Date: Monday, October 1st, 2018

Title: Purification: Third Passage Soil Sample B

Rationale: The purpose of today’s lab is to further passage the lysate for the third time in order to isolate a phage.

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. Plaque assays from the second purification passage were taken back out and a new plaque was chosen to be picked.
3. 100 microliters of phage buffer were transferred to a microcentrifuge tube.
4. A pipette tip was touched into the chosen plaque and swirled in the microcentrifuge tube to add phage to solution.
5. The microcentrifuge tube was vortexed to mix phage with buffer. This was set aside for later use.
6. Agar for 3 plates was made using the following recipe in a 50 mL conical vial:
   1. 6.0 mL LB broth
   2. 7.5 mL 2x Top Agar
   3. 67.5 microliters 1M CaCl2
7. The LB broth and 1M CaCl2 were added to the 50 mL conical.
8. 10 microliters of the phage buffer and phage solution in the microcentrifuge tube was transferred to a culture tube containing .5 mL arthrobacter.
9. The culture tube was set aside for 15 minutes to allow the phage to infect the arthro.
10. 7.5 mL 2x top agar was added to the 50 mL conical and pipetted to mix the solution.
11. 4.5 mL of the top agar solution was added to a top agar control plate.
12. 4.5 mL was added to the culture tube containing the arthro and phage sample.
13. This solution was added to an agar plate and moved around to cover the plate with solution.
14. The plates were left sitting to allow the agar to harden.
15. The plates were inverted and left to incubate.

Observations: The control plate from the second passage was contaminated. It’s still unclear what’s causing such consistent control plate contamination. The current theories are that either the pipette tips are being hit against a contaminated surface or that people are talking near the aseptic zone.

Results: This experiment yielded a new plaque assay that can be evaluated and either passaged again or begin to web a plate.

Next Step: Since this is the third passage for this sample, the next step is to prepare for amplification. However, if the titer is too low, the next step would be to passage the sample again.