Date: Wednesday, November 28th, 2018

Title: High Titer Plaque Assay Second Attempt

Rationale: The purpose of today’s lab is to calculate the titer of the lysate using a previous plaque assay and then to attempt to web a plate using the titer calculations.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

1. An aseptic zone was set up.
2. Plates from last lab were evaluated and found to have negative results.
3. A new 10^-7 dilution was made using the positive lysate.
4. 20 μL of 10^-7 diluted lysate was added to a culture tube containing .5 mL arthrobacter and left to infect for 15 minutes.
5. Top agar solution was made using the following recipe:
   1. 4 mL LB Broth
   2. 5 mL 2x top agar
   3. 45 μL 1M CaCl2
6. 4.5 mL of TA solution was added to a top agar control plate.
7. 4.5 mL of TA solution was added to the culture tube and pipetted to mix.
8. The contents of the culture tube were poured into a plate.
9. The plates were left to sit for 15 minutes before being inverted and placed in an incubator for the next 48 hours.

Observations: The plates from last lab yielded negative results. This is most likely due to the original 10^-7 dilution losing its infectivity. The newly made dilution should produce results, as it has a high titer.

Results: This experiment yielded a top agar control and a plaque assay that, according to titer calculations, should be a webbed plate.

Next Step: The next step is to evaluate the plates and flood it with phage buffer.