New England Biolabs, M0603S, EcoGII Methyltransferase

Available in higher concentration. Please Related Categories Methyltransferases for Epigenetics,, Base Modifying Enzymes Specification Unit Definition One unit is defined as the amount of enzyme required to protect 100 ng FAM-labeled dsDNA in 30 minutes at 37°C in a total reaction volume of 20 μl against cleavage by MboI restriction endonuclease. Reaction Conditions 1X rCutSmart™ Buffer Supplement with 160 µM S-adenosylmethionine (SAM) Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 250 mM NaCl 1 mM DTT 0.1 mM EDTA 0.15% Triton® X-100 180 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 10 minutes Unit Assay Conditions EcoGII Methyltransferase is incubated with 100 ng of a FAM-labeled dsDNA substrate (80 bp) in 20 μl of 1X rCutSmart Buffer supplemented with 160 µM SAM, for 30 minutes at 37°C followed by 10 minutes at 65°C. The extent of protection is determined by addition of 5 units of MboI restriction endonuclease (incubation at 37°C for 30 minutes, 65°C for 20 minutes) followed by capillary electrophoresis analysis to resolve the reaction products, assign the cleaved and uncleaved species and quantitate their abundance. FAQ 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. Q: What is the activity of EcoGII Methyltransferase at temperatures other than 37°C? A: 4°C=25% 16°C=25% 25°C=50% 30°C=100% 37°C=100% 42°C=100% 50°C=50% 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: Does EcoGII Methyltransferase require BSA? A: No, EcoGII Methyltransferase does not require BSA or Recombinant Albumin (rAlbumin). Q: Does EcoGII Methyltransferase require MgCl2? A: No, EcoGII Methyltransferase does not require MgCl2. Q: What is the activity of EcoGII Methyltransferase in other buffers? A: NEBuffer r1.1= 75% NEBuffer r2.1=100% NEBuffer r3.1=50% rCutSmart Buffer=100% Q: What is the molecular weight of EcoGII Methyltransferase? A: The molecular weight is 40,151 daltons. Q: Can EcoGII Methyltransferase be heat inactivated? A: Yes, heat inactivate at 65°C for 10 min. Q: Is EcoGII Methyltransferase sensitive to salt? A: Yes, EcoGII Methyltransferase is sensitive to salt. 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 EcoGII Methyltransferase methylate ssDNA? A: Yes, EcoGII Methyltransferase will methylate deoxyadenosines present in ssDNA molecules. Q: Will EcoGII Methyltransferase methylate DNA/RNA hybrids? A: Although adenine residues present in the RNA strand can and may be modified, EcoGII Methyltransferase will preferentially modify the adenine residues present in the DNA strand of a DNA/RNA hybrid molecule. Please see the table on the product description page for more details on methylation levels of various substrates by EcoGII Methyltransferase. Q: Will EcoGII Methyltransferase methylate gDNA? A: Yes, EcoGII Methyltransferase will methylate genomic DNA. Please see the table on the product description page for more details on methylation levels of various substrates by EcoGII Methyltransferase. Q: Will EcoGII Methyltransferase methylate RNA? A: Yes, EcoGII Methyltransferase will methylate adenine present in RNA molecules, however to a lesser extent than DNA. Please see the table on the product description page for more details on methylation levels of various substrates by EcoGII Methyltransferase. Q: Will EcoGII methylation block restriction enzymes? A: Methylation by EcoGII Methyltransferase will block many restriction endonucleases that recognize sequences containing dA bases. Please see the table on the product description page for more details on methylation levels of various substrates by EcoGII Methyltransferase. Q: How can I increase methylation of my substrate by EcoGII Methyltransferase? A: SAM is unstable at pH 7.5, 37 °C. For extended reactions adding more SAM after 4 hours can improve results. Methylation reactions, however, are also greatly affected by S-adenosylhomocysteine. S-adenosylhomocysteine, the byproduct of the methylation reaction binds more tightly to methylases than does SAM. Inhibition by S-adenosylhomocysteine greatly reduces the reaction rate as time passes. Please see the table on the product description page for more details on methylation levels of various substrates by EcoGII Methyltransferase. Q: Can I purchase large amounts of an existing Enzyme for Innovation? A: Yes, if you have use for large amounts of an Enzyme for Innovation, please contact NEB at EnzymesForInnovation@neb.com. Please note that there may be limited availability and thus extended lead times required for large orders. Additional QC testing may also be necessary to meet your specific application and needs. Q: What are Enzymes for Innovation? A: Enzymes for Innovation (EFI) is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities that are not commercially available elsewhere at the quality that you would expect from NEB. Enzymes for Innovation graduates are enzymes that have transitioned from having unknown or exploratory applications to becoming the cornerstone of a defined application. If you have an idea for an “Enzyme for Innovation” with a suggested application that may be useful, please email us at EnzymesForInnovation@neb.com. For more information, please view video. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.