Gel doctm eq system

Relaxed circular plasmid DNAs [pET-15b(+), pET-21a(+) and pET-28a(+)] were prepared using a Wizard Plus SV Miniprep kit (Promega). Recombinant HP0268 proteins (1, 2, 4 and 8 μM) were incubated at 37°C for 30 min with each circular DNA (10 ng/μl) in a final volume of 30 μl. The reaction was conducted in 20 mM NaH₂PO₄/Na₂HPO₄ buffer (pH 7.0) containing 150 mM NaCl. The reaction conditions included variable pH levels (pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5) and the presence of 1 mM metal ions (Ca²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Zn²⁺). The commercial enzymes Nt.BsmAI and NdeI (NEB, Inc.) were used as references to validate the DNA nicking and double-cutting activity of HP0268, respectively. Ten units of the two enzymes were used in a final volume of 30 μl. Reactions were stopped by the addition of 6 μl of a 6×loading buffer (100 mM EDTA, 0.7% SDS and 70% glycerol), and the reaction solutions were loaded onto a 1.0% agarose gel containing 0.5 mM TBE buffer (90 mM Tris borate and 2 mM EDTA) and electrophoresed in the same buffer. DNA was visualized under UV light at 254 nm using ethidium bromide staining. At least three independent experiments were performed for each protein and yielded similar results. The digested, nicked and relaxed circular plasmids were quantified using the Gel Doc^TM EQ system (Bio-Rad, Inc.).