Rationale:

To conduct five more plaque assays with 10^-2 lysate to create five webbed plates that can be flooded to produce a large volume of high titer lysate for later use.

Results from Monday:

Three plates had plaques but not to the extent of a webbed plate. Two plates appeared to be lysed. The top agar control showed some contamination.

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Procedures:

1. Setup an aseptic zone.
2. Added 20µL of CEW FLE to 180µL of phage buffer to create 10^-1 dilution.
3. Added 20µL of the 10^-1 dilution to 180µL of phage buffer to create 10^-2 dilution.
4. Added 20µL of “10^-2” to a .5mL of arthro, let infect for 15 minutes.
5. Added 2mL of LB broth to two vials, “PA CEW” and “TAC CEW”
6. Added 22.5µL of 1M CaCl2 to both vials.
7. Added infected arthro to “PA CEW”
8. Added 2.5mL of 2xTop Agar to both vials.
9. Shook and plated into respective plates, “CEW 11.7.18 PA” and “CEW 11.7.18 TAC-PA”
10. Let plates solidify for 15 minutes, incubated inverted until Thursday.
11. Created four more “CEW 11.7.18 PA”s using the same procedures listed in steps 4-10.

Observations:

The 10^-2 dilution that was used was freshly made, however, the lysate was fairly old which could have resulted in some degradation of the phage. One of the plate’s top agar’s slid on the plates.

Conclusions/Next Steps:

The next step will be to check to see if the plates are webbed and proceed to flood them with phage buffer if they are webbed.