Title: Webbed Plates and Lysate Amplification

Date: 19 November 2018

Rationale/Previous Results: A spot titer was done on the previously flooded lysate and countable growth was seen on the spot for a 10^-8 concentration of the previous flood lysate (“flood lysate 8”). 22 plaques were counted and the titer was calculated to be 2.2 x 10^11, which is considered a very high titer. Pictures of the spot titer are attached. Now that a high titer has been achieved, archiving, characterization, and investigation will begin. A certain amount (>42.8 mL) of lysate is needed for this to happen, so more lysate will have to be made by webbing and flooding plates. Therefore, plates will be webbed and flooded in order to have a high enough volume of lysate to continue the characterization process. (Note: the picture indicates 15 plaques counted, however this was an error and the plaques were counted a second time and the correct number was found to be 22 plaques.)

Procedure:

Under an aseptic zone:

• the following recipe was used to make 6 webbed plates (+control):
  • 14 mL LB broth
  • 17.5 mL 2x Top Agar
  • 157.5 uL CaCl2

~ 4.5 mL transferred to tube containing Arthrobacter culture + 10 uL “flood lysate 8” (high titer). The mixtures were plated, solidified, and incubated for ~24 hours.

Conclusions/Next Steps: A new lysate with a different titer will be made with these webbed plates. They will be flooded with 8 mL of Phage buffer each, and stored until the titer can be confirmed. The lysate will also be filtered in order to assure phage purity, and once enough lysate is acquired, DNA extraction, characterization, and archiving can occur.