New England Biolabs, M0660S, Nuclease P1

Related Categories Exonucleases and Non-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to liberate 1.0 µg of acid soluble nucleotides from Torula Yeast total RNA per min at 37°C in 1X Nuclease P1 Reaction Buffer. Reaction Conditions 1X Nuclease P1 Reaction Buffer Incubate at 37°C 1X Nuclease P1 Reaction Buffer 50 mM sodium acetate (pH 5.5 @ 25°C) Storage Buffer 25 mM Tris-HCl 50 mM NaCl 1 mM ZnCl2 50% Glycerol pH 7.2 @ 25°C Heat Inactivation 75°C for 10 minutes FAQ Q: Is Nuclease P1 sensitive to salt? A: Nuclease P1 is relatively insensitive to salt content, however it is recommended that the final salt concentration in the reaction remain ≤ 250 mM. Q: Can Nuclease P1 be inactivated? A: Yes, Nuclease P1 can be inactivated by the addition of EDTA (5 mM, final concentration) or by heat inactivation for 10 minutes at 75°C. Q: Does Nuclease P1 require ZnCl2? A: Yes, Nuclease P1 does require ZnCl2, and therefore can be inhibited/inactivated by the addition of EDTA. When using Nuclease P1, enough ZnCl2, is present in the storage buffer (1 mM) such that additional ZnCl2 does not need to be added to the reaction. Q: What applications are Nuclease P1 best suited for? A: Nuclease P1 can be used for the removal of single stranded tails from DNA molecules to create blunt-ended molecules, cleavage of hairpin loops, DNA or RNA base compositional analysis, and removal of nucleic acids during protein purification. Q: What substrates does Nuclease P1 digest? A: Substrate specificity for Nuclease P1 is as follows: 3′AMP > RNA > ssDNA >> dsDNA. Q: How much ssDNA or RNA can Nuclease P1 digest? A: Nuclease P1 (1 unit) can effectively liberate 1 µg of nucleotides from ssDNA or RNA per minute at 37°C. Q: What is the activity of Nuclease P1 in other buffers? A: Nuclease P1 is active over a wide range of pH values (optimally between pH 4.0-7.0). Additionally, since Nuclease P1 requires ZnCl2, reaction buffer should be free of significant amounts (< 5 mM) of chelating agents (i.e., EDTA). When using Nuclease P1, enough ZnCl2, is present in the storage buffer (5 mM) such that additional ZnCl2 does not need to be added to the reaction. NEBuffer 1.1 2.1 3.1 CutSmart® % Activity 100 50 25 50 Activity in other buffers: Buffer % Activity Nucleoside Digestion Mix Buffer 100% Std Taq Buffer 50% Thermopol Buffer 25% Q5 Buffer 50% DNase I Buffer 75% Endo III Buffer 75% Exo I Buffer 25% Exo VII Buffer 0% Lambda Exo Buffer 0% Micrococcal Nuclease Buffer 25% Mung Bean Nuclease Buffer 50% Q: How does the new unit definition relate to the historical unit definition for Nuclease P1? A: The historical unit definition for the 3′-5′-Phosphodiesterase activity Nuclease P1 is as follows: One unit is defined as the amount of enzyme required to liberate 1.0 μmole of acid soluble nucleotides from Torula Yeast total RNA per min at 37 °C in 1X Nuclease P1 Buffer. This unit definition has been updated to be directly comparable to the 3′-5′-Phosphodiesterase unit definition of Nuclease S1, such that the 3′-5′-Phosphodiesterase activity unit definition of NEB’s Nuclease P1 is as follows: One unit is defined as the amount of enzyme required to liberate 1.0 μg of acid soluble nucleotides from Torula Yeast total RNA per min at 37 °C in 1X Nuclease P1 Buffer. This updated unit definition makes NEB’s Nuclease P1 an easy drop-in replacement for Nuclease S1. Q: Will Nuclease P1 degrade double stranded DNA (dsDNA)? A: Although a single-strand specific nuclease (ssDNA and RNA-specific), Nuclease P1 does display some activity toward dsDNA in Nuclease P1 Reaction Buffer. If preferentially degrading single-stranded nucleic acids (ssDNA or RNA) in the presence of double stranded DNA (dsDNA), we recommend using Nuclease P1 in 1X NEBuffer 1.1 to limit activity on dsDNA while maintaining single-strand nuclease activity.