New England Biolabs, M0224S, HaeIII Methyltransferase

HaeIII Methyltransferase modifies the internal cytosine residue (C5) of the sequence GGCC. 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 HaeIII restriction endonuclease. Reaction Conditions 1X HaeIII Methyltransferase Reaction Buffer Supplement with 80 µM S-adenosylmethionine (SAM) Incubate at 37°C 1X HaeIII Methyltransferase Reaction Buffer 50 mM Tris-HCl 50 mM NaCl 10 mM DTT (pH 8.5 @ 25°C) Control Protection Assay HaeIII Methyltransferase is incubated with 1 µg of λ DNA in 10 μl of 1X HaeIII 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 is determined by the addition of 40 μl NEBuffer 2.1 and 10 units of HaeIII restriction endonuclease. Incubation for 1 hour at 37°C is followed by analysis on an agarose gel. Storage Buffer 50 mM Tris-HCl 1 mM DTT 10 mM EDTA 200 µg/ml BSA 50% Glycerol 50 mM KCl pH 7.4 @ 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: Does HaeIII Methyltransferase require DTT? A: Yes. Try fresh buffer if activity seems low. Q: Can HaeIII Methyltransferase be heat inactivated? A: Yes. Inactivate at 65°C for 20 minutes. Q: Does HaeIII Methyltransferase require MgCl2? A: No. Q: What is the molecular weight of HaeIII Methyltransferase? A: The molecular weight is 37,684 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.