Rationale: After my first round of purification I need to calculate the titer of my lysate. However, since not enough TA is available to make multiple plaque assays I conducted a spot test to calculate my titer.

Procedure:

1. Flooded plaque assay from Wednesday for 2 hours by pouring 8mL of phage buffer on the plate then putting it on a shaker.
2. Filtered the phage buffer with a .22µm syringe filter.
3. Diluted with phage buffer out to a dilution factor of 10^-8.
4. Marked a plate to spot for dilutions of 10^0 to 10^-7.
5. Made top agar with 2mL of LB broth, 2.5mL TA, 22.5µL of CaCl2, and 0.5mL of arthrobacter.
6. Let the plate sit until solidified.
7. Spotted each dilution in marked location with 4µL of corresponding lysate.
8. Let spot dry then inverted and placed into incubator.

Observations:

The section with a dilution factor of 10^-7 had 16 plaques. From that I calculated that my lysate has a titer of 4e10. Since  the plaques had a radius of 0.75mm it will take 8µL of 10^-5 lysate to web  a plate.

Conclusions and Next Steps: Since I have a high titer I now have to make a couple webbed plates so I can collect enough lysate to run DNA extraction and then archiving.