11-28-18

Objective:

To pick 3 plaques and make a plaque assay from the extracted phages.

Pre-Lab Observation:

The plaque assays prepared on 11-26-18 seemed to have a regrowth in Arthrobacter and can therefore not really be used. it is difficult to state whether plaques had formed. Because all the lysate was used, another plaque assay cannot be made. therefore, in a final attempt, an older plate was acquired and plaques were picked.

Procedure:

1. The aseptic zone was set up.
2. 3 plaques were picked from the old plaque  assay and the phages were deposited in a microcentrifuge tube with 30μl of phage buffer.
3. 30 μl of this phage extract was deposited in 0.5 ml of arthrobacter and was allowed to enrich for 15 minutes.
4. 1 TA mixture was prepared for three plates.
5. 6 ml of LB broth was transferred to a conical vial.
6. 67.5 μl of CaCl2 was added to the conical vial.
7. After the lysate was allowed to enrich for 15 minutes, 7.5 ml of 2X TA was added to the conical vial.
8. 4.5 ml of the TA mixture was added to the arthro tube.
9. The contents of the tube were then poured onto the agar plate.
10. 4.5 ml of the 2X TA mixture was plated on an agar plate for a control.
11. the plates were allowed to solidify for 15 minutes.
12. the plates were then placed in the incubator.

Analysis and Conclusion:

This is the final attempt. If the plate is webbed, it can be flooded and process for DNA extraction can be started.