Rationale

Today we will conduct TEM to image our phage. We will also make a precipitate using our lysate.

Procedure

• Established an aseptic zone.
• 10 mL of lysate was poured into a tube to begin making a precipitate. 40 µL of nuclease was added to the tube and it was inverted 10 times. 4 mL of phage precipitant solution was added and the tube was inverted once to mix. It was placed in the incubator for 25 minutes.
• TEM was performed during the incubation period and a grid was prepared.
• 1 square of parafilm was prepared by aliquotting 20 µL of lysate onto the square, 20 µL of D.I water, another 20 µL of D.I. water, and 20 µL of uranyl acetate in sequential drops.
• A TEM grid was placed into the lysate for 5 minutes, each D.I. water drop for 2.5 minutes, and the uranyl acetate for 1 minute.
• Filter paper was used to wick away the excess liquid from the grid and the grid was imaged.
• The precipitate preparation in the incubator was removed after 25 minutes and left at room temperature for 40 minutes.

Observations

The last spot titer test led to inconclusive results. The 10^-1, 10^-2, and 10^-3 quadrants had completely lysed, however, all the quadrants following did not produce any plaques. This resulted in the titer of our lysate being unknown. However, due to the titer of the current lysate being 1.2×10^7, it was decided to continue onto TEM.

After imaging, the head of the phage was recorded to be 53 nm in width. The tail was recorded to be 110 nm in length.

The centrifuge swinging bucket was not functioning, resulting in precipitate preparation to not be completed.

Conclusion/Next Steps

The precipitate preparation will be completed and DNA extraction will begin. A spot titer test will also be conducted to determine the titer of the lysate.