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Isolation Of Phages Lab Report

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Isolation of Bacteriophage from Mycobacterium smegmatis Abstract In our experiment, we wanted to isolate phage virus from Mycobacterium smegmatis. Phage viruses affect bacteria. Life cycles of phages are categorized into two; lytic and lysogenic. Lytic phages lyse of their host thereby killing them, while lysogenic phages integrate into host DNA. However, life cycle of phages cannot be easily predicted. In this experiment, mycobacteria phage DNA was isolated from a lysogenic phage. Plaques form when small dilution of phage was incubated with M. smeg and plated on L-agar plates is purified amplified and ran through gel electrophoresis to identify cluster. Phage therapy has proven to be a breakthrough to antibody resistant bacteria. This makes it important to isolate phage virus for specific bacteria. …show more content…

Gel is checked in 20 minutes to determine how far blue dye has travelled. Gel is carefully removed (with gloves) and placed under UV light to take pictures. Phage Cluster Identification and Agarose Gel Electrophoresis ll 20µL of sterile water and 5µL of phage genomic DNA is added to tube. Diluted DNA (2µL) was added to each PCR tube. Tubes was placed in thermo cycle and program started. 5µL of DNA loading dye was added to each PCR tubes. 20µL of each PCR sample is loaded into gel. Gel is started. Photograph of gel was taken. Results Concentration of phage was found to be 8.8 x 107 pfu/mL after phage titer assay activity. Plaque on phage was cloudy and turbid. Figure 1 shows plaque phage. Figure 1. Plaque phage. For this experiment a small dilution of phage was incubated with M. smeg and plated on L-agar plates. Mycobacteria phage had a cloudy morphology. Agarose Gel Electrophoresis Digest DNA with dye was loaded to loading dye. Movement of DNA fragments was determined to specify phage DNA. Figure 2 shows digest of phage

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This procedure requires plaque isolation, which enables characterization of a single phage population. A single plaque was chosen from the plate by circling the plaque with a sharpie and touching it with a sterile wooden applicator. The plaque was aseptically streaked onto a new agar plate. The wooden applicator was disposed, and a new applicator was obtained to start a new streak, starting from the end of the previous streak and repeated for a total of 3 streaks on the plate. The end of the third streak was marked with an “X” using a sharpie.

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Molecular typing and clonal relationships between isolates were determined by pulsed-field gel electrophoresis

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