Multiple Person Spot Test

11/5/18

Rationale: Since we’re waiting on Katherine to calculate the titer of her lysate we decided to run a quick spot test on our remaining soil samples.

Procedure:

1. Marked a plate in four sections with the  initials of each member running a plaque assay and one control section for phage buffer.
2. Top Agar was made for two plates by adding 4mL of LB Broth, 45µL of CaCl2, and 5mL of 2X TA.
3. Poured control plate with 4.5mL of solution
4. Added 0.5mL of arthrobacter to remaining solution and mixed by pipetting up and down.
5. Poured test plate and let sit for 15 minutes.
6. Added 10µL of each test lysate onto allocated areas and 10µL of phage buffer to control area.
7. Let sit for 10 minutes then placed in to incubator without inverting because agar did not adhere to plate.

Observations:

My section had a rather large plaque. Note that the control spot is in the area labeled EB and Emily’s spot is in the area labeled PB.

Conclusions and Next Steps:
It’s a relief finally getting a plaque, but also stressful since I don’t have much time to purify and amplify. Therefore, I’m going to conduct 3 plaque assays with 20, 40, and 80 µL of lysate in hopes of speeding up the process. The plaque is rather large for only a 10µL spot, this could mean that it is already of a high titer. If that is the case I could save a lot of time during amplification.