Micrococcal Nuclease cleaves single-stranded and double-stranded DNA and RNA. Complete digestion with Micrococcal Nuclease yields mono- and oligonucleotides with 3′-phosphates.
|Enzyme Commission Number||(E.C. 188.8.131.52)|
|Enzyme Inactivation||EDTA or EGTA in molar excess of CaCl2.|
|Storage||Store at -20°C for up to 12 months. Avoid repeated freeze/thawing. Prolonged storage should be ≤ -70 °C.|
|Unit Definition||One unit (U) will produce 1.0 µmole of acid soluble polynucleotides from native DNA per min at pH 8.8 at 37°C, based on EM/260 = 10,000 for the mixed nucleotides.|
Q1: What is the difference between Atlantis dsDNase and MNase?
• Atlantis dsDNase is a double-stranded DNA specific endonuclease that cleaves phosphodiester bonds in DNA to yield oligonucleotides with 5’-phosphate and 3’-hydroxyl termini. Increasing the incubation time with Atlantis dsDNAse will increase efficiency and generate more mono-nucleosomal DNA. • Micrococcal Nuclease (MNase) is a single and double-stranded DNA and RNA endonuclease. MNase is more specific for single-stranded nucleic acids, but cleavage is biased towards AT-rich and AU-rich sequences. MNase will result in more robust digestion compared to Atlantis dsDNase. Increasing the incubation time with MNase will increase efficiency of this enzyme and generate more mono-nucleosomal DNA. • The enzyme selection depends on desired downstream applications.
Q2: What is the sensitivity of the enzyme to different cell types?
Once the nuclei are isolated, the enzyme sensitivity should be similar between different cell types. However, some cells lines will be more sensitive to the detergent in the Nuclei Prep Buffer and thus result in a loss of nuclei. You can dilute the Nuclei Prep Buffer 1:1 with MN Digestion Buffer prior to nuclei isolation.
Q3: Is there information on whether the exonuclease activity of the MNase digest dsDNA from 3' to 5'?
MNase is an endo-exonuclease that preferentially cleaves at AT or AU-rich regions, yielding mono-nucleotides and oligonucleotides with terminal 3'-phosphates.