Rationale: Flood 3x plate performed 11/7. A new flood lysate will be created (flood x4) and diluted to calculate titer. A secondary plate will also be webbed with flood lysate x3 to try to obtain a high titer.

Procedure: Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. Plates were obtained from the incubator. 5mL of PB was added to the webbed plates to flood plates and shook for an hour. This solution was made to be x4 original flooded lysate. The formula below was used to make new plates that will be used to calculate titer (three plates for control and one for control):

• 2mL LB broth (x4)
• 2.5mL 2XTA (x4)
• 22.5µL CaCl2 (x4)
• Lysate
  • 1.15µl x 10^0
  • 10µL x 10^10-2
  • 10µL x 10^-3

Webbed plate using flooded original lysate x3 was used to make webbed plates for 11/14 if the experiment using x4 lysate failed, the same procedure would be used to create a newly flooded lysate using x3 flooded original lysate. The formula below was used to perform the experiment:

• 2mL LB Broth (x3)
• 2.5 2X TA  (x3)
• 22.5 CaCl2 (x3)
• Lysate x3 original lysate
  • plate 1 10µL
  • plate 2 20µL
  • plate 3 30µL

4.5mL of TA solutions were poured into test tubes containing 0.5mL Arthrobacter phage + lysate, which were quickly poured onto plates. Plates sat for 15 minutes and placed in the incubator at 27 degrees Celsius for 24 hours.

Observations: Control performed on 11/7 showed contaminations. Webbed plates x1 and x2 from 11/7 experiments showed contaminations, and x3 did not show contaminations. Webbed plate was used and flooded to create newly flooded lysate x4 original lysate. The lysate made 11/12 was used to create an x4 webbed plate. A secondary experiment was performed using flooded lysate x3 to be used as a back up if experiments from 11/12 fail.

Conclusions: If experiments performed 11/12 fail, flood x3 lysate plates created on 11/12 to make an x5 lysate. If experiments from 11/12 are successful, calculate titer. High titer will result in the next step of the procedure, and a failed experiment would result in recalculations and repeated experiments to obtain a high titer.