Spec vs Flourimeter for [DNA] ?

nishir at ohsu.edu nishir at ohsu.edu
Wed Dec 1 15:43:41 EST 1993


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 
>-- 
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>Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
>GeneSys Ltd.                        | Compuserve:  100015.1406 at compuserve.com
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We have both the 



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