DNA absorbance readings

Tyson mediablitz at home.com
Mon Dec 3 15:00:46 EST 2001


actually it's the other way around, phenol has an absorbance maximum of
around 275nm ....so phenol contamination will cause a LOW A260/280 ratio not
a high one.  A very high A260/280 ratio, like 3 or 5 would probably be
caused by using a an inappropriate buffer....try using TE, pH7.5 ...ionic
strength and pH have dramatic effects on the 260/280 ratio.  Another
possibility is that the samples are too dilute...the sensitivity of most
specs these days is A260>0.005 .

Hope this helps,
Tyson


<calliphora at cityweb.de> wrote in message
news:3c0bcbe6.8321271 at news.cityweb.de...
> On Tue, 27 Nov 2001 15:16:51 -0500, Tina Higgins <thiggins at wpi.edu>
> wrote:
>
>
> >(High A260/A280 in DNA-readings after Phenol/Chloroform-Extraction)
> >
>
> If you don´t remove Phenol carefully after
> Phenol/Chloroforn-extraction, traces of Phenol in Your sample give a
> strong absorption at 260nm - but not at 280nm. This results in the
> reported high A260/A280-ratio. In this case You mainly measure the
> concentration of Phenol in the sample, but not DNA, so that the common
> method to estimate the DNA-concentration multiplication of the A260
> with 50 will result in a high overestimation of the DNA-concentration.
> Ernst Kiehl
> Institut für Zoologie II
> Universität Düseldorf





More information about the Methods mailing list