New England Biolabs, R0661L, MspJI

Related Categories Methylation Sensitive and Methylation Dependent Restriction Enzymes for Epigenetics,, Restriction Enzymes for Epigenetic Analysis,, Restriction Endonucleases H M, Applications Restriction Enzyme Digestion Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 (dcm + ) DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Supplement with 1X Enzyme Activator Solution Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: <10% NEBuffer™ r2.1: <10% NEBuffer™ r3.1: <10% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent B Storage Buffer 10 mM Tris-HCl 300 mM NaCl 1 mM DTT 0.1 mM EDTA 500 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ Q: How has MspJI been used for epigenetic studies? A: This enzyme has been used for epigenetic analysis in which target DNA containing CpG sites methylated in both strands is cleaved by MspJI to generate 32 bp fragments containing the methylated CpG sites. The isolated 32 bp fragments can be sequenced to reveal 5-mC modification sites (Cohen-Karni et al., (2011) PNAS 108: 11040-11045). Q: How can I minimize off target or star activity? A: Star Activity, including digestion of non-methylated DNA substrates, may result from non-optimal reaction conditions such as extended digestion time, high enzyme or activator concentration or a glycerol concentration of > 5%. Optimization of the amount of enzyme needed to achieve efficient specific cutting while avoiding star activity may be required for each substrate DNA. Q: Should I add more MspJI to get more cutting? A: No, not necessarily. Unlike many common restriction enzymes, use of excess MspJI inhibits cleavage. Optimization of the amount of enzyme needed for complete digestion may be required for each substrate DNA. Q: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: No. This enzyme is not sensitive to dam, dcm, or mammalian CpG methylation. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Q: Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures? A: The recommended storage temperature for Gel Loading Dye, Purple 6X, is room temperature (25°C). Commonly, the SDS may precipitate out of solution when the dye is stored on cold temperatures. This slight precipitate can be dissipated by letting the vial sit at room temperature for an hour, or warming gently for 10 minutes. Mix the vial a few times before use, to bring the solution back to homogeneity.