Cleaning a contaminated primer stock

Dr. C. Dolphin cdolphin at crc.ac.uk
Sat Nov 20 06:34:39 EST 1993


>
>       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 cleanse them of the Foul Interloper
> DNA?
>
> --
> Michael
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> NPC rights activist  | Nameless Abominations are people too!
>
You could try 1) UV irradiating an aliquot of your contaminated primer stock, or
2) UV irradiating the PCR mix prior to addition of template, dNTPs, enzyme. I
was able to eliminate false positives in nested RT-PCRs by irradiating the PCR
mix (minus dNTPs, template & enzyme) in a clear 0.5ml tube for 2min in a UV
Stratalinker (Stratgene). This eliminated all false positives. More than 2/3min
will reduce product yield so the optimun irradiation time needs to be determined
empirically. Also see "Removal of DNA contamination in PCR reagents by UV
irradiation" Methods in Enzymology, 218, p381.

Colin Dolphin, Biochemistry, QMW, Uni of London. c.t.dolphin at qmw.ac.uk



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