Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

To sequence the phage DNA in the sample, the phages cultured from the webbed plates in the high titer lysate collected must be extracted from the phage buffer solution. The phage in the lysate will precipitate into a pellet and then it could be stored for DNA Extraction.

Pellet Precipitation for Sample (Gabe):

Materials:

• High Titer Lysate
• Nuclease Mix
• Phage Precipitation Solution

Procedure:

1. Added 40 ul of Nuclease Mix to 10 ml High Titer Lysate and mix gently
2. Added 4 ml of Phage Precipitation Solution and gently mix
3. Incubated at 37 degrees Celcius for 30 min and at room temp for 45 min
4. Centrifuged at 20 g for 20 min
5. Removed the supernatant, leaving the pellet and stored in freezer.

Observations, Results & Data:

The liquid after centrifuge had opaque cloud like substances suspended inside, and some were lost during the removal of the supernatant.

The imaging of the phage shows that it has a head about 47 nm and tail about 100 nm.

Interpretations & Conclusions:

The centrifuge was supposed to be set at 20000 g for 20 min according to the protocols yet was mistakingly set at 20 g, the results of this deviation of the protocol has yet to be seen.

The size of the phage raised interesting questions regarding the type of the phage, since that three different lysate from the sample yield different lengths of the tail, and since the tail length in phages are heavily conserved it may be likely that the sample contained multiple species of phages.

Next Step:

The next lab would be to start DNA extraction and calculate a concentration for the DNA collected.