New England Biolabs, M0227S, GpC Methyltransferase (M.CviPI)

Related Categories Methyltransferases for Epigenetics,, Chromatin Analysis,, Base Modifying Enzymes Specification Unit Definition One unit is defined as the amount of enzyme required to protect 1 µg of λ DNA in a total reaction volume of 20 μl in 1 hour at 37°C against cleavage by HaeIII restriction endonuclease. Reaction Conditions 1X GC Reaction Buffer Supplement with 160 µM S-adenosylmethionine (SAM) Incubate at 37°C 1X GC Reaction Buffer 50 mM NaCl 50 mM Tris-HCl 10 mM DTT (pH 8.5 @ 25°C) Control Protection Assay M.CviPI is incubated with 1 µg λ DNA in 20 μl 1X GC Reaction Buffer and 160 µM S-adenosylmethionine, for one hour at 37°C. The extent of protection by M.CviPI is determined by the addition of 30 μl NEBuffer 2 containing 10 units of HaeIII restriction endonuclease. Incubation for 1 hour at 37°C is followed by analysis on an agarose gel. Storage Buffer 15 mM Tris-HCl 0.2 M NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes FAQ Q: Will GpC Methyltransferase (M. CviPI) work in rCutSmart buffer? A: GpC Methyltransferase (M. CviPI) will work in rCutSmart Buffer, with the addition of DTT. To see its % functional activity in rCutSmart, and that of other DNA modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart. 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.