New England Biolabs, M0226M, CpG Methyltransferase (M.SssI)

Related Categories Methylation Sensitive and Methylation Dependent Restriction Enzymes for Epigenetics,, Methyltransferases for Epigenetics,, 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 BstUI restriction endonuclease. Reaction Conditions 1X NEBuffer™ 2 Supplement with 160 µM S-adenosylmethionine (SAM) Incubate at 37°C 1X NEBuffer™ 2 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 1 mM DTT (pH 7.9 @ 25°C) Control Protection Assay A 20 μl reaction in NEBuffer 2 containing 1 μg of Lambda DNA, supplemented with 160 uM SAM and 1 unit of M.Sss I incubated for 1 hour at 37°C, followed by heat inactivation at 65C for 15 minutes, results in 95% protection from digestion with 10 units of BstU I in rCutSmart buffer incubated at 60°C for 30 minutes as determined by agarose gel electrophoresis. Storage Buffer 10 mM Tris-HCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 200 µg/ml BSA pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes 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 using SssI Methyltransferase 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. If more than 1 μg of DNA is being methylated, increase the SAM concentration from the recommended 160 μM to 640 μM. A maximum of 4 μg per 20 μls reaction can be methylated using 640 μM SAM. Use 2-4 units per microgram. Use concentrated SssI M0226M to keep glycerol concentration low. Increase the reaction time to 4 hours. Due to SAM instability and the fact that the byproduct of the reaction (S-adenosylhomocysteine) is an inhibitor overnight reactions are not recommended. Clean up the DNA. Remove PCR primers and other small DNA using a spin column or similar procedure. Drop dialyze the sample to remove salts. Q: What is the activity of SssI Methyltransferase in other NEBuffers, including rCutSmart? A: The activity is 25% in NEBuffer 3 and 10% in NEBuffer 1 and 4. It can also be used in rCutsmart Buffer. 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: Can the SssI Methyltransferase methylate single stranded DNA? A: No. Q: Can SssI Methyltransferase be used for generating a positive control for methylation-specific PCR or bisulfate sequencing? A: Yes, SssI can be used to generate a positive control for the procedure. We suggest 4 units per μg of DNA for 2 hours. Bisulfate is used to convert unmethylated C to T leaving methylated C as C in the substrate DNA. A sample of modified and unmodified substrate is used in a PCR reaction. The differences seen in sequencing PCR products indicate methylated verses unmethylated regions (1). (1) Frommer, M. et al, Proc. Natl. Acad. Sci. USA (1992) Vol. 89, pp1827-1831. Q: What is the specific activity of SssI Methyltransferase? A: The specific activity is approximately 41,000 units/mg. Q: Does SssI Methyltransferase require magnesium in the buffer? A: No. With magnesium the enzyme reaction is distributive and the enzyme exhibits topoisomerase activity. Without magnesium the reaction is processive (2). (2) Matsuo et al, Nucl. Acids Res. (1994) Vol 22, 5354-5359. Q: Can DNA be radiolabeled with SssI Methyltransferase? A: Yes. Click for a complete protocol . Q: Can SssI methylated DNA be used to transform E. coli? A: SssI methylated DNA is mcr sensitive. An E. coli strain lacking mcrA, mcrBC and Mrr must be used or the DNA will be degraded. Q: What is the molecular weight of SssI (CpG) Methyltransferase? A: The molecular weight is 42,000 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.