11/07/2018

Objective:

Pick plaques and make plaque assay for the extracted phage and 10^-1 dilution of the lysate.

Pre Lab Observation:

The control plate from the plaque assays from 11/05/18 were contaminated and therefore reliable plaque assays were not obtained.

Procedure:

1. 30 plaques were picked from 3rd purification run and deposited in a microcentrifuge with 100μl of phage buffer.
2. After phages are mixed in the microcentrifuge tube, 10 μl of the extracted phage solution is transferred to a microcentrifuge tube with 90μl of phage buffer to acquire a 10^-1 serial dilution.
3. One top agar mixture was prepared for 5 plates.
4. 10 ml of LB broth was added to a conical vial
5. 112.5 μl of CaCl2 was added to the conical vial.
6. Two 0.5 ml of arthrobacter samples were enriched with the extracted phages and the 10^-1 dilution for 15 minutes.
7. 12.5 ml of 2X TA was added to the conical vial.
8. 4.5 ml of the TA mixtures was added to each enriched sample.
9. the samples and the mixture were then plated on two agar plates.
10. 4.5 ml of TA mixture was plated on another agar plate for a control plate.
11. after 15 minutes, all the plates were placed inverted in the incubator.

Analysis and Conclusion:

Most likely due to the contamination, the plates prepared on 11/05/18 were negative and a webbed plate was not produced. Due to the lack of lysate, another purification was done to acquire more phages. New titer will have to be calculated and a webbed plate will have to be prepared during the next lab.