Rationale:

Created two webbed plates using a previously known positive lysate to test and have as backup to calculate a high titer plate.  

Procedure: 

1. Created a aseptic zone using ethanol burner and cleaned with Cidecon and 70% ethanol.  
2. Obtained 20 uL of lysate and added 10 uL into two tubes containing 0.5 mL of Arthro.  
3. Let it sit for 10 minutes to infect.  
4. A 50 mL tube was obtained and 6 mL of LB Broth was added.  
5. 67.5 uL of CaCl2 was pipetted into the 50 mL tube as well.  
6. After 10 minutes were up, 7.5 mL of Top Agar was added was added into the 50 mL tube as well.  
7. Pipetted 4.5 mL of solution into each of the tubes containing Arthro and lysate.  
8. Poured each tube into plates and let it set.  
9. Poured remaining solution onto a plate to represent the control.  
10. Waited 15 minutes until the gels solidified.  
11. Inverted and placed into incubator to be viewed next class.  

Observation/ Results: 

The plate that was plated using 30 uL did not create a high titer. The new lysate created by our team members however produced a webbed plate. The control plate had contamination caused by the LB Broth used.  

Plaque Assay Plate using 30 uL of Adopted Lysate

Contaminated Control Plate

Next Steps/ Conclusion: 
Using the webbed plate, we will try to find the titer of the solution and if it’s a high enough titer then we will continue by viewing the phages and using a TEM. If the plates are not webbed or if they do not produce desired results, then we will try to web the plate again and find the titer of it.