11.26.2018 DNA Extraction Initiation

Rationale: Since results observed by Lathan and sent to me showed that the lysate possessed had a titer of 1e9, the lysate was officially a high titer. Therefore, it is possible to perform the DNA extraction procedure on the lysate to progress with analysis of bacteriophage as part of Claire’s group. Also, since the TEM was available for use, it was found to be beneficial to visualize the bacteriophage using the microscope. The DNA extraction procedure will allow for the genome of the phage to be analyzed if it is selected by the class.

Procedure:

1. Aseptic zone established
2. 10mL of lysate labelled “HMB from CEW 10^0 Lysate 11/14/18” was placed into a conical tube.
3. 40µL of nuclease mix was added to the lysate
4. 4mL of phage precipitant solution was added to the lysate
5. Lysate was placed on shaker at 35 degrees Celsius for 30 minutes
6. Lysate was let sit at room temperature for 45 minutes
7. Lysate was centrifuged at 7,500g for 20 minutes.
8. Eliminated supernatant from sample and rinsed sink.
9. Stored in refrigerator overnight.
10. Cleaned bench.

Results:

• These results date back to Monday, November 19 and the procedures performed on this day. As seen, the lysate was strong enough to completely lyse the spots through the 10^-5 dilution, and had plaques form through the 10^-7 dilution. After calculation, the lysate was found to be 1×10^9 pfu/mL. Therefore, a high titer lysate had been obtained! The control also showed no signs of contamination, which confirms that the results could be considered valid.
• The electron microscope revealed very present bacteriophages. The phages had a head with a diameter of 52nm and a tail of 100nm.

Observations:

• After the phage precipitant solution was added, the lysate appeared to have smaller particles or granules appeared to be seen. They dissipated more with shaking.
• Phages appear on the TEM in the shape of a lollipop with a dark head and banded tail.
• TEM showed phages with a grainy background. Small movements had large effects, and the images moved slightly due to how the electrons interacted with the plastic film.

Conclusions/Next Steps:

• Since there were plaques present on the 10^-7 dilution, it can be concluded through calculations that there is a high titer lysate present. This is helpful in furthering the processing of the lysate, and allowed the DNA extraction process to begin. The TEM also showed the morphology and size of the bacteriophages from the sample. Since procedures today did not show definitive results, no further conclusions are available to be reported. The next steps in this procedure would be to finish the DNA extraction process, which will be done on Wednesday.