Primer stocks that fail to PCR over time

Paul N Hengen pnh at
Tue Dec 6 13:26:03 EST 1994

Earlier I wrote:

: Some netters have noticed a loss in the ability to amplify DNA by the
: polymerase chain reaction when using the same lot of primers over a period of
: time.  On occasion, the failure of PCR amplification was directly linked
: to the primer stock going belly up for unknown reasons.
: PCR primers initially worked shortly after being purchased, but after storage
: for several months at -20 C as diluted stocks in water, these same primers
: failed to give the same amount of amplified product in identical reactions.
: Primers going bad also produced unreadable results in automated DNA sequencing
: reactions using the dye terminator chemistry combined with cycle sequencing and
: subsequent analysis on the ABI model 373A sequencer under the same conditions
: that previously gave good results.  If new primers were purchased, the reactions
: gave good results and acted as they did prior to storage at - 20 C.


This came from Andre-Denis Wright (awright at

| I read with interest your posting on primer stocks. This is the
| same problem we've been experiencing in our laboratory. This summer I was
| getting great PCR results, but for the past two months, I have not been
| able to get any amplifications. We ordered new primers, and i am using
| them with the other reagents i used with the old primers and now i am
| getting great amplifications again. We have been storing our
| concentrated primers, like everyone else, at -20 C. I can't figure this
| out.

: Dear Andre-Denis:
: The best response for explaining this problem was from Roger Aeschbacher.
: Roger Aeschbacher (teschba at suggested that some primer
: sets cause PCR failure by nonspecific priming because they are more likely to
: form primer-dimers or intrastrand secondary structures such as hairpins only
: after undergoing multiple cycles of warming and slow freezing. This would not
: happen to all sets of primers, explaining the reason why some go bad whilst
: others remain functional.  It also explains why some primers seem to work after
: a period of storage and others purchased from the same company on the same date
: do not.
: While Hot Start PCR may help reduce non-specific annealing to template DNA,
: denaturing the bad primer stock at 95 C to 100 C for 5 minutes and then quickly
: cooling it in a dry ice/ethanol bath or liquid nitrogen may help to restore
: these primers to their former selves.

| Dear Dr. Hengen,
| I took your advice about the primers and boiled two aliquots (100ul
| ea) at 100 C for 5 min before freezing quickly with liquid N2. The next
| day i thawed one of the samples and used it it a PCR reaction and
| VOILA!!! they worked. Thanks for the advice!
| Andre-Denis

Ahhhh. Excellent result! This topic will be discussed in my monthly column
in the January 1995 issue of TIBS.

author = "P. N. Hengen",
title = "Methods and Reagents - Wayward PCR primers",
journal = "Trends in Biochemical Sciences",
volume = "20",
number = "1",
pages = "???-???",
month = "January",
year = "1995"}


* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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