New England Biolabs, M7635L, Duplex DNase

Related Categories Exonucleases and Non-specific Endonucleases Applications cDNA Synthesis Specification Unit Definition One unit is defined as the amount of enzyme required to release 3 pmol of FAM from a 35-mer FAM-BHQ1 labeled hairpin oligonucleotide in 1 minute at 30°C in a 50 μl reaction volume. Reaction Conditions 1X NEBuffer™ r2.1 1X NEBuffer™ r2.1 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Usage Concentration 2,000 units/ml Storage Buffer 10 mM Tris-HCl 50 mM KCl 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 75°C for 10 minutes in the presence of 1 mM DTT. If the sample contains RNA, we recommend adding EDTA (10 mM final concentration) and DTT (1 mM final concentration), prior to heat inactivation. RNA may degrade at temperatures > 65°C in the presence of divalent metals such as Mg2+. FAQ Q: Can Duplex DNase be heat-inactivated? A: Duplex DNase can be heat inactivated by adding DTT or other reducing agent (1 mM final concentration) to a Duplex DNase reaction and incubating at 75°C for 10 minutes. If the sample contains RNA, we recommend adding EDTA (10 mM final concentration), along with DTT (1 mM final concentration) prior to heat inactivation as temperatures >65°C in the presence of divalent metals such as Mg2+ may degrade RNA. Q: Is Duplex DNase inhibited by salt? A: Duplex DNase is inhibited by increasing amounts of salt. We recommend keeping salt concentrations below 50 mM (final) in the Duplex DNase reaction. Duplex DNase Activity is inhibited by increasing salt concentration Duplex DNase activity was measured by monitoring the increase in fluorescence when a quenched 35 nt hairpin dsDNA substrate is cleaved in 1X Reaction Buffer (10 mM Tris-HCl, pH 7.6, 2.5 mM MgCl2, 0.5 mM CaCl2) with increasing salt concentration, as indicated. Q: At what temperature should I use Duplex DNase? A: Duplex DNase is active over a wide range of temperatures (25-65°C). Optimal reaction temperature will vary and may have to be determined empirically depending on reaction conditions. Duplex DNase preferentially degrades dsDNA in a mixture of dsDNA and ssDNA over a wide range of temperatures A mixture of 20 pmol of dsDNA (60 bp oligonucleotide) and 20 pmol of ssDNA (50 nt oligonucleotide) was incubated with 2 Units of Duplex DNase for 1 minute at 25°C, 37°C, 50°C or 65°C and then run on a 4% E-gel™ EX gel (Thermo Fisher Scientific®) stained with SYBR® Gold. Lane 1: dsDNA PCR Marker (NEB #N3234), Lane 2: ssDNA 50 nt, Lane 3: dsDNA 60 bp, Lanes 4–7: 25°C, 37°C, 50°C, 65°C. Duplex DNase displays increased activity and preference for double stranded DNA with increasing temperatures. FLUOR = Fluorophore Q: What is the minimal length of duplex DNA required for Duplex DNase to cleave? A: Duplex DNase requires at least 9-10 bp of duplex DNA for cleavage. Q: Does Duplex DNase require divalent cations? A: Yes, Duplex DNase requires the presence of divalent cations, specifically Mg2+. We do not recommend using Duplex DNase in the presence of calcium (Ca2+) as this will increase the enzyme’s promiscuity (activity toward ssDNA). Q: What are the optimal reaction conditions for Duplex DNase? A: Duplex DNase is most active in reactions performed at pH 7.0, containing less than 50 mM salt and a divalent cation of Mg2+ (not Ca2+). Duplex DNase is active over a wide range of temperatures (25-65°C) and requires at least 9-10 bp of duplex DNA for cleavage. Q: How does the unit definition for Duplex DNase compare to other duplex-specific DNases? A: Although the activity of Duplex DNase is not measured using the classical Kunitz unit definition, we estimate that, as with other duplex-specific DNases, the specific activity of Duplex DNase is 200,000 U/mg.