Journal: PLoS ONE

Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

doi: 10.1371/journal.pone.0114208

Figure Lengend Snippet: A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

Article Snippet: Portions of lysates were further heat inactivated (HI) at 95°C for 10 min. One µg of the commercial plasmid pECFP-N1 (Clonetech, CA, USA) was subjected to either MS11 P+ lysate or HI lysate together with CutSmart buffer (New England Biolabs, Ipswich, MA, USA) for 1 h. As controls, circular/uncut pECFP-N1 was used as well as HindIII (Roche, Mannheim, Germany) linearized pECFP-N1.

Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Positive Control, Purification, Negative Control, Incubation