Rationale: Calculate Justin’s titer by performing a series of serial dilutions. Justin has a medium titer, and a titer that is greater than 10^8 will be needed to extract the phage genome.

Procedure:  Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. Justin’s lysate was used to perform titer dilutions. The lysate was added into a microcentrifuge with 90µl of PB and 10µl of Justin’s lysate. The solution (lysate plus PB) was diluted from 10^0 to 10^-6. Once this was done, a 50mL vial was obtained to make the solution needed for the plaque assay. The formula below was used to make the solution for 7 plates (six for the experiment and one for the TA control).

• 2mL LB Booth (x7)
• 22.5 microliters of Calcium Chloride (x7
• 2.5mL 2X TA (x7)
• 500 microliters of Arthro

Added the 2X TA last to the 50mL vials. shook the vial, and quickly pipetted the solution onto each vial containing the Artho/lysate solution. Sat each plate for about 15mins to the solution solidify. The remaining solution that was left in the 50mL vial was used for the control. All the plates sat to solidify for 15 minutes, inverted, and then placed into an incubator at 27 degrees Celcius.

Observations: Justin has a medium titer and high titer calculations will be done on 11/7 lab experiment. The results from the titer dilution performed 11/5 will help determine how much lysate is needed to obtain a high titer of Justin’s lysate. The experiment performed on 10/31 with Justin’s lysate was contaminated. and the experiment was negative. The experiment performed on 10/31 was done to test the titer of Justin’s lysate, but the experiment performed 11/5 will do the same.

Conclusions: A titer will be calculated once the plaque assays develop from the 11/5 experiment (48 hours). A titer of 10^8 or greater is required or the experiment will have to be done again, and variables will be altered.