1. Dilution of the Nucleoside Digestion mix may result in a decrease of enzymatic activity and incomplete
digestion of substrate. Therefore, we recommend using 1 µL of the Nucleoside Digestion Mix for
digestion of samples containing < 1 µg of DNA or RNA substrate.
2. Samples containing a large number of modifications (particularly modifications at the ribose 2´-position)
may benefit from overnight incubation with the Nucleoside Digestion Mix in order to achieve complete
digestion. No signal deterioration has been observed by incubating DNA or RNA samples with the
Nucleoside Digestion Mix for up to 24 hours at 37°C. Alternatively, the ratio of Nucleoside Digestion
Mix:nucleic acid may be increased from 1 μl/μg substrate to 5-10 μl/μg substrate to ensure complete
digestion.
3. Although it is not necessary to stop the reaction prior to LC-MS, the mix can be inactivated by the
addition of EDTA (5 mM, final concentration).
4. In order to reduce the ion suppression effects of glycerol, the Nucleoside Digestion Mix has been
formulated to contain very little glycerol (<1%) and therefore the mix will freeze when stored at -20°C.
The mix is stable for >2 years when stored at -20°C and can withstand 50 freeze-thaw cycles without
significant activity loss.
5. As carryover of certain reaction components (e.g., EDTA, detergents, etc) from upstream steps may
result in incomplete digestion, it is highly recommended that DNA or RNA substrates be purified
(column purification/phenol chloroform extraction) before digestion with the Nucleoside Digestion
Mix.
