New England Biolabs, M0348L, NEBNext® dsDNA Fragmentase®

For most applications, especially where time-tunable fragmentation or preservation of methylation is needed, we recommend Related Categories DNA Fragmentation & RNA Fragmentation Specification Reaction Conditions 1X NEBNext® dsDNA Fragmentase® Reaction Buffer v2 1X NEBNext® dsDNA Fragmentase® Reaction Buffer v2 20 mM Tris-HCl 15 mM MgCl2 50 mM NaCl 0.1 mg/ml BSA 0.15% Triton® X-100 (pH 7.5 @ 25°C) FAQ Q: When dsDNA Fragmentase (NEB #M0348) was changed to include Fragmentase Buffer v2,  was there any change to the dsDNA Fragmentase enzyme itself? A: No, the formulation of the dsDNA Fragmentase enzyme has not changed, only the buffer supplied with the enzyme. Q: What sequencing platforms can DNA fragments produced using dsDNA Fragmentase (NEB #M0348) be used for? A: DNA produced by dsFragmentase is suitable for many downstream applications, including next generation sequencing. Since the ends created by dsDNA Fragmentase are not blunt, end repair will be needed for some applications. Q: What types of DNA are suitable for fragmentation using ds DNA Fragmentase (NEB #M0348)? A: dsDNA Fragmentase works on dsDNA that is free of enzymatic inhibitors and EDTA. If a DNA sample contains EDTA, the provided MgCl2 can be added to compensate. For consistent fragmentation the amount of EDTA and MgCl2 from sample to sample should be consistent. Q: What type of ends does dsDNA Fragmentase (NEB #M0348) create? A: While fragmentation is somewhat random, dsDNA Fragmentase typically generates 1-4 base overhangs on the 5´ end. Some blunt ends and 3´ overhangs will also be generated. Q: Can dsDNA Fragmentase (NEB #M0348) be heat inactivated? A: Yes, dsDNA Fragmentase can be heat inactivated by incubation at 65°C for 15 minutes in the presence of 50mM DTT. However, the sample must be purified for most downstream applications, even after heat denaturation. It is important to note that heating can damage the DNA. Stopping the reaction with EDTA and then removing EDTA is more gentle on the DNA and is therefore recommended. Q: Can I add ligase to my samples after using dsDNA Fragmentase (NEB #M0348) to seal any nicks? A: Yes, E. Coli DNA Ligase (NEB #M0205) can be added after a cleanup Q: Does the fragmentation reagent included in NEBNext Ultra® II FS DNA kits contain NEBNext dsDNA Fragmentase®? A: No, the Ultra II FS (Fragmentation System) products contain a unique mix of enzymes that fragment, end repair, and dA tail DNA, in a single tube. Fragmentase is not included in the FS enzyme mix.