Cleaning contaminated primer stocks (SUMMARY)

Michael Lush mjl17 at mbuc.bio.cam.ac.uk
Fri Nov 26 05:15:44 EST 1993


	Last Friday I posted a plea for help Re: Contaminated 
Primer stocks.  I recieved lots of email help which I have edited 
down a bit and am now posting with a big thankyou to everyone who 
sent advice....

	Also just as a little postscript the primer stocks
wern't contaminated (praise be!!),  it was probably one of
the buffers...   Anyway thanks again for the advice,  I just hope 
I never need it!

>	By dint of hard work and inexperience our primer stock
>solutions have become contaminated, probably with a 6kb DNA fragment,
>rarther than giveing them a 1 minute silance and a Viking funeral
>can anyone suggest a good way to clense them of the Foul Interloper
>DNA?

From: sbhattac at crc.ac.uk (Dr. S. Bhattacharya)
Date: Sun, 21 Nov 93 14:26:35 GMT

try uv light, cheers shoumo
-------------------------------------------------------
Shoumo Bhattacharya		
MRC Molecular Medicine Group
Royal Postgraduate Medical School
Hammersmith Hospital, LondonW12 0NN UK
Tel:081-7435566 ext.2343
Fax:081-7498341
-------------------------------------------------------

From: kong1 at husc.harvard.edu
Organization: Harvard University Science Center

Hi there!

Depending on how long the primers are and  how many you have, a possible
way yo can purify the buggers are by spin concentrators that have ~100kD
limit [eg. Amicon100].  Amicons have these concentrators that hold max 
of 500 microlitres, and around here cost ~$63 US. They are quite nifty and
can be used in the microcentrifuge.  If you have bigger volumes, there 
are also bigger concentrators.....

Cheers!
Steph

From: "JONG, SONG-MUH J" <SJJ at icbr.ifas.ufl.edu>
Subject: RE: Cleaning Contaminated Primers


How about running the primer stock on a polyacrylamide gel
and cut out the oligo band.

From: GARCIA02 at swmed.edu
Status: OR

you may try using a centricon (amicon) spin device choosing a cut-off 
that will let your primers go through but not your contaminating DNA.
good luck
Leon 


From: coady at ere.umontreal.ca (Michael Coady)
Organization: Universite de Montreal

Dear Michael,
	A few firms such as Millipore and Ambion, sell small filter
units that fit into microfuge tubes and allow size-selection of
nucleic acids.  The ones that I have work pretty well, and they 
have a cut-off limit of 100,000 daltons, which should work pretty
well at separating the primers from the 6 kb product.  Mind you,
the Viking funeral sounds like more fun....

Mike
-- 
Michael J. Coady
COADY at ERE.UMONTREAL.CA
The opinions expressed above are solely those of the author and do not,
in any way, shape or form, represent the Universite de Montreal.


From: George W Chacko <gchacko at magnus.acs.ohio-state.edu>
Organization: The Ohio State University

Use a 100,000 cut off spin filter and UV zap the stocks for a minute
or so.

GC

From: sbhattac at crc.ac.uk (Dr. S. Bhattacharya)

try uv light, cheers shoumo
-------------------------------------------------------
Shoumo Bhattacharya		
MRC Molecular Medicine Group
Royal Postgraduate Medical School
Hammersmith Hospital, LondonW12 0NN UK
Tel:081-7435566 ext.2343
Fax:081-7498341
-------------------------------------------------------

From: WILLIS at bcrssu.agr.ca

	You might simply try putting your primer solution through a spin
column (eg. Millipore MC or equivalent). Pick one with a suitable cutoff
so your contaminant remains behind and your primer goes through. If you 
only have one or a few contaminated samples I suggest you phone your local
supplier and ask for a free samples (they will almost always send you a 
few to try). This way you don't have to buy 50 or more and waste your 
money (they are quite expensive). Hope this helps. Les.

From: txpljfg at uabcvsr.cvsr.uab.edu

Once contaminated, we have never been able to clean up primers
sufficiently to be used again.  Break out the bugle and bury them at
sea.

Don't feel bad, this seems to happen to many people when they first
start using PCR, and before they appreciate how unbeleivably it is to
contaminate anything with anything.

==============================================================================
James F. George, Ph.D.              "Science is simply common sense at its
Department of Surgery                best -- that is, rigidly accurate in
University of Alabama at Birmingham  observation, and merciless to fallacy
205-934-4261 voice                   in logic"   --Thomas Huxley
txpljfg at uabcvsr.cvsr.uab.edu
===============================================================================

From: wra at biochem.dental.upenn.edu (Bill Abrams)
Organization: University of Pennsylvania

Ultrafiltration was suggested and that should work. An alternative would be
to use strong anion exchange chromatography or reverse phase chromatography
to repurify your primer.               

Bill Abrams, Ph.D.
University of Pennsylvania
School of Dental Medicine
wra at biochem.dental.upenn.edu
-- 
Michael
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
NPC rights activist  | Nameless Abominations are people too!
                   



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