Rationale: Perform a plaque assay to show that the plaques are indeed plaques. Passage of the plaques from plaque picking to phage buffer.

Procedure:

Before starting we had to create an aseptic zone to ensure that all bacteria were killed, and the working space would not contaminate our experiments. Cleaned off the workspace with CiDecon and applied the table with 70% ethanol solution. Wiped off the table after CiDecon was applied, same with the 70% ethanol solution, only, we let the ethanol solution evaporate. Then got an ethanol burner, and the aseptic zone was created. Used the 10^0 lysate that was made on Friday 9/28/18 since this passed the third round of purification. Added the 10^0 lysate to the 90 microliter PB into the 10^-1 solution, and pipetted/mixed well through the microcentrifuge. Labeled this solution as the 10^-1 solution on the microcentrifuge. Got one more microcentrifuge caps, labeled one cap 10^-2. Added 90 microliters of PB to the 10^-2 solution. Added 10 microliters of the 10^-1 solution to the 10^-2 solution. All microcentrifuge caps had the lysate solution, then added 10 microliters of Arthrophage to all three microcentrifuge caps (10^0,10^-1, and 10^-2). Once this was done, went to get a 50mL vial to make the solution needed for the plaque assay.

The formula below was used to make the solution for 9 plates (three for Michael and Justin, two for Cooper, and one for the control).

• 2mL LB Booth (x10)
• 22.5 microliters of Calcium Chloride (x10)
• 2.5mL 2X TA (x10)
• 500 microliters of Arthro

Added the 2XTA last to the 50mL vials. shook the vial, and quickly pipetted the solution onto each vial containing the Artho/lysate solution. Sat each plate for about 15mins to the solution solidify. The remaining solution that was left in the 50mL vial was used for the control. Added TA and poured that solution onto the last plate, which all the plates sat to solidify for 15 minutes.

Observations:

The last three experiments performed in the lab by group 4, the control had been negative. The class as a whole had also been experiencing contaminations in their controls, which affect the outcome of the experiments. The plaque assay procedure was not hard since the experiment has been done several times. With the experiment performed Wednesday 9/26/18, the 2XTA split. Why did this split? Group 4 thinks the reason why the 2XTA split was that after the pouring of the plates, the plates sat <15 minutes.

Next Steps:

Prepare for titer calculations, and to determine whether or not the third round of purification passes. Count the number of plaques present, and determine how much lysate will be needed to completely web a plate.