Hello Malin,

Indeed, you would expect the OD to increase instead of decrease. "Free" dNMPs have a higher absorbance value than dNMPs present in a helical DNA structure (hypochromicity effect).

You write that you use "6.25 mM MgSO4, 1M acetate buffer". Is it 1 M final concentration of acetate buffer? If so, this might cause your strange observation. DNAseI won't be very active under this high salt conditions and your DNA might slowly drop out of solution in this environment (especially if you work with highly polymerized size DNA). Otherwise I would have no other plausible explanation besides that there is something going wrong in the spectrophotometer. You might try different assay conditions? ....addition of CaCl2 (e.g. 2 mM) is known to benefit DNAseI activity.

Further, if I look at the preparation of the DNAseI solution, the kit says to dissolve the enzyme in reagent grade water...I don't know if this will be the best for the enzyme. Usually there are some buffering agents present to preserve enzyme stability.

By how much did the 260 nm absorbance decrease? Was it more than expected just from dilution of the sample?

I'd expect the OD to increase with cleavage as the pi-pi interaction (hyperchromicity) between the bases decreases. If you were my student I'd ask you to repeat the experiment with a series of DNA concentrations. (

Dnase hydrolyse the DNA and affecting ( decreasing ) its absorbance  at 260 nm.

Sorry @ Ben Amar Cheba you got this one wrong; 260nm Absorbance of DNA increases with cleavage. This is a  very well known phenomemum.