September
21
9/19/18 2nd Purification Plaque Assays – Soil B
Previous Results:
- Plaque assays from different dilutions created during the last lab (9/17) were all positive and the control did not have any contamination.
Objective:
- Pick a plaque from the plaque assay made during last lab
- Conduct a second series of serial dilutions and plate the diluted lysates with phage
Procedure:
- Aseptic Zone was prepared. Lab space was cleaned using CiDeon and wiped dry with paper towel. Ethanol (70%) was then sprayed, wiped, and evaporated. Ethanol burner was then lit on the table.
- A plaque was picked from the 10^0 plate by placing the tip of the pipet into a clearing on the plate. The tip was then put in 100 microL phage buffer and pipetted up and down 15 times to release phage in buffer, becoming a new 10^0 dilution
- 10 microL of new 10^0 was transferred to a microcentrifuge tube containing 90 microL of PB, creating 10^-1 dilution
- Step 3 was repeated to create a 10^-2 dilution
- The 3 dilutions were poured into separate tubes of 0.5 mL Arthrobacter to allow phage to interact with bacteria
- A mixture of Overlay Agar for 4 plates was made using 8 mL of LB broth, 90 microL CaCl2, 10 mL 2xTA. Then the mixture was separated into 4 50 mL tubes, with 4.5 mL in each.
- The mixture of Arthrobacter and dilutions were poured in their respective tubes, 10^0, 10^-1, and 10^-2
- All experimental tubes and the control tube were plated and left to harden for 15 min
- Plates were placed in incubator for 48 hours.
Results:
- Results will not be available until next lab (9/24) to see if the phage were plated correctly and there are positive results.
Conclusion:
- The results from the previous lab were positive
- The serial dilution was correctly conducted using a picked plaque from the positive plate
Next Steps:
- During the next lab (9/24) the plates with the second round of serial dilutions will be examined for phage. A third series of dilutions will be done to ensure only one strain of phage is being tested. The same steps will be conducted.