10.22.18 PCR Test of Soil Sample E

Rationale: Since more inconclusive test results were obtained and no filters were available to test another soil sample, it was found to be important to use PCR to definitively determine whether or not phage particles were present in Soil Sample E.

Procedure:

1. Aseptic zone was established
2. 4mL of soil sample F was added to a conical tube
3. 10mL of LB Broth was added to tube
4. Tube was shaken and vortexed for 10 minutes
5. Tube was centrifuged for 5 minutes to separate soil from supernatant
6. Attempted to filter using a 3 mL syringe and a 22µm filter. Filter did not fit, and no filters of proper size were present. Tube was labeled “HMB Soil F” and stored in refrigerator.
7. Obtained enriched lysate from soil sample E
8. 1mL enriched lysate E was added to a microcentrifuge tube
9. Microcentrifuge tube placed in 37°C container for 10 minutes to boil
10. Obtained 3 tubes with 12.5µL of Taq Polymerase in them. Tops labeled 1,2,3.
11. Added 7.5µL DDI water to each tube
12. Added 4µL Primer 1, Primer 2, and Primer 3 to corresponding tubes
13. Added 1µL of the boiled enriched lysate sample to each tube.
14. Thermocycled all tubes.
15. Cleaned and tidied bench.

Observations:

• While using the improper filter, it was easy to distinguish that the filter broke due to pressure. After the filter broke, white foam was observed on top of the sample that was forced into the conical tube. Therefore, it would be relatively safe to assume the foam was from the filter breaking. This foam was previously of unknown origin and was seen in a prior experiment.
• Mass of sample in the centrifuge was 22.767g.
• Soil added to conical tube was wet when placed in the freezer. Thus, it still retained a clay texture and while shaking, it was difficult to get all parts of the soil sample to be mixed with the LB Broth.

Results:

• The plates prepared after flooding the previous plate with unknown clearings showed no clearings other than tears in the top agar overlay. The control plate also showed signs of a different type of contamination than normal.

Conclusions:

• Since no clearings were seen on the flooded plate, it is generally thought that the plaques seen on the previous plate were due to contamination. However, since the control plate was yet again contaminated, results cannot be fully supported or disproved. Therefore, the PCR test should be useful in determining whether or not the plaques were due to phage in a setting where contamination will not have such a prevalent effect. The next step will be processing the PCR sample and running a gel to determine presence of phage.