New England Biolabs, M0219S, TaqI Methyltransferase

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 65°C in a total reaction volume of 20 μl against cleavage by Taq I restriction endonuclease. Reaction Conditions 1X rCutSmart™ Buffer Supplement with 80 µM S-adenosylmethionine (SAM) Incubate at 65°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) Control Protection Assay Protection Assay Conditions: Taq I Methyltransferase is incubated with 1 µg λ DNA in 20 μl 1X rCutSmart Buffer and 80 µM S-adenosylmethionine, for 1 hour at 65°C. The extent of protection by Taq I Methyltransferase is determined by the addition of 30 μl 1X rCutSmart Buffer and 10 units of Taq I restriction endonuclease. Incubation for 15 minutes at 65°C is followed by analysis on an agarose gel. Storage Buffer 10 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No Molecular Weight Theoretical: 47845 daltons 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: What is the activity of TaqI Methyltransferase at 37°C? A: Taq I Methyltransferase gives 25% activity at 37°C. Q: What is the activity of TaqI Methyltransferase at 50°C? A: The activity is nearly 100%. The enzyme can be used on DNA embedded in low melting agarose at this temperature. The plug won't melt. Q: Does TaqI Methyltransferase require MgCl2? A: No. Q: Does TaqI Methyltransferase require BSA? A: Yes, BSA or Recombinant Albumin (rAlbumin) increase activity of TaqI Methyltransferase by 4-fold. Q: What is the activity of TaqI Methyltransferase in other buffers? A: The activity of TaqI Methyltransferase is approximately 50% in NEBuffer 3. Q: What is the molecular weight of TaqI Methyltransferase? A: The molecular weight is 47,845 daltons. Q: Is TaqI Methyltransferase used in any special techniques? A: Yes. TaqI Methyltransferase (TCGA*) can be used to create the 8 base recognition site TCGA*TCGA that can be cleaved with DpnI. DpnI recognizes GATC and cleaves when the DNA is methylated. Do not overdigest the DNA because DpnI will cut hemimethylated DNA slowly (TCGATC). 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: 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.