New England Biolabs, M0222S, dam Methyltransferase

Related Categories Methyltransferases for Epigenetics Specification Unit Definition One unit is defined as the amount of enzyme required to protect 1 µg ( dam -) Lambda DNA in 1 hour at 37°C in a total reaction volume of 10 μl against cleavage by MboI restriction endonuclease. Reaction Conditions 1X dam Methyltransferase Reaction Buffer Supplement with 80 µM S-adenosylmethionine (SAM) Incubate at 37°C 1X dam Methyltransferase Reaction Buffer 50 mM Tris-HCl 5 mM β-ME 10 mM EDTA (pH 7.5 @ 25°C) Control Protection Assay dam Methyltransferase is incubated with 1 µg of ( dam- ) Lambda DNA in 10 μl of 1X dam Methyltransferase Buffer, supplemented with 80 µM S-adenosylmethionine, for 1 hour at 37°C followed by 15 minutes at 65°C. The extent of protection is determined by addition of 40 μl 1X NEBuffer 3 supplemented with 10 mM MgCl 2 and 10 units of MboI restriction endonuclease. Incubation at 37°C for 1 hour is followed by analysis on an agarose gel. Storage Buffer 50 mM Tris-HCl 50 mM KCl 1 mM DTT 10 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 20 minutes 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: Can dam Methyltransferase be heat inactivated? A: Yes. Inactivate at 65°C for 20 minutes. Q: Does dam Methyltransferase require MgCl2? A: No. Q: Is dam Methyltransferase sensitive to salt? A: Yes. Make sure the DNA solution is low in salt concentration or that it makes up only a small percentage of the final reaction volume. If salt is a problem, reduce the salt concentration by drop dialysis. Q: Will DpnI cleave dam methylated DNA? A: Yes. 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.