5 November 2018 ✷ Adoption

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 assay was run (pick a plaque) to amplify present phage.

Procedure

• The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
• 70 µL of phage buffer was placed into a microcentrifuge tube and a micropipette tip was used to “pick” a plaque from a plaque assay run by Melissa from her soil sample’s lysate and the tip was swirled around the tube to add phage to the buffer.
• A plate for a plaque assay and a control plate was made. However, because the lab was short on arthrobacter for this test, only Rachel’s picked plaque was run. The concentrations and volumes can be seen below:

• component	volume	final concentration
  LB Broth	6 mL	–
  2X Top Agar	7.5 mL	1X
  1M Calcium Chloride	68 µL	4.5 µM

• NOTE: the mixture shown above was meant for three plates, but because of the limited resources, only 2 plates could be made.
• The mixture was left in the hot water bath so the TA wouldn’t harden while Rachel’s lysate infected the arthrobacter for 15 minutes. Then, 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 Wednesday at 27 degrees celsius.

Observations, results, data

The control plate showed contamination but plaques were present on the test plate.

Interpretations, conclusion, next steps

The plaque assay will need to be repeated because of the contamination. After a clean plate and positive plaque assay are seen, the plate will be flooded in order to amplify the phage presence/titer.

Other groups’ control plates had the same contamination, so this likely wasn’t a result of human error.