Rationale:

To create a high titer lysate for use in transmission electron microscopy and DNA analysis.

Procedures:

1. Established an aseptic zone.
2. Added 10µL “CEW Lysate” to 90µL phage buffer to make 10^-1 dilution, repeated to make 10^-2 and 10^-3.
3. Added 10µL of each dilution plus 10µL of 10^0 to 4 tubes of arthro, let infect 10 minutes.
4. Added 10mL of LB broth and 112.5µL of CaCl2 to a vial.
5. Added 2mL of this solution to each of the vials of arthro.
6. Added 2.5mL of 2xtop agar to all five vials.
7. Plated all vials in plates following the pattern “NMN 10^x(CEW) 11.12.18”, plated the top agar control.
8. Let solidify and incubated inverted.

Observations:

From the previous plating of this lysate on 11.8.18 it was observed that the entire plate had been lysed and it appeared that arthro had regrown over the plate in a lesser amount, however, it could have also been contamination.

Conclusions/Next Steps:

The next step will be to check on Wednesday to see which plate(s) has been webbed and which are lysed and which are spotted with plaques. We can then use this to determine the titer of the lysate and flood the webbed plate to create a higher titer lysate if needed.