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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