Previous Results:

• No plaque assays were created during last lab, only titer calculation and lysate collection was conducted, therefore there are no results to report.

Objective:

• To correctly plate a 10^0 lysate webbed plate for phage extraction later on
• Correctly plate 10^0 lysate plaque assays to be prepared if the 10^0 webbed plate is not webbed

Procedure: Webbed Plate

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and Ethanol burner
2. 15 mL tube containing “10^0 #1” lysate was obtained
3. Using titer/volume calculations from last lab, 64 microL of lysate was added to 0.5 mL of Arthrobacter and left to infect for 10 min
4. Overlay Agar was created using 2 mL LB broth, 2 mL 2xTA, 22.5 microL CaCl2, and 0.5 mL Arthrobacter infected with phage
5. Mixture was mixed then poured on a plate labeled “Webbed 10^0 #1” (since the lysate was from the 10^0 #1 lysate)
6. Plate was left to harden for 15 min then incubated for 48 hours

Procedure: Plaque Assays

1. Aseptic technique was used
2. 15 mL tubes containing “10^0 #2” and “10^0 #3” lysate were obtained
3. 10 microL of each lysate were pipetted into separate tubes 0f 0.5 mL Arthrobacter and left to infect for 10 min
4. Overlay Agar was created for the 2 experimental plates and a control plate using 6 mL LB broth, 6 mL 2xTA, and 67.5 microL CaCl2
5. The Overlay Agar was separated equally into 3 separate 50 mL tubes
6. 2 infected Arthrobacter tubes were poured into respected Overlay Agar mixtures
7. The 3 plates were poured and left to harden for 15 min, then incubated for 48 hours

Results:

• The experiment was not completed during this lab, therefore there are no results to report.

Next Steps:

• During the next lab the plates will be examined. If the ” webbed 10^0 #1″ plate is completely webbed, then the phage will be extracted and the next step of the project will be conducted. If the webbed plate is not completely webbed, then the titers of the 2 10^0 plaque assays will calculated and webbed plates will be created using those calculations.