DNA Degradase Plus
E2020 / E2021
- 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
|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?
Q2: Can I let the reaction go longer than stated in the protocol?
Q3: Can I add excess enzyme to the reaction?
Q4: Does this work with RNA/ ssDNA?
Q5: Can I scale up/ down the reaction volumes? How do I do that?
The authors used DNA Degradase Plus to determine total methylation in genomic DNA from developing mouse embryonic tissue by mass spectrometry.Oda M, Oxley D, Dean W, Reik W. 2013. Regulation of Lineage Specific DNA Hypomethylation in Mouse Trophectoderm. Plos One DOI: 10.1371/journal.pone.0068846.
The authors sought to quantify levels of 5-mC in a number of yeast species using DNA Degradase Plus and LC-MS/MS.Capuano F, Mülleder M, Kok R, Blom HJ, Ralser M. 2014. Cytosine DNA Methylation Is Found in Drosophila melanogaster but Absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Other Yeast Species. Anal. Chem. 86(8): 3697–3702.
DNA Degradase Plus was used to quantify the levels of 5-hmC and 5-mC in genomic DNA of tumor cells or cultured cell lines using a sensitive liquid chromatography-tandem quadrupole mass spectrometric method.Tsuji M, Matsunaga H, Jinno D, Tsukamoto H, Suzuki N, Tomioka Y. 2014. A validated quantitative liquid chromatography-tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic DNA. J Chro