{"product_id":"neb-m0224s","title":"New England Biolabs, M0224S, HaeIII Methyltransferase","description":"HaeIII Methyltransferase modifies the internal cytosine residue (C5) of the sequence GGCC.  \u003cb\u003eRelated Categories\u003c\/b\u003e Methyltransferases for Epigenetics,, Base Modifying Enzymes  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eUnit Definition\u003c\/b\u003e 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.  \u003cb\u003eReaction Conditions\u003c\/b\u003e 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)  \u003cb\u003eControl Protection Assay\u003c\/b\u003e 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.  \u003cb\u003eStorage Buffer\u003c\/b\u003e 50 mM Tris-HCl 1 mM DTT 10 mM EDTA 200 µg\/ml BSA 50% Glycerol 50 mM KCl pH 7.4 @ 25°C  \u003cb\u003eHeat Inactivation\u003c\/b\u003e 65°C for 20 minutes  \u003cb\u003eFAQ\u003c\/b\u003e 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. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835497762985,"sku":"M0224S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0224s","provider":"Iright","version":"1.0","type":"link"}