DNA assay when RNA present

Sun Feb 21 23:51:14 EST 1993

Baker at mouse at lamar.colostate.edu:
>My DNA prep results in RNA remaining with the DNA. I don't 
>want to use RNAse because I will lose some DNA going through 
>phenol-chloroform-etoh ppt. What alterntives might be used? 
>I have access to Hoefer TK0 102 standard kit, TKO 100 minifluorometer
>that is supposed to work with Hoechst dye and be accurate for 
>0.1 ng/uL DNA - contaminants that are ignored include RNA
>and protein etc. I don't know if this is bis benzimide dye or not; 
>don't know how accurate readings are.

Fluorometric assay of DNA using DNA-specific dyes such as Hoechst 33258
(bis-benzimidazole) and diaminophenylindole (DAPI) are by far the most
sensitive quantitation method I know that is widely accessible to most
lab. Sensitivity ranges from 10 ng to 15 5g per ml for H33258. Compare
this to 1-50 5g/ml and 100 ng-10 5g/ml for absorbance (260 nm) and 
ethidium staining, respectively. Since both H33258 and DAPI binds
specifically to DNA, interference from most contaminating species
such as RNA and protein is negligible. In fact, crude cell lysates can
be used directly for DNA quantitation due to high dye-binding
specificity as long as particulate matters are kept to a minimum. The
only drawback with the use of DAPI and H33258 comes when one is
dealing with a sample with unusually high GC content since these dyes
preferably associate with DNA at AT-rich domains. In such case one
may opt to switch to ethidium-staining of minigels where RNA has been
resolved electrophoretically from the sample DNA and do visual 
comparison with adjacent standard DNA lanes.

Have a nice day. :-)

Genetics Lab, Pathology Div., Intl Rice Res Inst
e-mail: rscott at irri.cgnet.com

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