Journal: Scientific Reports
Article Title: Expression and purification of a single-chain Type IV restriction enzyme Eco94GmrSD and determination of its substrate preference
Figure Lengend Snippet: Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in NEB buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).
Article Snippet: GmrSD enzyme activity assay T4 (glc-5hmC), T4gt (5hmC), and λ DNA (Dam+ Dcm+ ) or 5hmC-modified PCR DNA were digested with purified GmrSD enzyme in NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT) supplemented with 1 mM ATP at 37°C for 1 h unless specified otherwise.
Techniques: Polymerase Chain Reaction, Generated, Modification