Rationale:

The purpose of today’s lab was to prep a TEM grid for microscopy due to a high titer lysate finally being acquired. In addition to TEM, DNA extraction procedure will continue with the pelleting of the phage particles through centrifuge.

Results from 11/16/18:

• HTL was successfully prepared for DNA extraction with the addition of the nuclease mix and phage precipitant solution. Also, 100 µL of the HTL had been set aside for TEM and will be used to get phage particles onto the grid.

Materials:

• High Titer Lysate (HTL)
• 400 Mesh Copper Grid
• Grid Box for Storage
• TEM forceps
• DI Water
• Uranyl Acetate

Procedure for TEM:

1. Plates were lined with parafilm and 15 µL of the HTL was spotted onto the parafilm.
2. Added two 20 µL drops of DI water onto the plate as well, keeping them all separated.
3. Then a drop of liquid uranyl acetate was spotted onto the plate as well.
4. A 400 mesh copper grid was removed from the B6 slot on the grid box with the TEM forceps and was left in the HTL shiny side down for 5 minutes.
5. Once 5 minutes were up, the grid was transferred to the DI water, where it rested for 2.5 minutes and was repeated with the second DI water spot as well.
6. Once removed from the second DI water spot, the grid was placed on the uranyl acetate spot for 1 minute to stain.
7. After a minute had passed, the grid was placed into the corresponding B6 slot on a new grid box that contained prepped TEM grids.
8. Grid box was then stored until ready for TEM.

Procedure for DNA Extraction:

1. Since the DNA was already prepared and balanced with another conical vial full of water, all that was left was to place the vials in the centrifuge.
2. Both conical vials were placed in the centrifuge to spin at 10,400 rcf for about 20 minutes at 4 degrees Celsius.
3. After the 2o minutes had passed, the pelleted lysate was removed from the centrifuge, decanted of any remaining supernatant, and left to freeze to prevent any degradation.

Results:

• A fair amount of pellet had formed on the bottom of the conical vial, but unaware if it is considered a “large” pellet or not. Also pellet will be left in the freezer for over a week, and it is typically only supposed to be left for 3 days.
• 
• Ring on the bottom of the conical vial is the pelleted phage particles.

Conclusions:

The amount of phage pellet could be a result of the titer of lysate present. The titer of the lysate is on the lower end of the spectrum of the high titer category, so that could determine a low pellet count. Also, the TEM grid preparation process could yield some over-dyed grids as the TEM forceps had to be shared amongst 8 students, and some had to leave their grids for longer than recommended.

Next Steps:

The next steps for the experiment are to analyze the phage morphology under TEM using the previously prepared grid and continue with the extraction of DNA from the pelleted phage particles.