10/22/18

Objectives:

• to test for the presence of bacteriophages in enriched lysate using PCR

Pre-Lab Observations:

the plaque assays and top agar control prepared on 10/17/18 were contaminated. Therefore the results were inconclusive. PCR and gel electrophoresis will be used for testing for the presence of Phages in enriched lysate.

Procedure:

1. Set up aseptic zone.
2. transferred 4 ml of enriched lysate for soil sample C into a 15 ml conical vial
3. centrifuged the conical vial for 5 minutes
4. transferred 1 ml of centrifuged enriched lysate into a micro-centrifuged tube.
5. the microcentrifuge tube was then boiled
6. picked up 3 green vials with 12.5 μl of Taq polymerase.
7. 4 μl each of primer 1, primer 2 and primer 3 were added to the three green vials and they were labeled 1,2 and 3 according to the primer added.
8. 6.5 μl of DDI water was added to each of the three green vials.
9. 1 μl each of the boiled enriched lysate sample from 2 students (one of them being me) was added to each of the three green vials.
10. the vials were allowed to rest for some time and were then placed in the thermocycler.

Analysis and Conclusion:

judging from the results obtained from the plaque assays, there seem to be no phages in this soil sample. PCR and gel electrophoresis ( the latter will be performed on /10/24/18) will perhaps present a more definitive answer.