Title: Spot Titer for New Lysate

Date: 26 November 2018

Rationale/Past Results: About 43 mL of new lysate was acquired through plate flooding (“Flood Lysate #9”) that will be spot titered and run under a TEM in order to discover the official titer and investigate the individual phage, respectively. If the titer is confirmed high, then the lysate will be archived and prepped for DNA extraction.

Procedure: Under an aseptic zone…

• One spot plate was run in order to determine titer
• Dilutions of “Flood Lysate #9” were done through the following method
  • 10 uL of original flood lysate was transferred to a new microcentrifuge tube containing 90 uL Phage Buffer. This mixture was designated as the 10^-1 concentration dilution
  • 10 uL of the 10^-1 concentration was then transferred to another microcentrifuge tube containing 90 uL phage buffer. This mixture was designated as the 10^-2 concentration dilution.
  • This process was repeated until a 10^-9 concentration was made.
• The plate was divided into 6 regions: a control, 10^-5, 10^-6, 10^-7, 10^-8, 10^-9 and labelled accordingly
• The following recipe was used for the spot plate:
  • 2 mL LB Broth
  • 2.5 mL 2x Top Agar
  • 22.5 uL CaCl2
  • 0.5 mL Arthrobacter culture

~5 mL of mixture was then plated and cooled. 10 uL of phage buffer was spotted onto the control spot while 10 uL each of the 10^-5 through 10^-9 dilutions were spotted in their respective locations. The plate was placed into the incubator.

Conclusions: If the titer is high enough (≥1.0 x 10^8), then the lysate can be named and archived, and its phage DNA extracted through pellets. A TEM grid can also be run on the phage in order to investigate more closely its characteristics.