Previous Results:

• The webbed plate created on 10/22 was not completely webbed, therefore the volume used to create it was not high enough
• The Plaque Assay of Lysate #4 was positive, but had too many plaques to count and calculate the titer. It was realized that serial dilutions should have created at the time of plating Lysate #4 to ensure the titer could be calculated

“Lysate #4” Plaque Assay using 10 microliters lysate

“Lysate #2” Webbed plate using 64 microliters lysate

Objective:

• To replate Lysate #4 and dilute it out to 10^-4 to ensure plaques can be counted for titer calculations
• To corrected make plaque assays with contaminations

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. The “Lysate #4” tube was obtained and 10 microliters was added to a microcentrifuge tube containing 90 microliters phage buffer. This lysate was labeled “10^-1”
3. Step 2 was repeated until “Lysate #4 was diluted out to 10^-4
4. 10 microliters of each dilution (10^0 – 10^-4) were added to individual tubes containing 0.5 mL Arthrobacter and left to infect for 10 min
5. Overlay Agar was created for all experimental plates (5 plates) and a control plate in a 50 mL tube using 12 mL LB broth, 15 mL 2x TA, and 135 microliters CaCl2
6. 4.5 mL of Overlay Agar was added to each Arthrobacter tube with lysate and mixed. 5 mL Overlay Agar was left in the 50 mL and used for the control plate
7. All 6 agars were plated and left to harden for 15 min, and incubated for 48 hours

Results:

• Experiment was not completed, therefore there are no results to report

Next Steps:

• During the next lab, the plates will be examined and the plaque assay with less than 100 plaques will be used in a titer calculation. Once the titer is known the volume needed to web a plate using “Lysate #4” will be calculated and a plate will be made using that result.