DNA QUANTITATION

dadler at milton.u.washington.edu dadler at milton.u.washington.edu
Wed Oct 2 13:54:39 EST 1991


In article <0094F789.374DC180 at aclcb.purdue.edu> muriana at aclcb.purdue.edu  
(murianap) writes:
> We are presently isolating DNA via ethanol precipitation (without CsCl 
> purification) and have gotten rid of residual RNA by RNAse digestion. 
> However, I presume that the presence of individual RNA bases would still 
> give absorbance and mask the true levels of DNA quantitation by absorbance
> at 260nm.  Am I correct? and would this be alleviated by ethanol 
> precipitation, whereby the digested bases would not be precipitated by 
> ethanol, so the DNA can be revovered again for quantitation.
> 
> Thanks in advance,
> Peter M. Muriana
>     Name: murianap
> Internet: muriana at aclcb.purdue.edu
>    Phone: 317-494-8284
I have found that the most reliable technique for DNA quantification is by  
using fluorometry - we use Hoechst 33258 and an Aminco Bowman  
spectrophotofluorometer - (see for ex. LaBarca and Paigen, Anal.Biochem, 1978).  
The technique is sensitive to about 5 ng/ul and is not effected by presence of  
RNA or proteins, i.e. can be done on crude extracts as well as purified  
samples.
--
David A. Adler                  Pathology SM-30
University of Washington        Seattle, WA 98195
"Science is nothing but trained and organized common sense"
T.H.Huxley



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