Rationale:

The purpose of today’s lab was to run a spot test with several serial dilutions on a single plate to quickly determine a titer without running multiple plaque assays for each dilution.

Results from 11/12/18

• 25 mL of a new combined lysate was gathered and stored in a fridge. This lysate was combined to quickly move towards getting a high titer and volume of phage as time is running short for TEM and DNA extraction. A high titer is required by the next week and it was decided to spot titer out the new lysate with several serial dilutions.

Materials:

• LB Broth
• Phage Buffer
• Calcium Chloride
• New Combined Flooded Lysate
• 2X TA
• Agar Plates

Procedure:

1. Began by establishing an aseptic zone.
2. Next, began the process of diluting the lysate out to the 10^-8.
3. To do this, 100 µL of phage buffer was added to a micro centrifuge tube, and 90 µL of phage buffer was added to 8 more micro centrifuge tubes to dilute the lysate, each was labeled with their respective dilution.
4. Then, 10 µL of the combined lysate was added to the 100 µL micro centrifuge tube, making the 10^0 dilution.
5. After this, 10 µL was removed from the 10^0 and added to the firs 90 µL tube, making it the 10^-1 dilution, this process of removing 10 µL of dilution from the previous centrifuge tube and adding to the next was repeated until the lysate had been diluted all the way out to 10^-8.
6. Once serial dilutions were complete, two agar plates were acquired and one had a 3×3 grid drawn on it with each grid being labeled with a dilution factor.
7. A spot test was then performed and began with adding 2.0 mL of LB broth to a conical vial 1 and 2.5 mL to a top agar control vial.
8. Then 22.5 µL of Calcium Chloride was added to both of the conical vials.
9. 0.5 mL of Arthrobacter was added to conical vial one without lysate infection.
10. After, 2.5 mL of 2X TA was added to each conical vials and then the top agars were plated immediately.
11. Plates were then left to solidify for 20 minutes.
12. After the time was up, 10 µL of each diluted lysate was spotted onto their respective and labeled grid on the top agar plate, and that was left to sit for 15 minutes before flipping and leaving in the incubator.

Results/Data:

• Plate was not flipped fast enough and spotted lysate ran out from the marked area on the agar plate. Also, due to the high volumes of lysate, an increased titer is expected from the amplification process. Top agar seemed hazier than normal, but due to the low amounts of top agar that were available in lab and the constant contamination of LB broth and TA in the lab, no other alternative was any better.

Conclusions

It can be concluded and expected that the spot titer will yield plaques past than the 10^-4 dilution due to the amplification process. The target titer of the lysate has to be above 10^8, so TEM and DNA extraction can be made possible. The hazy top agar could lead to possible contamination of the top agar control plate as well.

Next Steps:

The next steps for this experiment are to calculate the titer from the spot titer test. If the titer is a high titer, then the lysate will be prepped for DNA extraction and TEM. If not, the amplification process will be continued, so that a high titer can be achieved.