isolation of small DNA fragments

Jeff Smerage smerage at cmg.health.ufl.edu
Fri Mar 22 16:50:50 EST 1996


In article <CDMENU.22.314E66F8 at ccs1.cc.monash.edu.au>,
CDMENU at ccs1.cc.monash.edu.au wrote:
> 
> Hi Netters,
> l was wondering if anyone can help me with the following problem. l need to 
> isolate small DNA fragments (110-300bp) that are derived from the
digestion of 
> a PCR product. l have tried etOH precipitation as well as butanol 
> precipitation of the digest, but when l read the OD260 on the spectro to 
> determine the DNA concentration, l get a very low reading. l don't understand 
> how l could be losing so much DNA through the precipitation. The OD readings 
> are still really low even when l pool several digests together. Can any tell 
> me if there is a way of isolating the DNA without a significant loss? l can't 
> use Geneclean to isolate the DNA, since l will need to DIG label the
fragments.
> l would be grateful for any advice. Thanks!!
> 
> Jackie C

The molarity of your DNA fragments may be high but the number of µg will
be low due the small size of your fragments.  Even when using a
microcuvette I have a hard time quantitating such small fragments of DNA
accurately.  When I need to quantitate small DNA fragments I run them on a
gel next to a size standard of known concentration.  Then I convert the
weight of size standard to moles of DNA per band.  You can then estimate
the number of moles of DNA in your band and convert this to µg.

I hope this helps,

Jeff

-- 
Jeff Smerage
University of Florida
Smerage at cmg.health.ufl.edu



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