Rationale:

The purpose of todays lab was to analyze the plaque assay results from the previous lab. If plaques were present, amplification and purification processes were to be continued, but if no plaques were present, plaques would have to be picked to be diluted again.

Results from 10/15/18:

• 
• Top Agar had split due to not fully solidifying before being put in the incubator, this can be examined by the sliding of the top agar from the side of the plate.
• Top Agar control was uncontaminated.
• Plaques were not present, however plates were cracked as well.

Materials:

• Phage Buffer
• Plaques to Pick
• LB Broth
• 2X TA
• Calcium Chloride

Procedure:

1. To pick a plaque, used a micropipette tip to poke 10 plaques separately the top agar and lifted out (avoid bacterial lawn beneath) and inserted tip into 100-μL of phage buffer.  This was the 10^0 dilution
2. Added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
3. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
4. Once diluted, 3 separate plaque assays were run with each dilution.
5. 2.0-mL of LB broth was added into 3 separate conical vials.
6. Next, 22.5-μL of calcium chloride was added to the conical vials.
7. 10-μL of each of the dilutions was combined with 3 separate 0.5 mL quantities of Arthrobacter and left to infect.
8. After about 15 minutes, the diluted lysates were combined with their LB Broth mixtures and 2.5-mL of 2X TA was added to the broths.
9. Swirled and plated top agars immediately.
10. Once solidified, they were added to the incubator until the next lab.

Results:

Top Agar had solidified the moment the top agar had been plated, causing it to slide around the side of the plate without fully solidifying. This caused the top agar to solidify unevenly. This is going to cause the top agar to split, not allowing phage to infect.

Conclusions:

Top Agar will most likely split due to the improper solidifying that took place, which will not allow plaques to form. The top agar was not hot enough to be poured and plated effectively.

Next Steps:

The next steps will be to analyze the plaque assay results for plaque presence and morphology. Another plaque assay procedure will most likely be performed as the top agar did not solidify properly, causing the top agar to split.