UV treatment of PCR contamination

Linn, Charles Ernest linn at utkvx.utk.edu
Wed Dec 15 23:05:00 EST 1993


In article <2e5hah$bti at news.bu.edu>, rocket1 at bu.edu (Richard Near) writes...
>I am interested in decontaminating PCR
>reagents with UV light using a UV oven
>ordinarily used for crosslinking.  I've
>been told that only one-two minutes is
>required since these UV boxes have sharply
>defined wavelengths.  I would like to know
>others' experiences with UV treatment to
>eliminate contamination and how much time/
>exposure is needed?  
>  Also any interesting tales as to sources
>of contamination would be useful or at least
>"fun" to hear about.
>  Another question.  Are areosol resistant
>pipet tips themselves, effective in 
>preventing contamination.
> 
>Thanks
> 
>Rick
>rocket1 at acs.bu.edu
> 
> 
Stratagene will gladly send you their protocol for using their UV Stratalinker
for decontamination of PCR components.  I have several references concerning
PCR decontamination (not only with UV) at work and will E-mail them to you.

Tales of contamination sources, let's see.  The one that comes most to mind is
the DNA that comes withevery DNA polymerase.  Because DNA polymerase is a DNAA
binding enzyme, it is IMPOSSIBLE to remove all DNA during the purification
process.  Almost all the available polymerases are grown up using E. coli, so
if you are amplifying any bacterial DNA with any sort of sensitivity using
primers that aren't extremely specific, you will get conflicting amplification
from the DNA PRESENT IN THE ENZYME.  Perkin-Elmer's LD enzyme is the only one
that is actually tested for the presence of contaminating DNA, and their QC
protocol (which they will provide to you) can only assure that a normal assay
will contain fewer than 10 copies of 16S bacterial DNA. With the average
bacterium having 7 copies per cell, I'm not sure why there are some reports of
detecting 10 or less cells with non-specific 16S primers.  In my experience,
this is probably wishful thinking.

With that in mind, does anyone know of DNA polymerases grown in anything but a
bacterial strain?




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