Plaque Assay

11/12/18

Rationale: My plate from the previous plaque assay came back with contaminant, and PCR doesn’t seem to be able to detect even known positives. Therefore, I will be performing another plaque assay in hopes of finding the phage from the spot test last week.

Procedure:

1. Enriched 10µL of McClane soil lysate and Lauren’s Bookstore soil in 0.5mL each of arthrobacter for 30 minutes. Also used 10µL of phage buffer and 0.5mL of arthrobacter for a arthrobacter control.
2. Made top agar by mixing 8mL of LB Broth, 90µL of CaCl2, and 10mL of 2X TA.
3. Put 4.5mL of top agar into control plate then pipetted 4.5mL into each tube of arthrobacter.
4. Plated each tube of solution and let sit for 15 minutes then inverted and placed in incubator for 24 hours.

Observations:

My plate was fully lysed. The arthrobacter lawn looked fine and the TA control was also uncontaminated. Lauren’s sample didn’t have any plaques.

Conclusions and Next Steps: My phage seems to be extremely aggressive so I will have to dilute quit far. I will have to conduct a spot test or multiple plaque assays for each dilution in hopes of finding a decent plaque or webbed plate to either flood or purify from. Also I believe that the increased enriching time definitely had some effect on the ability for me to perceive the presence of my phage in my sample.