10/05/18

Objective:

• To make a webbed plate from adopted lysate
• To pick a plaque and make another plaque assay

Procedure:

Webbed Plate:

1. Add 300 μl of lysate to 0.5 ml of arthrobacter culture and allow the sample to enrich for 15 minutes
2. 1 top agar is made for 2 lab partners.
3. While the tube enriches, add 4.5ml of LB broth to a conical vial
4. Add 67.5 μl of CaCl2 to the vial.
5. After the 15 minutes have passed, add 4 ml of TA mixture to the vial with arthrobacter and lysate.
6. pour contents of the vial onto an agar plate.
7. pour 4 ml of TA mixture onto another Agar plate for a control plate,
8. Allow the plates to rest for 15 minutes and then place them inverted in the incubator.

Purification:

1. Pick a plaque of the latest plaque assay using a micropipette and dip the tip in a microcentrifuge tube with 70μl of phage buffer.
2. Vortex microcentrifuge tube for 15 seconds.
3. Transfer 30μl of this phage extract to a 0.5 ml of arthrobacter and allow the sample to enrich for 15 minutes.
4. 1 plaque assay is made using the same LB broth and 2X TA as before.
5. Meanwhile, add 2 ml of LB broth to a conical vial.
6. Add 22.5μl of CaCl2 to the conical vial.
7. After 15 minutes have passed, add 2.5 ml of 2X TA to the conical vial.
8. Transfer all the contents of the conical vial to the test tube with the Enriched Arthrobacter.
9. Pour the contents of the test tube onto an Agar plate.
10. Allow TA to solidify for 15 minutes and then place the plate inverted in the incubator.

Analysis and Conclusion;

A webbed plate and a 4th purification run was done so as to preserve time. If this procedure does not result in a webbed plate, same procedures will be repeated for the new phage extract, titer will be calculated and another webbed plate will be attempted. The current lysate has a low titer so further purification might me needed, even if webbed plate is flooded.