Primer Degradation?

Kevin Gilbride

unread,

Sep 30, 1997, 3:00:00 AM9/30/97

to

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
gilb...@bu.edu

Z. Zhao

unread,

Oct 1, 1997, 3:00:00 AM10/1/97

to

You suspect that degraded primers were used as primers and
the original oligos were used as templates in your PCR. How
long is the largest PCR product in your water controls? If
it is longer than 25 bp, I would guess that your PCR was
contaminated, which might be in your primer stock.

Pascal Mertens

unread,

Oct 1, 1997, 3:00:00 AM10/1/97

to

In article (Dans l'article) <gilbride-300...@ppp-10x1-5.bu.edu>,
gilb...@bu.edu (Kevin Gilbride) wrote (écrivait) :

> I need some advise regarding the stabilty of primers.

Not directly linked to your problem:
long term (in)stability: a (previously good) primer stored 6 years at -20
as a 100X stock and reused in sequencing does not work anymore.
It seems to be the same for a plasmid (maxiprep, 6 years old): no more
readable sequence (tested of course with a new primer that works on a new
DNA).

Pascal

--
Pascal Mertens
Laboratoire d'Immunologie et Microbiologie
URBM-FUNDP
61 Rue de Bruxelles
5000 Namur, BELGIUM
Phone: 32 81 724438
Fax: 32 81 724420

Pam Snyder

unread,

Oct 3, 1997, 3:00:00 AM10/3/97

to

In article <gilbride-300...@ppp-10x1-5.bu.edu>, gilb...@bu.edu
(Kevin Gilbride) wrote:

"clean" primers are stable. Do you store master (concentrated) stocks
separate from your working stocks? Then anytime you see something odd
you go back to you masters and see where the problem is starting.

It also greatly limits the # of times primers master stocks are
frozen/thawed/pipeted. We make multiple working stocks from the masters
in 1 ml vols, and further aliquots these to 200ul aliquots and use them
one at a time. If we have any reason to suspect an aliquot, we pitch it.
We have masters that have remained clean and intact for 8 years.

Why do you think that contamination is not a problem?

Paul Rohde

unread,

Oct 4, 1997, 3:00:00 AM10/4/97

to

snyd...@osu.edu (Pam Snyder) writes: > In article <gilbride-300...@ppp-10x1-5.bu.edu>, gilb...@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
> > gilb...@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?