New England Biolabs, M0302L, T7 Endonuclease I

Related Categories Validation of CRISPR-based Gene Editing,, Exonucleases and Non-specific Endonucleases,, DNA Repair Enzymes and Structure-specific Endonucleases Applications Whole Genome Amplification & Multiple Displacement Amplification Specification Unit Definition One unit is defined as the amount of enzyme required to convert > 90% of 1 μg of supercoiled cruciform pUC(AT) to > 90% linear form in a total reaction volume of 50 μl in 1 hour at 37°C. Reaction Conditions 1X NEBuffer™ 2 Incubate at 37°C 1X NEBuffer™ 2 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 1 mM DTT (pH 7.9 @ 25°C) Storage Buffer 20 mM Tris-HCl 200 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.15% Triton® X-100 pH 7.5 @ 25°C Heat Inactivation No FAQ Q: Is there a way to inactivate T7 Endonuclease I? A: Yes, to inactivate T7 Endonuclase I, we recommend using Proteinase K and incubating at 37°C for 5 minutes. Please see the Protocol: "T7 Endonuclase I-based Mutation Detection with the EnGen® Mutation Detection Kit” for more details. Q: Does T7 Endonuclease I recognize single base pair mismatches? 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. Depending on the mismatches in your experiment, you may observe different cleavage efficiencies (Guan, C. et al. (2004) Biochemistry, 43, 4313-4322). T7 Endonuclease I may not be the best choice for detecting mismatches of unknown composition. However, if your mismatch is known to contain C, you can use this enzyme for your assay. We recommend titrating the enzyme to optimize cleavage. Q: What common additives inhibit T7 Endonuclease I? A: T7 Endonuclease I requires Mg2+ or Mn2+ for activity. A chelating agent, such as EDTA, will inhibit the reaction. Q: What is the molecular weight of T7 Endonuclease I? A: The T7 Endonuclease I supplied is fused to the E. coli maltose binding protein. The molecular weight of the entire fusion protein is 60,302 daltons. Q: What PCR reagents do you recommend for DNA amplification in genome editing (CRIPSR/Cas9,TALEN,ZFN) mismatch detection assays? A: We recommend Q5® High-Fidelity DNA Polymerase with Q5® Reaction Buffer or Q5 Hot Start High Fidelity 2X Master Mix because of Q5 DNA Polymerases’ high fidelity and robustness. The EnGen Mutation Detection Kit provides reagents for detection of on-target genome editing events. The kit comes with optimized enzymes (including PCR reagents), protocols and includes a control. Notes: T7 Endonuclease I is a structure-selective enzyme. It acts on a variety of DNA substrates with different specific activities. It is important to control the amount of enzyme and the reaction time used for cleavage of a particular substrate. Temperatures above 42°C cause an increase in nonspecific nuclease activity and should be avoided. pUC(AT) is derived from pUC19 with a modification of the polylinker between the EcoRI site and the PstI site. Q: Can I use T7 Endonuclease I genome editing (CRIPSR/Cas9,TALEN,ZFN) mismatch detection assays using unpurified PCR products? A: We recommend using 1-4 μL of the appropriate PCR reaction directly in a 50 ul T7 Endonuclease I reaction when the following PCR reagents are used: -Q5® High-Fidelity DNA Polymerase with Q5® Reaction Buffer -OneTaq® DNA Polymerase. Under these conditions, purification of PCR products before digestion with T7 Endonuclease I is optional. For purification of PCR products we recommend the Monarch PCR & DNA Cleanup kit. Q: Can I use T7 Endonuclease I for genome editing applications? A: Yes. T7 Endonuclease I can be used to detect indel formation (on-target editing events) in genome editing workflows.The EnGen® Mutation Detection Kit provides reagents for detection of on-target genome editing events. The kit contains T7 Endonuclease I in an optimized formulation, as well as the necessary PCR reagents, control substrate and protocols. T7 Endonuclease I can also be purchased as a stand-alone reagent. Please refer to this protocol when using this product. Q: What is the molecular weight of T7 Endonuclease I? A: The T7 Endonuclease I supplied is fused to the E. coli maltose binding protein. The molecular weight of the entire fusion protein is 60,302 daltons. Q: What PCR reagents do you recommend for DNA amplification in genome editing (CRIPSR/Cas9,TALEN,ZFN) mismatch detection assays? A: We recommend Q5® High-Fidelity DNA Polymerase with Q5® Reaction Buffer or Q5 Hot Start High Fidelity 2X Master Mix. The high-fidelity and robustness of Q5 DNA Polymerase makes it a good choice for this assay. The EnGen® Mutation Detection Kit provides reagents for detection of on-target genome editing events. The kit comes with optimized enzyme (including PCR reagents), protocols and includes a control.