New England Biolabs, M0223S, BamHI Methyltransferase

The large size of this product was discontinued on 12/31/2020. The small size will continue to be available. 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 BamHI restriction endonuclease. Reaction Conditions 1X BamHI Methyltransferase Reaction Buffer Supplement with 80 µM S-adenosylmethionine (SAM) Incubate at 37°C Control Protection Assay BamHI Methyltransferase is incubated with 1 µg λ DNA in 10 μl 1X BamHI Methyltransferase 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 BamHI Methyltransferase is determined by the addition of 40 μl NEBuffer 1 supplemented with 10 mM MgCl 2 and 10 units of BamHI restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on agarose gels. Storage Buffer 50 mM Tris-HCl 1 mM DTT 10 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation No 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 BamHI Methyltransferase? A: The molecular weight is 49,223 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.