Rationale:

To make plaque assays with the plaques picked last class and to also pick new plaques from the latest plaque assay made.

Procedure:

• First, the lab table was wiped down with ethanol and CiDecon and an aseptic zone was set up to prevent contamination.
• Using the most recent plaque assay, phages were spotted, picked, then put into 90 µL of phage buffer, making the 10ˆ0 dilution.
• Then, 10 µL of lysate and phage buffer from the 10ˆ0 dilution was added to 90 µL of phage buffer to make 10ˆ-1 dilution.
• Then, 0.5 mL of Arthrobacter and 100 µL of 10ˆ0 dilution were mixed together and left alone for 10 minutes.
• The same process occurred for the 10ˆ-1 dilution.
• Next, two sets of combined LB broth (2 mL) and CaCl2 (22.5 µL) were made.
• After 10 minutes, the 2x Top Agar (2.5 mL) was added to the top agar solutions along with the lysate and Arthrobacter.
• The solutions were then poured onto their respective plate and given enough time to solidify.
• After solidification, the plates were placed into the incubator and left there for 48 hours.

Results and Analysis:

The possible plaques are the small, clear areas in the center of the plate.

Conclusion:

Due to the possibility of plaques on the recent plaque assay, those plaques were picked and placed in phage buffer, becoming 10ˆ0 dilution. Then, 10 µl of lysate was transferred from the 10ˆ0 dilution to another vile of phage buffer which made 10ˆ-1 dilution. Arthrobacter was added to both dilutions and left alone for 10 minutes to infect. While infection, two sets of LB broth and calcium chloride were mixed. After 10 minutes, the dilutions along with the Arthrobacter were added to their culture. 2X Top Agar was added then poured onto their respective plates.

Future Plans:

In the future (10/15/18), the plates will be checked for the presence of plaques. If there are plaques, then dilutions of the plaque will be continued. If not, another picked plaques will be tested to determine whether or not it is a plaque.