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:

By implementing Electrophoresis, the image captured on the gel after could help determine if there is any trace of DNA amplified after PCR in the filtered Enrichment Lysate sample. A Plaque Assay helps us determine if there is a presence of bacteriophages by adding Arthrobacter directly to the lysate. We can tell the existence of bacteriophages by checking the presence of plaques on the agar plate.

Electrophoresis for sample (10.24.18):

Materials:

• 1x TBE
• Agarose powder
• EtBr
• Electrophoresis apparatus and power supply
• Heat source
• DNA loading dye
• DNA ladder
• Gel Imager

Procedure:

1. prepare a solution with 40 ml of 1x TBE and 0.8 g Agarose
2. microwave until the solution boils and retrieves it for cooling
3. Add EtBr to the solution and have a final concentration of 0.5 %
4. While the solution is still warm, pour it into the preassembled gel apparatus
5. wait for solidification
6. After the gel had solidified, fill the apparatus with 1x TBE
7. Load the samples(10 ul), DNA dye(10 ul) and DNA ladder(5 ul) into the wells
8. Run the gel at 100 volts 45 min

Plaque Assay for Soil (10.1.18):

Materials:

• Micropipette
• Serological pipette
• Centrifuge tube(1.5ml)
• 50 ml conical tube
• LB Broth
• CaCl2(aq)
• Sample (Gabe) Picked Plaque 10^0
• 2x Top Agar
• Agar plate
• Arthrobacter

Procedure:

1. Set up an Aseptic zone.
2. Add 0.5 ml Arthrobacter and 10^0 lysate (100ul) to a Centrifuge tube 10 min for infection.
3. Add 2 ml of LB Broth, 22.5 ul Calcium Chloride (aq) to a tube and repeat to another tube.
4. Add the infected lysate to their respective tubes.
5. Add 2.5 ml of 2x Top Agar, pipette up and down then decant the solution to an agar plate and repeat.
6. Wait for 10 min to solidify (slightly shooked during) and place into the incubator.

Observations, Results & Data:

The results of gel imaging shows negative for phage DNA for all three lanes, there are three bands in the three sample lanes at the same distance.

Interpretations & Conclusions:

The bands on the gel are located at the region where it might be the primers self-replicating, due to there is no DNA to replicate so it is clear that the three bands are located at the region indicating the size of the DNA are very small, conforming to the size of the primers.

Next Step:

The next step would be to check the results of the plaque assay ran on sample (Gabe), if it yields positive results.