HaeIII Methyltransferase

Protocol tips

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	S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.

Protocol tips
S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.

Publication protocol

Analysis of methylation by M.CbeI
To identify the site of methylation by M.CbeI, unmodified DNA was compared to that after methylation and the changes were determined by direct visualization in automated DNA sequencing chromatograms , . methylation of pUC18 DNA isolated from DH5α () was carried out using M.HaeIII (NEB) and purified M.CbeI. The efficiency of methylation was determined by cleavage of the methylated and unmethylated DNA with HaeIII (NEB), purified CbeI, and cell free extracts (CFE). Digested DNA was displayed by agarose gel electrophoresis and visualization using ethidium bromide staining. Automatic sequencing was performed using primers M13F(−20) and M13R(−20) in an ABI automated PRISM big-dye-terminator system (Macrogen, Inc, Maryland). Sequences were analyzed using the Chromas Lite v2.01 (Technelysium Pty Ltd.) and ABI chromatograms were compared by aligning the Sequencing traces and using SeqDoc .

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