November
8
11.7.18 Plaque Assay for High Titer
11.7.18 Plaque Assay for High Titer
Rationale: Since the results from Monday (11/5) showed inconsistent results and problems with slipping top agar overlays, it was found necessary to redo the procedure to get five new plates that will be used to begin to raise the titer of the lysate.
Procedure:
- Established an aseptic zone.
- Added 20µL of 10^0 titer to 180µL of phage buffer to create 10^-1 lysate. Repeated with 10^-1 lysate to create 10^-2.
- Added 20μL of 10^-2 lysate to 5 different 0.5mL Arthrobacter. Let sit for 10 minutes.
- Obtained 5 plates and labeled “CEW 11/7 10^-2”.
- Added 12mL LB Broth to conical tube.
- Added 135 μL CaCl2 to conical tube. Swished to mix.
- Added 2mL of solution from conical tube to each tube with 0.5mL arthrobacter andlysate.
- Added 2.5 mL 2X Top Agar to each tube. Swished and poured onto 5 separate plates.
- Poured all tubes onto 6 separate plates (1 plate was top agar control and was labeledaccordingly).
- Let sit for 10 minutes before placing them in the incubator.
Results and Observations:
- The plates created displayed very inconsistent results. Two plates displayed the expected results, two plates showed severe overlay slipping, and the last experimental plate showed complete lysing of arthrobacter. The complete killing of arthrobacter was thought to be because the lysate was not mixed, so a small pocket of highly concentrated phage was used erroneously. The control plate showed smaller signs of contamination that had not been observed before in the lab – small areas of discoloration with one small, highly-colored center.
- One of the plates that was created today had a top agar overlay that slipped. The rest appeared to set correctly and should display correct results.
Conclusions and Next Steps:
- The next step for this procedure will be to flood the plates and use the new lysate of higher concentration to run another plaque assay. This will likely result in a high-titer lysate, which is the end goal of the procedures that have been run. Based on results from Monday, the inconsistent amounts of LB Broth did have a negative effect on the ability of Top Agar overlay to set properly on the plate. This will be accounted for and be done more carefully in future experiments using the Plaque Assay procedure.