Team:BYU Provo/Notebook/SmallPhage/Springexp/Period2/PR

Overview

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We infected E. coli BL21 with different concentrations (1ul, 10ul, and 100ul) of phage to see which would yield the highest titer. Plaques formed up to -8 on all the spot tests, showing that the concentration difference had no effect on the titer. However, the plates were badly contaminated.

5.13 Determining E. coli Concentration With Spectrophotometer

To check if we could determine E. coli concentration with a spectrophotometer, we created a 7/5 dilution series of E. coli. We then measured the absorption at 600nm of each E. coli dilution. When plotted, these measurements showed a linear relationship between E. coli concentration and the absorption reading.

5.15 Titer Test on 5.3 T7 new Phage Stock

Created a 1:10 dilution series with 5.3 T7 stock. We then created titers of -5, -6, -7, and -8. -5, -6, and -7 plates all had overlapping plaques. However, the -8 plate had 7 plaques, giving us an estimated phage concentration of 7E8 particles/20ul.

5.20 Mutagen Concentration Experiment

In order to determine the best concentration of mutagen to use, we infected the E. coli in 5 tubes with 0ul, 10ul, 50ul, 100ul, and 200ul of our mutagen, 5-bromodeoxyuridine. We then added 6uL of 5.3 phage stock to each tube, allowed it to incubate for 20 minutes, and purified the phage. Next, we made a dilution series and performed a spot test and found that when the mutagen concentration is increased, the concentration of phage is decreased. We then performed titers from -6, -7, and -8 of phage from our dilution series with x8 top agar. There are a couple plaques on each plate.

3. Next Steps