Genomics and Proteomics of Mycobacteriophage Patience, an Accidental Tourist in the Mycobacterium Neighborhood

Pope, Welkin H.; Jacobs-Sera, Deborah; Russell, Daniel A.; Rubin, Daniel H. F.; Kajee, Afsana; Msibi, Zama N. P.; Larsen, Michelle H.; Jacobs, William R.; Lawrence, Jeffrey G.; Hendrix, Roger W.; Hatfull, Graham F.

Electron microscopy. CsCl gradient-purified Patience particles were applied to glow-discharged Formvar-and carbon-coated copper grids (400 mesh) (Ted Pella). They were stained with 1% uranyl acetate and imaged with a Morgagni 268 transmission electron microscope fitted with a Hamamatsu Orca HR side-model digital camera and AMT540 software. SDS-PAGE. Patience particles were concentrated and purified via CsCl gradient and ultracentrifugation. The visible phage band was dialyzed against two changes of phage buffer; 500 l of the dialyzed CsCl band was pelleted by a 30-min spin at 14,000 rpm in a microcentrifuge. The pellet was resuspended in 75 l of 20 mM dithiothreitol (DTT), and then 2 l of 0.5 M EDTA and 1 l of 1 M MgSO 4 were added. The phage was disrupted by being heated to 75°C for 2 min and then sonicated on ice six times for 30 s to disrupt the DNA. The sample was then mixed with 25 l of 4ϫ SDS sample buffer and heated in a boiling bath for 3 min at 95°C. The sample was electrophoresed through a 12% polyacrylamide gel containing SDS and stained with Coomassie brilliant blue in methanol.