Rationale: Created a plaque assay with newly enriched Soil Sample E, and collect remaining soil metadata.  

Procedure: 

1. Wiped down table with 70% Ethanol and Cidecon, and created an aseptic zone with ethanol burner 
2. Obtained enriched lysate from refrigerator and filtered the lysate into a 50 mL tube using a syringe.  
3. Ended up with about 7 mL of enriched lysate.  
4. Added 10 uL of enriched lysate into the red capped vial containing the 0.5 mL of Arthro, and waited 10 minutes.  
5. While waiting, collected soil sample E metadata. 
6. Obtained weigh boat and from hood to obtain results for percent sand, silt, clay, and water. 
7. After 10 minutes, added 67.5 uL of CaCl2, 6 mL of LB Broth, 7.5 mL of Top Agar into a 50 mL tube.  
8. Mixed the solution using a pipette and pipetted 4.5 mL of solution into red vial containing enriched lysate and Arthro.  
9. Poured the 5 mL solution into each plate, and let it set for 10 minutes.  
10. The remaining Top Agar solution was poured into the control plate.  
11. Let plates sit for 15 minutes to harden.  
12. Placed plates inverted in the incubator to be observed next class period.

Observations/Results: 

Soil Metadata: 

• % sand silt clay 
  • % sand = (3.5 mL) / (5.5 mL) = 63.63% 
  • % silt = (1.5 mL) / (5.5 mL) = 27.27% 
  • % clay = (0.5 mL) / (5.5 mL) = 9.1 % 

• % water :  
  • Weigh boat = 2.475 g 
  • Weigh boat + soil (before) = 5.817 g 
  • Weigh boat + soil (after) = 5.084 g  
  • % water = 21.93% 

Plaque Assay:  

While plating the plaque assay today, noticed that bubbles formed near the edges of the plates.  

Next steps: 

Next class, I will observe the plaque assay plates. If the plate is negative then I will perform a new method, PCR. If the plate is positive, then I  will pick the plaque and start on the amplification and purification process.