Title: Webbed Plate and Flooding

Date: 12 November 2018

Rationale: The 3x plate from the previous experiment contained the most plaques, so it will be flooded in this experiment. A new flood lysate will be created and diluted in order to calculate a new amplified titer. A secondary plate will  be webbed in order to immediately flood in case the flood titer fails to reach the acceptable limit.

Procedure: Under an aseptic zone,

• 5 mL phage buffer added to plate and shaken for 1 hour
• Transferred to 15 mL conical vial to create a flood lysate
• Dilutions made by transferring 10 uL flood lysate into a micro-centrifuge tube containing 90 uL Phage Buffer
  • Dilution process repeated until a 10^-3 dilution was created
• 3 plaque assays (+control) were made using the following recipe:
  •  8 mL LB Broth
  • 10 mL 2x Top Agar
  • 90 uL CaCl2

~ 4.5 mL transferred to tube containing 0.5 mL Arthrobacter + Plate 1) 50 uL 10^0 lysate

Plate 2) 10 uL 10^-2 lysate

Plate 3) 10 uL 10^-3 lysate

• 3 more plates were made in order to ensure a plate would be webbed in the next lab in case of titer failure using the following recipe/procedure:
  • 6 mL LB Broth
  • 7.5 mL 2x Top Agar
  • 67.5 uL CaCl2

~ 4.5 mL transferred to tube containing 0.5 mL of Arthrobacter + Plate 1) 10 uL original flood lysate

Plate 2) 20 uL original flood lysate

Plate 3) 30 uL original flood lysate

Conclusions/Results: Only the 3x web calculation plate from the previous experiment yielded a decent amount of plaques (pictured). If the flood lysate fails to yield a high titer, a plate will be webbed and ready to flood to try and get a high titer and in order to save time.