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.

10/15/18

Objective:

• To make a plaque assay to test for the presence of phages in soil sample C

Pre- Lab Observations and Data:

The metadata for soil sample was collected from the results of the procedures performed on 10/10/18

% water = mass of water/mass of soil and water x 100= 1.038/5.095 x 100= 20.37%

pH of Soil= 6

%sand= 9.1/10 x 100= 91%

%silt= 0.4/10 x 100=4%

%clay=0.5/10 x 100=5%

Procedure:

1. The aseptic zone was set up.
2. Some of the enriched lysate prepared on 10/10/18 was filtered into a microcentrifuge tube.
3. 0.5 ml arthrobacter was retrieved and enriched by filtered lysate.
4. The vial was then allowed to rest on the test tube rack for 15 minutes
5. While the vial was resting, one Top Agar mixture was made for the group.
6.  8 ml of LB broth was transferred to a conical vial.
7. 90 microliters of the CaCl2  was transferred to the conical vial with the LB broth.
8. The vial was then set on the rack.
9. 10 ml of the 2X TA was added to the LB broth and Cacl2 after the sample was allowed to enrich the lysate for 15 minutes.
10. 4.5 ml of the top agar mixture was transferred to the test tube with the enriched lysate.
11. The contents of the test tube were then poured onto the agar plate.
12.  Part of the top agar mixture was poured into the top agar control plate for the group.
13. To let the top agar solidify, the plates were allowed to rest for 20 minutes.
14. The plates were placed upside down in the incubator, where they will remain for 48 hours.

Analysis:

analysis of the soil metadata leads to the conclusion that the soil that was collected loamy sandy

10/17/18

Objectives:

• Make a plaque assay for the enriched lysate extracted from soil sample C

Pre-Lab Observations:

the plaque assays prepared on 10/15/18 seemed to have not properly solidified and had some air bubbles. So, although there seemed to be plaques on the plate, another plaque assay will be made to verify the presence of phage in the lysate.

Procedure:

1. The aseptic zone was set up.
2. Some of the enriched lysate prepared on 10/10/18 was filtered into a microcentrifuge tube.
3. 0.5 ml arthrobacter was retrieved and enriched by filtered lysate.
4. The vial was then allowed to rest on the test tube rack for 15 minutes
5. While the vial was resting, one Top Agar mixture was made for the group.
6.  8 ml of LB broth was transferred to a conical vial.
7. 90 microliters of the CaCl2  was transferred to the conical vial with the LB broth.
8. The vial was then set on the rack.
9. 10 ml of the 2X TA was added to the LB broth and Cacl2 after the sample was allowed to enrich the lysate for 15 minutes.
10. 4.5 ml of the top agar mixture was transferred to the test tube with the enriched lysate.
11. The contents of the test tube were then poured onto the agar plate.
12.  Part of the top agar mixture was poured into the top agar control plate for the group.
13. To let the top agar solidify, the plates were allowed to rest for 20 minutes.
14. The plates were placed upside down in the incubator, where they will remain for 48 hours.

Analysis and Conclusion 

Although there seemed to be plaques on the plate form 10/17/18, it seemed wise to redo the plaque assay to verify and avoid waste of time that may result from picking plaques. There was no contamination and all procedures were properly performed in the aseptic zone.