New England Biolabs, M0214S, HpaII Methyltransferase

HpaII Methyltransferase recognizes the same sequence as the MspI Methyltransferase, but modifies the internal cytosine residue (C 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 HpaII restriction endonuclease. Reaction Conditions 1X rCutSmart™ Buffer Supplement with 80 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) Control Protection Assay HpaII Methyltransferase is incubated with 1 µg of λ DNA in 10 μl 1X rCutSmart Buffer, supplemented with 80 µM S-adenosylmethionine, for one hour at 37°C followed by 15 minutes at 65°C. The extent of protection by HpaII Methyltransferase is determined by the addition of 40 μl NEBuffer 1 supplemented with 10 mM MgCl 2 and 10 units of HpaII restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on agarose gels. Storage Buffer 50 mM Tris-HCl 150 mM NaCl 0.1 mM EDTA 5 mM TCEP 200 µg/ml BSA 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 20 minutes Molecular Weight Theoretical: 40397 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: Can HpaII Methyltransferase be heat inactivated? A: Yes. Inactivate at 65°C for 20 minutes. Q: Does HpaII Methyltransferase require MgCl2? A: No. Q: What is the molecular weight of HpaII Methyltransferase? A: The molecular weight is 40,397 daltons. Q: Can HpaII methylated DNA be used to transform E. coli? A: HpaII methylated DNA is mcrA sensitive. An E. coli strain lacking mcrA must be used or the DNA will be degraded. 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.