Quantification of DNA in presence of NADPH

Emir Khatipov ekhatipo at NOSPAMmidway.uchicago.edu
Thu Mar 29 11:25:40 EST 2001


What about
1) Gel filtration? - NAP or other brand prepacked Sephadex columns. DNA is
eluted in void volume, NADPH is retained in the column.
2) Quantitation of DNA with ethidium bromide - check Maniatis or other
reference for protocol.

-Emir


""nauduri dhananjaya"" <nauduri at hotmail.com> wrote in message
news:LAW2-F103FapNuT1FM100003be5 at hotmail.com...
> I working on microsomal activation of benzene. For the in vitro reaction,
I
> use Microsomes, NADPH (co-factor), Calf thymus DNA and benzene. After
> completion of reaction NADPH is also co-precipitating along with DNA.
Since,
> both DNA and NADPH show absorption at 260 nm, quantification of DNA is not
> possible to measure DNA concentration at 260 nm. (A conventional method of
> separating DNA from the reaction mixture by isoamyl alcohol+chloroform and
> precipitation by cold ethanol would give both DNA+NADPH.)
>
> Diphenylamine method is not also possible because perchloric acid break
both
> DNA and NADPH to produce deoxyribose moiety.
>
> I request anybody suggest me a method(s). I appreciate any easy way to
> eliminate NADPH without disturbing the DNA or simple spectrophotometric
> estimation would be great. I have no access to fluorescence
> spectrophotometer.  I sincerely appreciate for your advise. Email me
> :nauduri at hotmail.com
> _________________________________________________________________
> Get your FREE download of MSN Explorer at http://explorer.msn.com
>
> ---





More information about the Proteins mailing list