Rationale:

To conduct another plaque assay with lysate from Soil E to positively confirm the presence of phage in Soil E.

Results from 10.10.18:

My plaque assay was negative and the top agar control for it was free of contamination. My spot test was negative for defined plaques, however, there was abnormal bacteria growth and an abnormal spot where the enriched lysate was spotted. The top agar control for it was severely contaminated.

Procedures:

1. Setup an aseptic zone.
2. Added 10µL of “FLE” to a .5mL of arthro, let infect for 15 minutes.
3. Added 2mL of LB broth to two vials, “PA-E2” and “TAC”
4. Added 22.5µL of 1M CaCl2 to both vials.
5. Added infected arthro to “PA-E2”
6. Added 2.5mL of 2xTop Agar to both vials.
7. Shook and plated into respective plates, “NMN 10.12.18 PA-E2” and “HMB NMN 10.12.18 TAC-PA”
8. Let plates solidify for 15 minutes, incubated inverted until Monday.

Observations/Data:

I observed LB we used was remarkably clear compared to LB previously used.

Conclusions/Next-Steps:

The next step will be to check on Monday for the presence of plaques, and if negative another soil sample will be collected, however, the new sample will be initially tested with PCR due to a lack of time. If positive the plaque(s) will be picked and another plaque assay will be conducted to begin purification.