Previous Results:

• On 11/13 the plaque assays from 11/12 were flooded by Dr. Adair. The plates were completely lysed, meaning the lysate may be a high titer

Objective:

• The lysate from the flooded plates will be filtered and a spot test of dilutions will be made to later calculate the titer of Lysate #8

Procedure:

1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
2. Flooded plaque assays from 11/12 were obtained and lysate was filtered using a top filter and vacuum. 25 mL of Lysate #8 was collected from 4 plates
3. Overlay agar for spot test was mixed using 2 mL LB broth, 2.5 mL 2x TA, 0.5 mL Arthrobacter, and 22.5 microliters CaCl2
4. Overlay was plated and left to harden for 15 min. grid was drawn on plate, making 9 spaces
5. Lysate #8 was obtained and dilutions were made out to 10^-8. 90 microliters phage buffer was added to microcentrifuge tube labeled “10^-1” and 10 microliters of lysate was added. Then 10 microliters of “10^-1” was added to tube containing 90 microliters phage buffer, labeled “10^-2.” Process was repeated through 10^-8
6. 10 microliters of each dilution (10^0 – 10^-8) was pipetted onto the hardened plate, in their separate spaces in the grid.
7. The plate and dilutions were left to sit for 10 min. Then incubated for 48 hours

Results:

• The experiment was not completed, therefore there are no results to report

Next Steps:

• During next lab, the spot test will be examined. It will be determined which dilution to make a plaque assay with to calculate the titer. After the titer of the lysate is known, TEM will be conducted.