Cloudy DNA

Michael Kawalek kawalekm at earthlink.net
Mon Oct 4 20:52:33 EST 2004


I've seen cloudiness before and it is something I believe is polysaccaride contamination.
Phenol/choroform extractions will not remove this.  An excellent agent for removing polysaccarides
is CTAB, who's protocol is listed in the plant-DNA extraction section of Current Protocols.  You mix
in the CTAB solution with your DNA, heat for a period of time, then remove the CTAB with a choroform
extraction.  Pay attention to the warnings about the NaCl concentration.  If too low, the DNA will
coprecipitate with the CTAB.  The pH warning for the phenol also has merit.  If the pH is too low
the DNA will remain in the phenol phase.  Make sure the phenol is well equilibrated with TE.
Michael

On 23 Aug 2004 02:23:43 -0700, aaron.engelhart at gmail.com (Aaron Engelhart) wrote:

>We do some work with calf thymus DNA at high concentrations (3-7
>millimolar) in milliliter quantities for spectrophotometry/transient
>absorption spectroscopy purposes. One problem is that it seems to be
>fairly cloudy above the millimolar level. This is the cheapest source
>of DNA, so I like to use it for obvious reasons before doing
>experiments on synthetic oligos or polyG-polyC, etc, but the
>cloudiness is a problem. I have done some work with phenol extraction,
>which seems to help a bit, but with significant product loss. Every
>time I do a phenol extraction, there is a bit of whitish gunk at the
>interface. I don't understand the mechanics of this completely, or
>exactly what it is. There's some obvious product loss from leaving a
>bit of the supernatant behind to avoid taking up the impurity, but my
>understanding is that the dsDNA stays in the aqeuous phase and the
>proteins go into the organic phase. A260/280 ratio looks fine (and
>unchanged!) before and after extraction. Does anyone have a good way
>to clear up CTDNA/know what this is? TIA,
>
>Aaron




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