How do you quantitate DNA with a flourimeter?

John J. Shaskus shaskusj at ohsu.edu
Tue Jul 23 10:37:28 EST 1996


There is a procedure for using Hoechst 33258 fluorochrome to quantitate 
DNA in Short Protocols in Molecular Biology 3rd edition p.A3-13.  It's 
undoubtably in Current Protocols also.  The only caveat I can think off 
right off is that the method is sensitive to GC% and you need to use an 
appropriate control to get an accurate quantitation.
I find UV to be a particularly bad way to quantitate nucleic acids and 
DNA in particular - unless you're sure of the purity and have several 
micrograms you can spare.  There are just too many things which 
interfere at 260 nm:  RNA, phenol, carbohydrate, protein....
Concentration of linear DNA can quickly and easily estimated by running 
a minigel and staining in ethidium bromide solution.  If you use Hind 
III cut lambda as a marker at 1 ug per lane the largest fragment 
contains about 0.5 ug and the 2.0 and 2.3 kb about 50 ng each. 





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