Title: Plaque Dilution

Date: 1 October 2018

Rationale: Possible plaques were found on both plaque assays (with split top agar). A suspected plaque was picked and diluted to be passaged and purified.

Procedure: Aseptic zone created by washing bench with CiDecon and Ethanol, and lighting an ethanol flame.

• Previous plaque assay picked for a 10^0 lysate and placed into 100 microliters phage buffer. 10 microliters of the 10^0 lysate was transferred to another tube with 90 microliters phage buffer to create a 10^-1 lysate.
• The above process was repeated to create a 10^-2 lysate, generating 3 different lysates for a plaque assay.

The following recipe was used for 10 plaque assays (9 + 1 Top Agar control)

• 20 mL LB Broth
• 225 microliters CaCl2
• 25 mL 2X Top Agar

~ 4.5 mL pipetted into tube containing 0.5 mL Arthrobacter culture + 10 micro liters diluted lysate (one plaque assay for each dilution), cooled for 15 minutes, and incubated.

Conclusions: The suspected plaques are not confirmed, and if the dilutions fail, then new soil will be collected. If the plaques pass, then the passaging sequence will continue to purify the phage.