Rationale

Performed a spot test from the newly enriched soil lysate to test whether or not group 6 as a phage. Performed a plaque assay by using another classmate’s enriched lysate sample which does have phage. I had to perform the plaque assay because I need to be able to start creating a high titer soon.

Detailed Procedure: Spot Test

1. Took two vials labeled as 1X and 2X.
2. For the 1X vial, took 2.5 mL of 2X TA, 2 mL LB Broth, and 23 microliters of 1M CaCl2.
3. Poured 1X vial onto plate labeled as control.
4. For the 2X vial, took 5.0 mL 2X TA, 4.0 mL of LB Broth, 4.5 microliters of 1M CaCl2, and 1.0 mL of Arthro.
5. Took half of the 2X vial and poured equally onto two separate experiment plates.
6. Waited 15 min for plates to solidify
7. Took 10 microliters of each sample and PB and spot tested onto designated plates.

Detailed Procedure: Plaque Assay

1. Took 10 microliters of filtered enriched lysate and added to 0.5 mL Arthro. Sat for 15 mins.
2. During 15 mins, took 4.0 mL LB Broth, 45 microliters of 1M CaCl2, and 5.0 mL of 2X TA into separate 50 mL vial. Mixed through pipetting.
3. Used sterile pipette and took half of solution from step 3 into another separate 15 mL vial. Labeled as control.
4. After 15 mins were done, took solution from step 2 and mixed through pipetting into previous 50 mL vial.
5. Took both vials into water bath and waited for plate to be ready.
6. Labeled plates as “Plaque Assay” and Plaque Assay Control.”
7. Took control vial and poured into control plate and same with other plate+vial.
8. Waited 15 mins to solidify.

Conclusion

On the control plate for the plaque assay, the mixture did not cover the entire plate so it solidified unequally on the plate. Until next lab day, group 6 will check results on the spot test from the new enriched soil and start picking the plaque and creating a high titer from the plaque assay.