Previous Results:

• Plaque assays from second purification that were created during the last lab (9/19) were all positive and the control did not have any contamination.

Objective:

• Pick a plaque from the plaque assay made during last lab
• Conduct a third series of serial dilutions and plate the diluted lysates with phage

Procedure:

1. Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
2. A plaque was picked from the 10^0 plate by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming a new 10^0 dilution
3. 10 microL of new 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
4. Step 3 was repeated to create a 10^-2 dilution
5. The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
6. A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
7. The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
8. All experimental tubes and the control tube were plated and left to harden for 15 min
9. Plates were placed in incubator for 48 hours.

Results:

• Results will not be available until next lab (9/26) to see if the phage were plated correctly and there are positive results.

Conclusion:

• The results from the previous lab were positive
• The serial dilution was correctly conducted using a picked plaque from the positive plate

Next Steps:

• During the next lab (9/26) the plates with the third round of serial dilutions will be examined for phage. If positive, then the experiment will move on to the next steps of calculating a high titer to create a webbed plate.