19 November 2018 ✷ Pick a Plaque

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque was picked from the prior plaque assay and run in a plaque assay again.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• The lone plaque on the plate from the prior lab session was picked and swirled into 30 µL of  phage buffer. 50 µL of this mixture was added to 0.5 mL of arthrobacter and allowed to sit for 15 minutes while the plates were made.
• 
• A plate for 1 plaque assay and a control plate were made with the following concentrations and volumes:

• component	volume	final concentration
  LB Broth	4 mL	–
  2X Top Agar	5 mL	1X
  1M Calcium Chloride	50 µL	4.5 µM

• The arthro/lysate mix was combined with 5 mL of the above plate mix and the plate was poured, allowed to harden, and then inverted and incubated until Tuesday at 37 degrees celsius.
• On Tuesday, the plate was removed from the incubator, wrapped with parafilm and frozen until the following Monday.

Observations, results, data

The plate had several plaques following the plaque assay, but they were quite small and far from being a webbed plate. This sample will need to be amplified quite a bit in order to get a webbed plate.

Interpretations, conclusion, next steps

The sample seems weak because it produced a very small amount of plaques (that were very small themselves). Additionally, after picking a plaque the first time, the plaques were lost, so the phage sample in this soil isn’t the strongest phage. The sample will need to be amplified by picking plaques until the plate is webbed and there is a high titer.