New England Biolabs, M0215S, MspI Methyltransferase

Related Categories Methyltransferases for Epigenetics,, Base Modifying Enzymes Specification Unit Definition One unit is defined as the amount of enzyme required to protect 1 µg λ DNA in 1 hour at 37°C in a total reaction volume of 10 μl against cleavage by MspI restriction endonuclease. Reaction Conditions 1X MspI Methylase Reaction Buffer Supplement with 80 µM S-adenosylmethionine (SAM) Incubate at 37°C 1X MspI Methylase Reaction Buffer 50 mM Tris-HCl 100 mM NaCl 5 mM β-ME 10 mM EDTA (pH 7.5 @ 25°C) Control Protection Assay MspI Methyltransferase is incubated with 1 µg of λ DNA in 10 μl 1X MspI Methyltransferase Reaction Buffer, supplemented with 80 µM S-adenosylmethionine, for one hour at 37°C followed by 15 minutes at 65°C. The extent of protection by MspI Methyltransferase is determined by the addition of 40 μl NEBuffer 2 supplemented with 10 mM MgCl 2 and 5 units of MspI restriction endonuclease. Incubation for MspI at 37°C is followed by analysis on agarose gels. Storage Buffer 50 mM Tris-HCl 50 mM NaCl 5 mM β-ME 10 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation No Molecular Weight Theoretical: 40397 daltons FAQ Q: Is S-adenosylmethionine (SAM) supplied with the Methyltransferase? A: Yes, a stock solution is provided at 32mM. It can also be ordered separately as product NEB# B9003S. The SAM is the most labile component of the reaction. If the stock solution is older than 9 months it should be replaced. Q: What should be considered if the methylation is not going to completion? A: The SAM is the most labile component of the reaction. If the stock solution is older than 6 months it should be replaced. Q: What is the molecular weight of MspI Methyltransferase? A: The molecular weight is 47,654 daltons. Q: Will any NEB methyltransferases (methylases) work on RNA? A: Yes, EcoGII Methyltransferase (NEB #M0603) produces 5-10% methylated adenosine residues on RNA substrates. However, other methyltransferases do not. Please note: NEB has tested the following transferases/methylases (MT): dam Methyltransferase (NEB #M0222), AluI Methyltransferase (NEB #M0220), BamHI Methyltransferase (NEB #M0223), M.SssI Methyltransferase (NEB #M0226), EcoRI Methyltransferase (NEB #M0211), M.CviPI Methyltransferase (NEB #M0227), HaeIII Methyltransferase (NEB #M0224), HhaI Methyltransferase (NEB #M0217), HpaII Methyltransferase (NEB #M0214), MspI Methyltransferase (NEB #M0215) and TaqI Methyltransferase (NEB #M0219) using their supplied reaction buffers on two substrates:1) An in vitro transcribed mRNA encoding Firefly luciferase (1 µg) and 2) total HeLa RNA (250 ng). Each substrate was incubated with two amounts of enzyme (1 and 5 µl) for 1 hour at 37°C (65°C for Taq I MT), cleaned up using a spin column, digested to individual nucleosides using an enzyme cocktail and analyzed by LC/MS to identify modified and unmodified residues. None of the methylases examined were able to transfer a methyl group to the RNA substrates examined.