PCR CONTAMINATION

Brian Hjelle bhjelle at vaxine.unm.edu
Fri Apr 24 08:55:05 EST 1992


In article <k?o.703966394 at soma.umdnj.edu> byrne at soma.umdnj.edu (Bruce Byrne) writes:
>
>followup to massive contamination problems:
>
>1.  Get someone else to synthesize your primers for you; buy or trade 
>services.  Working with PCR products in the same room where any of the 
>sensitive reagents that go into the reaction mix is very touchy.  When
>you get the new oligos, set up the reaction mixtures in a colleague's
>lab.  

Yes! Yes! Trying to purify your own oligos in the lab is a prescription
for disaster. We buy ours from Oligos Etc, which are pretty good and
run about $4.50 per base, no setup charge, and no purification
charge (1 umol scale is $6.50/base). Maybe this is more expensive
than purifying it in your own lab, but the first, what, 100 contamination
episodes gets pretty expensive too :-(.
>
>2.  Decontaminate pipettes, glassware, evaproating equipment, etc. with
>1 N HCl or dilute clorox.  Check out minimim PCR carry-over protocols   
>and follow them with great care.  See, for instance Nature 339:237-238
>(1989)

Does anyone know if dilute clorox really destroys DNA? I thought so
for some time but then ran into a paper on forensic use of DNA by
the FBI that showed some pretty good Southern blots of DNA that
had been deliberately exposed to bleach. Reference (probably)
available upon request...

1N HCl sounds reasonable, assuming a depurinated template is a
poor template.

We rely a lot on flame (for scissors, Pasteur pipets, etc), and
UV light (Pipetmen, other reusables) in addition to thoroughly
segregating pre-PCR from post-PCR space and equipment.

Brian



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