Rationale: Pick plaque from a past successful plaque assay, that has been passaged twice.

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 9/28/18 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 (x3, 2 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.

Observations: 

Control came back negative once again, and all the plaque assays performed came back negative. Why? Possible conclusions:

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

Control 10/15/17

Conclusions: 

Perform PCR 10/22/18 to see if phage is in fact present. This test will show extract the phage’s DNA using PCR and Gel. PCR will be used to make multiple copies of DNA at a specific region. PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest. Gel electrophoresis will be used to see the DNA from PCR, which will prove if phage is in the soil sample in less than 2 hours, Multiple factors could have affected the outcome of the result, but the two predominant factors are the two mentioned above. For the past three labs, the same 2X TA/LB broth was used, and all results were negative.