11.28.18 Finishing DNA Extraction

Rationale: Since a pellet had been obtained through the first part of DNA extraction, it was possible to finish the process of DNA extraction. Therefore, the next logical step was found to be the second half of the procedure that ended with measuring the sample using the nanodrop.

Procedure:

1. 0.5mL sterile water added to thawed phage pellet
2. Pellet was resuspended
3. 2mL of resin was added to the pellet, then the tube was inverted many times to mix
4. Resin and re-suspended phage pellet was added to two distinct microcentrifuge tubes (split evenly)
5. Tubes were centrifuged at 12,500g for 3 minutes
6. Supernatant was removed with pipette and placed in waste
7. 1mL of 80% isopropanol was added to each microcentrifuge tube
8. Steps 5-7 were repeated twice
9. 1mL of 80% isopropanol was added to the pellet to re-suspend
10. Contents of tubes were added to a column in a vacuum to move DNA to the filter
11. Column was added to microcentrifuge tube, then centrifuged for 5 minutes at 12,000g
12. Column was moved to new microcentrifuge tube
13. 100µL of Elution Buffer (85 degrees Celsius) was added to column, then centrifuged for 1 minute at 12,000g
14. DNA quantified on nanodrop
15. Cleaned station

Observations

• Pellet was initially very difficult to see, which led to doubt about whether or not DNA was strongly present
• Nanodrop was very sensitive and difficult to place drop on platform without missing or creating air bubbles
• Pellets after centrifuging formed more of a cloud shape rather than solid in bottom of tube, which made it more difficult to remove the supernatant clanly.

Results

• The DNA concentration present was 65.69µg/µL. The 260/280 ratio was 1.89, and 260/230 was 0.16.

Conclusions

• Since the concentration of DNA was above 50, the sample theoretically has a chance to work using the PCR procedure to cluster the phages. The ideal number would have been closer to 500µg/µL, but the sample should still be able to proceed. Furthermore, the ratios found in the results section illustrate that the sample is relatively pure and that the isolation was done reasonably accurately, as the 260/280 ratio should have been close to 2, and it was. The next step will be to process this sample using PCR clustering to see whether or not the procedure will work with the lower concentration and to determine specifics about the phage’s DNA.