Rationale:

To conduct five 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.

Procedures:

1. Setup an aseptic zone.
2. Added 10µL of “10^-2” to a .5mL of arthro, let infect for 15 minutes.
3. Added 2mL of LB broth to two vials, “PA CEW” and “TAC”
4. Added 22.5µL of 1M CaCl2 to both vials.
5. Added infected arthro to “PA CEW”
6. Added 2.5mL of 2xTop Agar to both vials.
7. Shook and plated into respective plates, “CEW 11.5.18 PA” and “CEW 11.5.18 TAC-PA”
8. Let plates solidify for 15 minutes, incubated inverted until Wednesday.
9. Created four more “CEW 11.5.18 PA”s using the same procedures listed in steps 2-8.

Observations:

The 10^-2 dilution that was used was three days old which could have resulted in some degradation of the phage. Two of the plate’s top agar’s slid on the plates, they were still incubated but right side up. The 4 out of the five tubes of arthro was from a batch of arthro produced in October.

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.