Rationale

The two plates from previous lab day will be flooded and filtered out. Plaque assays will be performed using 10 µL of 10⁰, 10 µL of 10^-1, and 10 µL of 10^-2 of the new lysate with a goal to receive a webbed plate and get a high titer.

Detailed Procedure

1. Add 5 mL of PB to each plates from previous lab day.
2. The plates was shaken on an incubator for one hour.
3. Filtered lysate from flooded plate through a 0.22 µm syringe filter into a conical vial labeled “SJ 11/12/18 FS lysate 10⁰.”
4. 10 µL of “SJ 11/12/18 FS lysate 10⁰” was added with 90 µL of phage buffer to create 10^-1 dilution which was vortexed.
5. 10 µL of 10^-1 was added with 90 µL of phage buffer to create 10^-2 dilution which was vortexed.
6. 10 µL of “SJ 11/12/18 FS lysate 10⁰“, 10 µL of 10^-1, and 10 µL of 10^-2 of the new lysate were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them. Sat for 15 mins.
7. 8 mL of LB Broth, 90 µL of CaCl₂, and 10 mL of 2X TA were combined into a conical vial.
8. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into each test tube.
9. Each test tube was poured onto their correlating plate, and the remaining 4.5 mL of the TA mixture was poured onto a plate labeled “SJ 11/2/18 Control.”
10. Waited 15 mins to solidify ad inverted into incubator for 48 hours.

Observations/Results/Next Step

Control plate from previous lab day had no contamination and size of plaques all seem to remain the same size. Although not fully webbed, it was enough to flood the plate. For the new plates, air bubbles were formed. For the next lab day, a new titer will be calculated and hope to reach a high titer by Wednesday.

results from lab day 23

new plates after flooding and serial dilutions