Plaque Isolation and Sequencing of Mycobacteriophages

Lysates were made from plaques forming on M. tuberculosis strains. Lysates on M. smegmatis were amplified under BSL3 conditions and were filtered twice using 0.2-μm filters. Aliquots of lysates (1 ml) were serially diluted and plated onto agar lawns for isolated plaques. Isolated plaques (n = 8 to16) were picked using a 0.2 to 10 μl micropipette tip into 50 μl of phage buffer (21 (link)) in 0.2-ml PCR strip tubes. An aliquot of 5 μl containing phage particles picked from agar was used as the template for PCR utilizing Muddy gp24-specific primers (Table S2) along with Q5 master mix (New England BioLabs) following PCR according to the manufacturer’s enzyme conditions. Amplicons were verified by gel electrophoresis and were sequenced (Genewiz).