CpG Methylase (M.SssI)   • For complete in vitro methylation of DNA for methylation analysis. • Methylation of chromatin DNA for DNA accessibility studies. • Inhibition of endonucleases with overlapping CpG sequence recognition. • [3H]-labeling of DNA.   • Storage: Store at -20°C for up to 12 months. Avoid repeated freeze/thawing of reagents. Prolonged storage is at ≤ -70°C. • Enzyme Concentration: 4 units/µl • Unit Definition: One unit is defined as the amount of enzyme required to “protect” 1 µg of Λ DNA against cleavage by BstUI restriction endonuclease in a total reaction volume of 20 µl for 1 hour at 37°C. • Reaction Conditions: CpG Methylase in 1X CpG Reaction Buffer w/ 600 µM SAM. Incubate reaction mixtures at 30°C (Performing the Methylase reaction at 30°C rather than 37°C promotes optimal reaction kinetics). • Inactivation: Heat-inactivate the enzyme at 65°C for 20 minutes. • Standard Reaction Setup: The setup (below) is an example of a typical CpG methylase reaction in a 20 µl final reaction volume. The reaction volumes can be adjusted accordingly depending on experimental requirements (see protocol).   2 µl 10X CpG Reaction Buffer 1 µl 20X SAM (S-adenosylmethionine), 12 mM 4 µl DNA at 100-250 ng/µl 1 µl CpG Methylase (4 units/µl) 12 µl Water    Incubate at 30°C for 2 hours.   The CpG Methylase (EC 2.1.1.37) from Zymo Research completely methylates all cytosine residues (C5) in double-stranded, non-methylated and hemi-methylated DNA having the dinucleotide sequence 5…CpG…3. The recombinant CpG Methylase is isolated from an E. coli strain that expresses the Methylase gene from Spiroplasma sp. strain MQ1. The reaction conditions are optimized to maximize the processivity of the enzyme to ensure rapid, complete, and reproducible methylation of DNA for accurate DNA methylation analysis.   The CpG Methylase from Zymo Research catalyzes complete methylation of the CpG sites in DNA. Methylase activities of CpG Methylase from Zymo Research versus that of another supplier were tested for complete methylation of equivalent amounts of a linearized plasmid DNA using reaction conditions recommended by the supplier. “Completion” of CpG methylation was assessed by resistance to digestion with a methylation-specific endonuclease (HpaII) and subsequently analyzed in an agarose gel. As shown in the figure above, the CpG Methylase from Zymo Research completely methylated the CpG sites in the DNA whereas that of the other supplier did not. Samples were assayed in duplicate.

ORDERING INFORMATION

Product                      Catalog No.           Size             Price(RMB)

CpG Methylase (M.SssI)     E2010                 200 Units       

                             E2011                 400 Units