Rationale:

The purpose of today’s lab was to observe the purified phage sample under a TEM, and extract the DNA from the pellet acquired previously.

Results from 11/19/18:

• A TEM grid was prepared to be ready for viewing.
• Phage particles were pelleted and extracted from a centrifuge, they were left to freeze for the week to preserve the phage structure.

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  Phage Pellet and Supernatant 11/19/18

Materials:

• TEM Grid
• Pellet of Phage Material
• Sterile H2O
• DNA Resin at 37 °C
• 80% Isopropanol
• Elution Buffer
• Vacuum Manif0ld

TEM Procedure:

1. Previously prepped TEM grid was loaded into the microscope.
2. The sample was then adjusted and analyzed until acceptable phage was found.
3. Measurements were taken of the head and tail of the phage, and a picture was taken for data.

DNA Extraction Procedure:

1. Began with re-suspending the phage pellet using 500 µl of sterile H2O.
2. Once re-suspended, 2 ml of DNA clean-up resin was added to the pellet and gently mixed.
3. After adding the resin, the mixture was equally separated into 2 microcentrifuge tubes and set into the centrifuge at 12,500 xg for 3 minutes to spin.
4. Microcentrifuge tubes were then removed and contained a cloud of genetic material at the bottom, with a clear liquid on top. The clear liquid (resin) was removed using various micropipettes, careful not to disturb the DNA cloud.
5. Once the liquid was removed from both tubes, 1 ml of 80% isopropanol was added to each tube and shaken to combine before adding the tubes back into the centrifuge for 3 minutes at 12,500 xg.
6. The tubes were removed and step 4 was repeated with the DNA sample
7. 1 ml of 80% isopropanol was then again added to each tube and combined, and poured the mixture into two separate vacuum columns under the fume hood to filter the DNA and resin out from the mixture.
8. Once filtered, the DNA and resin were added back to the centrifuge to spin, this time for 5 minutes at 12,000 xg.
9. After removing the microcentrifuge tubes, 50 µl of elution buffer was added to each of the tubes, and they were spun again for 5 minutes at 12,000 xg.
10. The tubes were removed, and the extracted DNA was combined into one microcentrifuge tube before being taken to the Nanodrop machine.

Nanodrop Procedure:

1. First the Nanodrop machine was cleaned with sterile wipes, both the bottom and top sensors.
2. An elution buffer blank was run first by spotting 2 µl of elution buffer onto the Nanodrop sensor, then the arm was slowly lowered and the blank was run.
3. After running the blank, the machine was cleaned again, and 2 µl of the extracted DNA was spotted onto the sensor and the machine was run to analyze the DNA.

Results/Data:

• For TEM, it appeared that the mass majority of the phages found in the sample had their heads separated from the tails.
• For the intact phage that was found, it had a capsid size of approximately 50 nanometers, with a tail size of 174 nanometers.
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• DNA extracted was a clear liquid, could not see any individual parts of it within the elution buffer.
• The Nanodrop results for the DNA extracted was 805.8 micrograms per ml of DNA

Conclusions:

It is unclear what exactly is causing the damage to the phages which is separating the heads from their bodies. It could be due to improper storage or poor TEM prep that is damaging the phages before they can be seen under the microscope. Several phage were found with portions of their tails missing, but the head size was consistent throughout. Also, the 805.8 micrograms per ml of DNA suggest their is a significant amount of genetic material extracted, it is unclear if it is phage or bacterial DNA, and PCR and gel electrophoresis is needed.

Next Steps:

The next steps for this experiment are to run a PCR on the extracted DNA and prep a gel for gel electrophoresis.