A260 or Fluoromete ??? Which is correct

Shaun Tyler styler at HPB.HWC.CA
Mon Mar 18 19:03:15 EST 1996

We have recently started using a fluorometer (from Hoeffer) to quantitate 
our DNA and often find HUGE discrepancies between our spec readings and 
those of the fluorometer.  We work with a wide range of bacteria and are 
using a standard phenol-chloroform extraction to purify the DNA (I guess 
NA would be more appropriate).  The A260/A280 ratios look good (1.8 - 
2.0) but according to the A260's the concentration should be much higher 
than what the fluorometer gives us.  Our preps are resuspended in TE + 
RNase so I had at first contributed the difference to digested RNA in the 
prep adding to the A260 reading.  However we have found that the 
differences in concentration can be more than 1000 fold and I am starting 
to question if RNA copurifying with the DNA can explain the difference.  
For years I have quantitated DNA by A260 and have trusted the results but 
I am now starting to question if my faith has been misplaced.  If you 
have any comment on this I would appreciate hearing from you.

Shaun Tyler
Head, DNA Core Facility
Laboratory Centre for Disease Control
Health Canada

Ph#:  (613) 941-6441
FAX#: (613) 957-1358

E-mail:  styler at hpb.hwc.ca

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