Rationale: Picked plaque assays from soil sample A, that have been passed for three rounds, but lost during the second-third round of purification.

Procedure:

Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone. Three plaque assays were obtained from the walk-in fridge to pick the plates once more. 90 microns of PB was added to three separate microcentrifuge caps, and picked plaques added to the solution, which was then added to 0.5mL of arthrobacter.  The formula below was used to make the solution for 4 plates (three for the experiment and one positive control).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro
• (made in a 50mL vial)

4.5mL of the solution made in the 50mL vial was added to the Arthrobacter + phage solution, which was then poured onto a plate. Plates sate for 15 minutes and then inverted and placed in an incubator at 30 degrees Celsius.

Control 10/15/18

Observations: Soil sample B came back negative. Control was contaminated as wells as soil sample A and B plates. Plates that had a successful passage of phage were obtained to continue the experiment. The three plates were passaged twice but failed the third time of purification due to many factors including contamination.

Control 9/10/18

10^0 Soil A

Soil A Plaque Assay

Soil B Plaque Assay

Conclusions/Next steps: Passage and calculate titter on Wednesday. After the third round of purification, calculate the titter needed to obtain a high titer. If the results of this experiment come back negative, the next step would to pick the plaque again, and perform another plaque assay.