Rationale: In preparation for PCR testing, soil A was washed to determine if phage are present in the soil. Plaque assay was performed to purify possible phage.

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. Plate labeled 10/17/18 was picked, added to 90 microns of PB, and then added to 0.5 microns of Arthrobacter (solution sat for about 15 mins). In a 50mL vial, the TA solution was made following the formula below (x2, 1 for the experiment and one for the 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 this solution was pippeted into the Arthrobacter + plaque solution, which was then quickly poured onto plates. Plates sat for about 15 minutes and then placed in the incubator at 30 degrees Celsius.

Soil Washing

Soil obtained from the walk-in fridge was poured into a 15mL vial (4mL mark). LB broth added and the solution was shaken for 15 minutes. The solution was centrifuged and then placed into the fridge since no filter was available in the lab.\

Observations: 

Plaques were present from 10/17/18 lab experiment. One of the plates was picked and purified in 10/22/18 experiments. The groups control was negative once again. Plaque Assay 10/17/18

Control 10/17/18

Conclusions: 

Washed soil will be used to run PCR. PCR will be run 10/29/18 since filters will be in the lab. Possible contaminations reasons:

• LB broth and 2X TA are contaminated
• Opening/Closing of microcentrifuge caps/things not done in an aseptic zone.

Positive experiments from plaque assays will be used to calculate titer.