Spec vs Flourimeter for [DNA] ?

Derek M. Harkins harkinsd at solix.fiu.edu
Wed Dec 1 17:43:54 EST 1993

nishir at ohsu.edu wrote:
: In article <754686028snz at genesys.demon.co.uk> Duncan at genesys.demon.co.uk
: (Duncan Clark) writes:
: >In article <19931130120206.bloksber at thomashaw-at.css.msu.edu>
: bloksber at pilot.msu.edu writes:
: >
: >>Question about the Hoefer TK100 DNA flourimeter.
: >>.
: >>I have 3 samples of plasmid DNA, all are subclones of the same clone in 
: >>pBlueScriptKS- or empty vector, all double CsCl banded, look great on gels,
: >>etc.  When I measure my DNA concentration on an LKB Biochrom Ultrospec II
: >>spectrophotometer as A260, I get 1 set of readings and when I measure on the 
: >>Hoefer TK100 DNA flourimeter I get another.  The 260/280 ratios are all 1.80.
: >>The spec give a very consistent reading of 3x the flourimeter reading.  Which

: >>do I believe and why?  Can anyone offer some insight or warnings?  The 
: >>exact concentration is important to me for titrating DNA binding proteins.  
: >>Thanks in advance.  I will post a summary of important points if warrented.
: >
: >I can't give am answer either way but try the following.
: >
: >Add a set amount of bona fide DNA ( ie commercial lambda DNA etc )to your 
: >samples and reassay. The spec reading should only go increase by the extra
: >DNA. From memory the fluorimeter is using Hoescht dye staining. If I'm right
: >that is specific for DNA and DNA alone whilst the spec will pick up RNA and 
: >oligos that will not be seen by the fluorimeter and may not be visible on
: gels.
: >I would tend to believe the fluorimeter unless the addition of extra DNA still
: >gives a relative 3 fold increase in spec reading. Final thought, anyone 
: >accidently swapped your quartz cuvettes for something else?
: >
: >Happy hunting.
: >
: >Duncan 
: >-- 
: >-----------------------------------------------------------------------------
: >Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
: >GeneSys Ltd.                        | Compuserve:  100015.1406 at compuserve.com
: >-----------------------------------------------------------------------------
: We have both the 

When I first read the question I thought the same thing Duncan did--that the
spectrophotometer was reading total nucleic acids and not only DNA, as the 
fluorometer does.                                    
But then I re-read the initial question and the plasmid nucleic acids
have been spun through CsCL--therefore no contaminating RNA should be present
to skew the readings of the spectrophotometer.  I don't know what IS causing
the difference but I don't think it's RNA.  I wish I had an answer.  Duncan
has a good idea though- you could try adding the known amount of DNA,
and re-read them.  In either case I would be interested in hearing the 
results.  Good luck.



* Derek M. Harkins <harkinsd at solix.fiu.edu> | All opinions here stated, if  *
* United States Department of Agriculture   | correct and lucid, are solely *
* Agricultural Research Service             | my own.  All others you can   * 
* Miami, FL  33158    USA    (305) 238-9321 | blame on my evil twin.        *

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