned before, it could be contamination, slight conatmination
is just one step up from no cantamination so it may not show with all
primers (especially when contaminated with a single vector or PCR
product.)

But if you are getting a ladder on your -ve control only, this is my
educated judgement:

This is an artifact from primer dimers.  And a ladder is occuring because
a PCR product itself is acting as a primer (megaprimer), so your obtain
PCR products that get bigger and bigger, and all stages up to it: a
ladder.

Check your primers carefully for self annealing sites.  And try raising
the annealing temps and seperatly & together try lowering the Mg++.  Also
try a hot start PCR.  Do this on a good sample and -ve control

Why is it occuring in just a -ve control?  The primers have nothing to
do there so theres no competion.

Is this answer Ok?



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