Results:

No contamination appeared on either the control or spot test plate. The first three dilutions were completely lysed. Plaques formed on the 10^-4to 10^-7dilutions, but not on the 10^-8dilution as shown below.

Rationale:

From the spot plate, the 10^-6dilution will be used to calculate the titer of the lysate. If there is a high titer, some of the lysate will be archived and DNA extraction procedures will begin. If not, more calculations will be performed to determine how much lysate should be used to web a plate.

Procedure:

1. Counted plaques and performed calculations to determine the titer.
2. Once an aseptic zone was established, 2.8 mL of the high titer lysate (HTL) was archived in a conical vial labeled “KEA 11/14/18 Ferranti HTL – 1.3× 10⁹pfu/mL.”
3. 100 µL was placed in microcentrifuge tube labeled “KEA 11/14 TEM.”
4. Diluted the remaining 7 mL of HTL with 3 mL of phage buffer.
5. 40 µL of nuclease and 4 mL of polyethylene glycol (PEG 8000) were combined with the diluted HTL into a conical vial labeled “KEA 11/14/18 Ferranti DNA Ex.”
6. Inverted the conical vial multiple times to mix the solution.
7. Incubated the conical vial for 30 minutes at 37ºC and then at room temperature for 25 minutes.
8. Stored “KEA 11/14/18 Ferranti DNA Ex.” conical vial at 8.2ºC.

Observations:

• 13 plaques were counted under a light microscope as shown in the picture below.

Calculations:

• The titer of the HTL was determined to be 1.3× 10⁹pfu/mL.
• There was barely 10 mL of the HTL. Since DNA extraction requires 10 mL, it was determined that 2.8 mL would be archived, 100 µL would be used for TEM, and the remaining 7 mL would be diluted to 10 mL.
• The titer of the diluted HTL used for DNA extraction was determined to be 9.1× 10⁸pfu/mL.
• After adding the nuclease and PEG 8000, small particles were suspended throughout the lysate.

Next Steps:

Tomorrow, “KEA 11/14/18 Ferranti DNA Ex.” will be centrifuged and the supernatant will be decanted.