Rationale:

The goal of today’s lab was to continue the purification process and work to getting a high titer lysate as soon as possible. The lab as a whole has not yielded very many positive phage results and does not have high enough titer lysates for TEM and sequencing.

Results from 10/29/18:

• Plaque assays for the enriched lysate, 10^0, and 10^-1 dilutions were positive for plaques.
• The 10^-2 dilution plaque assay, as expected, was contaminated due to the different top agar used in the experiment.
• Enriched lysate was littered with plaques, 10^0 had roughly 15 small plaques, and the 10^-1 had 1 plaque.
• 

Materials:

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

Procedure: 

1. Established an aseptic zone.
2. To pick a plaque, used a micropipette tip to poke 1 plaque from the 10^-1 plaque assay (avoiding bacterial lawn beneath) and inserted tip into 100-μL of phage buffer to release phage particles.  This was the new 10^0 dilution.
3. Added 10-μL of the 10^0 dilution to 90-μL of phage buffer, making a 10^-1 dilution.
4. Made a 10^-2 dilution by repeating the dilution process with the 10^-1 dilution and adding it to 90-μL of phage buffer.
5. Once diluted, 3 separate plaque assays were run with each dilution.
6. 2.0-mL of LB broth was added into 3 separate conical vials and 2.5-mL was added to a fourth top agar control.
7. Next, 22.5-μL of calcium chloride was added to each of the conical vials.
8. 30-μL of each of the diluted lysates were combined with 3 separate 500-µL quantities of Arthrobacter and left to infect for approximately 15 minutes..
9. After about 15 minutes, the diluted lysates were combined with their respective LB Broth mixtures and 2.5-mL of 2X TA was added to each of the conical vials.
10. Swirled and plated top agars immediately.
11. Once solidified, they were added to the incubator until the next lab.

Results/Data:

• The procedure went smoothly, and top agar did not solidify before plating. Lysate was decreased slightly from the previous experiment, but still should be able to yield enough phage particles to show on a plaque assay.

Conclusions:

• Phage concentration in the original lysate was not very high to begin with, and this explains the repeated negative plaque assay results with diluted lysates. All of the phage had been diluted out and there simply was not enough to form a plaque. Although the plaque amount was relatively small on these dilutions, there were still plaques to be picked to continue the purification process to help work towards amplification and flooding plates.

Next Steps:

• The next steps for this experiment are to continue with 1-2 more rounds of purification until phage yields consistent plaque morphologies. The ultimate goal is to work towards gathering a high titer lysate as quickly as possible as time is running out in the semester and a high titer lysate is needed for TEM.