Rationale: Since I need at least 12.8mL of lysate for DNA extraction and archiving I need to make some webbed plates to flood. I calculated that I need 8µL of lysate diluted to the 10^-5 to  web a plate, so I will plate three  plates with  that amount.

Procedure:

1. Put parafilm on a plate and pipetted 15µL of lysate onto the parafilm.
2. Pipetted 20µL of water twice onto the parafilm.
3. A TA aliquotted uracyl acetate onto the parafilm
4. Placed a TEM stage from section A10 onto the lysate and let sit for 5 minutes.
5. Transferred stage to water and let sit for 2.5 minutes.
6. Repeated for the second drop of water.
7. Transferred stage to uracyl acetate for 1 minute then took off and placed back into container on the side to be used for TEM.
8. Pipetted 8µL of 10^-5 lysate into 0.5mL of arthrobacter 3 times and let sit for 30 minutes.
9. Made top agar with 8mL of LB broth, 90µL of CaCl2, and 10 mL of 2X TA.
10. Plated control with 4.5mL of solution.
11. Aliquot 4.5mL of solution into each tube of enriched lysate and arthrobacter.
12. Mixed by pipetting  up and down then plated each.
13. After letting solidify inverted and placed into incubator for 24 hours.

Observations:

The plate wasn’t fully lysed because the plaque morphology isn’t uniform. I placed back in the incubator over break in hopes of getting a slightly more webbed plate.

Conclusions and Next Steps: This means that there is more than one type of phage in  my  lysate. However, I do not have time to run another round of purification. This means that I will just have to keep running with my current lysate and acknowledge that there are two phages in my lysate when I extract DNA. From there I can differentiate between the two phages.