Figure 1
From: Plasmid DNA nicking- a Novel Activity of Soybean Trypsin Inhibitor and Bovine Aprotinin
Soybean Trypsin Inhibitor (STI) affects migration pattern of plasmid pGL3. pGL3 plasmid (0.5 ug) in Buffer A (25 mM Tris-acetate (pH 7.5), 100 mM potassium acetate, 10 mM magnesium acetate) was incubated with 0–4 ug of STI (A), for 0–2 h (B), in the presence of 0-0.9 M NaCl (C) or 0-15 mM EDTA (D). (E) prior to incubation with STI (S), the plasmid was linearized with KpnI (Kp/S), or vice-versa (S/Kp) or co-incubated with both (SKp). (F) longer run of STI or KpnI treated plasmid. (G) STI treated or untreated pGL3 was purified using a PCR reaction purification kit or phenol-chloroform extraction prior to run on a gel. All samples were analyzed on 2% agarose gels stained with ethidium bromide. The intensity of the slower moving bands was quantified using NIH ImageJ. The intensity of the control (− or 0 lane) was set to 1.0. The intensity of the slower migrating band increases with STI amount and incubation time, but decreases in the presence of NaCl, while presence of EDTA has no effect. The STI induced slower migrating band disappears with linearization using digestion with Kpn I, however, remains intact following purification by phenol-chloroform extraction or other method. Samples in each gel were prepared in parallel. All gels were processed using Microsoft Word “Corrections” for brightness and contrast applied equally across the entire image including controls. The full length gels are presented as Supplementary Information with corresponding figure numbers as in the main article.
