Rationale:

Due to the possible presence of plaques on the plate, two will be picked and put into phage buffer and a plaque assay will be made with the original lysate.

Procedure:

• First, the previous plaque assays were examined to find some contamination in the negative control and some presence of phage in the plates.
• The lab table was wiped down with CiDecon and Ethanol, after which an aseptic zone was set up to prevent contamination.
• The phages were located and picked using a pipette tip, then inserted into 90 µL of phage buffer and stored away.
• Then, 0.5 mL of Arthrobacter and 1 µL were mixed together and left alone for 10 minutes.
• Next, LB broth (2 mL) and CaCl2 (22.5 µL) were combined.
• After 10 minutes, the 2x Top Agar (2.5 mL) was added to the top agar solution along with the original lysate and Arthrobacter.
• The solution was then poured onto the 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 amount of contamination in the negative control has significantly decreased compared to other plaque assays conducted in the past.

The phages in the plaque assay are located in the center and are very small.

Conclusion:

First, the tables were cleaned with CiDecon and ethanol along with an aseptic zone. Plaques were spotted, picked (2), then put into 90 µL of phage buffer. However, due to the contamination in the negative control, another plaque assay was made. The lysate and Arthrobacter were mixed together to infect. The LB broth and CaCl2 were added together. After 1o minutes, the 2X top agar was added along with the lysate and Arthrobacter onto the plate. It was left alone to solidify then put into the incubator.

Future Plans:

In the future (10/10/18), the plaques that were picked and put into phage buffer will be used to make a plaque assay. Before doing that, the lysates will be diluted.