Rationale

Today we will perform a spot titer test on the flooded lysate to determine if our lysate is a high titer.

Procedure

• Established an aseptic zone.
• 90 µL of phage buffer was aliquotted into 8 microcentrifuge tubes. 10 µL of flooded lysate was pipetted into the microcentrifuge tube labeled 10^-1. The tube was vortexed for a few seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-1 were pipetted into the microcentrifuge tube labeled 10^-2. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-2 were pipetted into the microcentrifuge tube labeled 10^-3. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-3 were pipetted into the microcentrifuge tube labeled 10^-4. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-4 were pipetted into the microcentrifuge tube labeled 10^-5. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-5 were pipetted into the microcentrifuge tube labeled 10^-6. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-6 were pipetted into the microcentrifuge tube labeled 10^-7. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-7 were pipetted into the microcentrifuge tube labeled 10^-8. The tube was vortexted for 10 seconds to mix.
• 4 mL of LB Broth, 5 mL of 2X TA, and 45 µL of CaCl2 were combined in a vial. 4.5 mL of the mixture was combined with 0.5 mL of Arthrobacter and was poured onto a plate. The remaining 4.5 mL of the mixture was combined with another 0.5 mL of Arthrobacter and was poured onto another plate. The plates sat aside for 10 minutes to solidify.
• The 5 µL of each microcentrifuge tube was spotted onto a designated quadrant. 5 µL of phage buffer was spotted onto a control quadrant.
• The plates set aside for 10 minutes and then placed into the incubator.

Observations

No air bubbles were observed in the process of making the plates.

Conclusions/Next Steps

The results of the plate will be observed on 11/28/18 and the titer of our lysate will be calculated.