Previous Results:

• The webbed plate created 10/26, labeled “Lysate #4 Webbed”, was not completely webbed, meaning the volume used to make the web was not high enough
• The plate, “10^0 #4”, that was flooded (10/26) had completed the 48 hour time period with phage buffer, the experiment will be continued using this collected lysate

Objective:

• Correctly filter and obtain the lysate from the flooded plate
• To make plaque assays and obtain at least one webbed plate to calculate the titer of the lysate

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. The lysate on the plate from last lab (10/26) that was flooded, labeled “10^0 #4”, was obtained using a syringe and filter. It was filtered into a 15 mL tube and labeled “Lysate #5”
3. Serial dilutions were made using Lysate #5, 10 microliters were added to a microcentrifuge tube containing 90 microliters phage buffer. This lysate was labeled “10^-1”
4. Step 2 was repeated until “Lysate #5″ was diluted out to 10^-2
5. 10 microliters of each dilution (10^0 – 10^-2) were added to individual tubes containing 0.5 mL Arthrobacter and left to infect for 10 min
6. Overlay Agar was created for all experimental plates (3 plates) and a control plate in a 50 mL tube using 8 mL LB broth, 10 mL 2x TA, and 90 microliters CaCl2
7. 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
8. All 4 agars were plated and left to harden for 15 min, and incubated for 48 hours

Results:

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

Next Steps:

• During the next lab the plaque assays will be examined and if webbed, then the plates will be flooded for a future calculation of a high titer