11/26/18

Objective:

• Make a plaque assay from a picked plaque

Pre-Lab Observation:

The spot test prepared on 11/14/18 resulted in a weak titer or an absolute lack of plaque. Another group member was assigned to extract lysate from the same soil and make a plaque assay. plaques were picked from the plaque assay in this lab in an effort to restart purification.

Procedure:

1. The aseptic zone was set up.
2. A plaque was picked from the group members plaque  assay and the phages were deposited in a microcentrifuge tube with 50μl of phage buffer.
3. 50 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

50 μl of the extract was used to possibly get a webbed plate. the probability of such an outcome is small. due to the lack of time, this would be the last chance for possibly getting to DNA extraction, which seems unlikely at this time.