- Quick and simple procedure for generating single nucleosides from DNA for quantitative analysis via LC/MS
- Convienent 2 hour, single enzyme digest v. conventional 6-16 hr multi-step enzyme digestion protocol
DNA Degradase Plus from Zymo Research is a nuclease mix that quickly and efficiently degrades DNA to its individual nucleoside components. Since nucleosides lack negatively charged phosphate, DNA Degradase Plus is ideal for whole-genome DNA methylation analysis by LC/MS. Digestion with the enzyme is performed via a one-step procedure that is faster and simpler than other available methods.
|Assay Condition||DNA Degradase Plus in 1X DNA Degradase Reaction Buffer. Incubate reaction mixtures at 37C for =1 hour|
|Enzyme Inactivation||Heat inactivate enzyme at 70C for 20 minutes|
|Storage||Store at -20C for up to 12 months. Avoid repeated freeze/thawing of reagents. Prolonged storage is at = -70C.|
|Unit Definition||5 U of enzyme will digest 1 µg of genomic DNA in 25 µl reaction volume at 37C for = 1 hr|
Q1: Can I visualize the treated samples/ do I need to purify after Degradase treatment?
The best way to confirm degradation is to run the sample on a gel. Nothing should be visible for the Degradase-treated samples. You do not need to purify the reaction.
Q2: Can I let the reaction go longer than stated in the protocol?
Yes, the protocol time is a suggestion for sufficient digestion. Additional incubation time can be added to completely ensure that all sample has been digested. There is no harm in letting the reaction proceed longer.
Q3: Can I add excess enzyme to the reaction?
Yes, you can add excess enzyme to ensure full digestion.
Q4: Does this work with RNA/ ssDNA?
The preferred substrate for Degradase and Degradase Plus is double stranded DNA. There will be only minor degradation of single stranded DNA template and no degradation of RNA template.
Q5: Can I scale up/ down the reaction volumes? How do I do that?
Yes, you can scale the reaction up or down as necessary. For the best results, digest no more than 1 µg of DNA with 5 U (1 µl) of enzyme in 25 µl reaction volume. The reaction volume is just as important as the amount of DNA, and we recommend scaling up the volume of enzyme accordingly. For example, if the reaction volume is 100 µl, use 4 µl of DNA Degradase (Plus).
DNA Degradase Plus