Rationale: The purpose of this lab is to pick a plaque and conduct a plaque assay for soil C plaque purification, and to conduct a plaque assay for soil C repeat.

Description of Procedures:

1. The workstation was cleaned using aseptic technique and an aseptic zone was created.
2. A plaque was picked from the plaque assay 1 and mixed with 100 ul of phage buffer and vortexed.
3. The enriched lysate from soil  C repeat was spun again in the centrifuge for 10 minutes at 5000 x g.
4. Percent soil calculations were conducted. 4 ml of soil were measured. Sand was found to be 25%, silt was found to be 50%, and clay was found to be 25% of the soil.
5. The percent water was then found for soil C repeat. 0.684 g were lost from the original 4.058 g, calculating a percent water of 16.86%.
6. Soil C repeat supernatant was filtered with a 0.22 um syringe filter.
7. 10 ul of soil c repeat lysate was mixed with 0.5 ml of arthrobacter and was allowed to sit for 10 minutes. 10 ul of the phage buffer containing the phage from soil C was also mixed with 0.5 ml of artrobacter and allowed to sit.
8. The top agar solution was made for 5 plates. 10 ml of LB broth, 112.5 ml of CaCl2, and 12.5 ml of 2x TA were added to a tube. 4.5 ml of this solution was then poured into the 0.5 ml of arthrobacter and immediately poured onto plates labeled LIP 10-8-18 PA 2, LIP EAG SS Control 10-8-18, and LIP SS EAG Soil C repeat.
9. The plates were allowed to sit for 10 minutes and then inverted and stored in the incubator until the next lab.
10. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations/Results:

• Observations:
  • Plaque Assay 1 had plaques
  • Enriched lysate from soil C repeat was foggy, so it was respun and refiltered
  • Control plate had bubbles
  • Metadata for soil c repeat was very different from the original soil c metadata
• Results:
  • % Water- 16.86%
  • % Sand- 25%
  • % Silt- 50%
  • % Clay- 25%

Interpretations/Next Steps:
The procedures were complete for soil C repeat and the plaque from soil C. The next steps will be to complete a plaque assay for soil C unless it is webbed, and will then be flooded, and to check for plaques on soil C repeat.