New England Biolabs, M0678S, Mismatch Endonuclease I

Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to cleave 50% of 0.2 pmol of a fluorescently labeled 60mer oligonucleotide duplex containing a single T:T mismatch in 30 minutes at 37°C in a total reaction volume of 20 μl in 1X NEBuffer r2.1. Reaction Conditions 1X NEBuffer™ r2.1 Incubate at 37°C NEBuffer™ r2.1 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 500 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 70°C for 5 minutes FAQ Q: What is the activity of Mismatch Endonuclease I in other buffers? A: The relative activity of Mismatch Endonuclease I in the following buffers is indicated in the table below: Buffer % Activity1 NEBuffer r1.1 50% NEBuffer r2.1 100% NEBuffer r3.1 25% rCutSmart™ Buffer 75% Q5® Reaction Buffer2 0% ThermoPol Reaction Buffer2 25% Standard Taq Reaction Buffer2 75% Phusion HF Buffer2 100% 1 % Activity is listed in comparison to the optimal buffer, NEBuffer r2.1. 2 Supplemented with 10 mM MgCl2 Q: Can Mismatch Endonuclease I be inactivated? A: Yes, Mismatch Endonuclease I can be inactivated by the addition of EDTA (10 mM, final concentration) or by heat inactivation for 10 minutes at 70°C. Q: What is the activity of Mismatch Endonuclease I at temperatures other than 37°C? A: 30°C = 75% 37°C = 100% 42°C = 100% 50°C = 75% Q: Does Mismatch Endonuclease I require MgCl2? A: Yes, Mismatch Endonuclease I requires MgCl2. MgCl2 is present in NEBuffer r2.1, so if Mismatch Endonuclease I is used with the supplied NEBuffer r2.1, no additional magnesium needs to be added to the reaction. If using Mismatch Endonuclease I in a buffer other than NEBuffer r2.1, supplementation with magnesium may be required for optimal enzyme function. Q: Can Mismatch Endonuclease I function in the presence of other divalent cations? A: Other divalent cations, such as Mn2+, Co2+, Ni2+ and Zn2+ will displace the Mg2+ required for activity and inhibit the activity of Mismatch Endonuclease I. The activity of Mismatch Endonuclease I is unaffected by the presence of Ca2+ ions. Q: Is Mismatch Endonuclease I sensitive to salt? A: No, Mismatch Endonuclease I is relatively insensitive to salt. However, concentrations of salt > 250 mM can decrease the activity of Mismatch Endonuclease I. It is important to note that Mismatch Endonuclease I is stored in a high salt (500 mM) diluent, so some salt is carried over into the reaction from the storage buffer and should be accounted for in the final salt concentration of any reaction. Q: What mismatched substrates will Mismatch Endonuclease I cleave? A: Mismatch Endonuclease I prefers to cleave T:T, G:G and T:G DNA mismatches, but will also readily cleave T:I, G:I and G:U DNA mismatches. Additionally, Mismatch Endonuclease I will nick the thymine containing strand of a T:C DNA mismatch, and cleave T:G and T:U (DNA:RNA) mismatches albeit to a lesser extent than DNA:DNA mismatches. Q: Will Mismatch Endonuclease I cleave consecutive mismatches? A: Yes, Mismatch Endonuclease I will cleave a substrate with sequential T:T, G:G or T:G DNA mismatches. However, Mismatch Endonuclease I cannot cleave a bulged DNA substrate with multiple (>2) consecutive mismatches. Additionally, if your DNA contains multiple sequential mismatches, Mismatch Endonuclease I will preferentially cleave at a T:T mismatch over a G:G or T:G mismatch. Q: Does Mismatch Endonuclease I cleave mismatches in a DNA/RNA hybrid? A: Yes, Mismatch Endonuclease I will cleave T:G and T:U (DNA:RNA) mismatches. Q: What mismatches will Mismatch Endonuclease I not cleave? A: Mismatch Endonuclease I will not cleave A:A, A:C, A:G, C:C DNA mismatches or Watson-Crick base-paired DNA. Q: How many picomoles of mismatch DNA can Mismatch Endonuclease I cleave? A: One unit is defined as the amount of enzyme required to cleave 50% of 0.2 pmol of a single T:T mismatch in 30 minutes at 37°C in a total reaction volume of 20 µl in 1X NEBuffer r2.1. 1 μl of Mismatch Endonclease I contains 80 U, therefore, 1 µl of Mismatch Endonuclease I is able to completely cleave 8 pmol of mismatched DNA. Q: How is Mismatch Endonuclease I different than T7 Endonuclease I? A: Although T7 Endonuclease I has activity on mismatched DNA, it is not an ideal enzyme for cleaving all single base pair mismatches in heteroduplex DNA. However, if your mismatch is known to contain C, you can use this enzyme for your assay. Mismatch Endonuclease I does not recognize and cleave all mismatches in DNA. Mismatch Endonuclease I is ideal for cleaving T:T, G:G and T:G mismatches in DNA, but will also cleave T:I, G:I, G:U DNA mismatches, T:G and T:U DNA:RNA mismatches and will nick the thymine containing strand of T:C mismatches in DNA. Q: What are the differences between Mismatch Endonuclease I (NEB #M0678) and Authenticase (NEB #M0689)? A: Mismatch Endonuclease I only cleaves the following DNA mismatches: G/G, G/T and T/T. Authenticase will cleave all single base mismatches (except for G/A) and all 2+ base pair DNA mismatches and indels (insertion and/or deletion).