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need: info on A260-A280 absorbance measurements

Michael Benedik benedik at uh.edu
Mon Oct 14 17:44:44 EST 1996


In article <53tt16$9gc at info.service.rug.nl>
g.j.e.j.hooiveld at farm.rug.nl (Guido Hooiveld) writes:

> Hi everybody!
> 
> After performing a prep I estimate the amount and purity of my plasmid DNA sample by measuring 
> the A260 and A280. I use the ratio of A at 260 and 280 nm to get an indication of the purity of 
> my sample. 
> 
> I was told an A280/A260 ratio of 1.8 to 1.9 indicates a highly purified DNA preparation and its 
> theoretically limit is 2.0. However, I often find a ratio larger than 2.0; like 2.1 to 2.2. So, 
> is this (theoretically) value of 2.0 correct, if yes, does anybody have a suggestion why I find 
> values >2; if not, what is this value? 
> 
> And what about the factor you have to use to determine the concentration of DNA present: 
> multiply A260 with 50 to obtain the concentration (ug/ml), or use 35? Which value is correct?
> 
> Any comments would be appreciated!
> 
> Thanks in advance,
> 
> Guido
> 
> -- 
> ***************************************************************************
> * Guido Hooiveld                              |                           *
> * Groningen University Center for Pharmacy    |phone: (+)31 50 3637565    *
> * dep. of Pharmacokinetics and Drug Delivery  |       (+)31 50 3633272    *
> * A. Deusinglaan 1                            |fax:   (+)31 50 3633247    *
> * 9713 AV Groningen                           |                           *
> * The Netherlands                             |                           *   
> ***************************************************************************
> 


The value of 50 ug/ml per OD is for double strand DNA and 35 is for
single strand DNA. 

The proper ratio is 260/280, not 280/260. Recalculate and see what you
have.


Michael Benedik
Department of Biochemical Sciences
University of Houston
benedik at uh.edu



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