Rationale

Today we will conduct TEM to image our phage. We will also make a precipitate using our lysate.

Procedure

• Established an aseptic zone.
• 10 mL of lysate was poured into a tube to begin making a precipitate. 40 µL of nuclease was added to the tube and it was inverted 10 times. 4 mL of phage precipitant solution was added and the tube was inverted once to mix. It was placed in the incubator for 25 minutes.
• TEM was performed during the incubation period and a grid was prepared.
• 1 square of parafilm was prepared by aliquotting 20 µL of lysate onto the square, 20 µL of D.I water, another 20 µL of D.I. water, and 20 µL of uranyl acetate in sequential drops.
• A TEM grid was placed into the lysate for 5 minutes, each D.I. water drop for 2.5 minutes, and the uranyl acetate for 1 minute.
• Filter paper was used to wick away the excess liquid from the grid and the grid was imaged.
• The precipitate preparation in the incubator was removed after 25 minutes and left at room temperature for 40 minutes.

Observations

The last spot titer test led to inconclusive results. The 10^-1, 10^-2, and 10^-3 quadrants had completely lysed, however, all the quadrants following did not produce any plaques. This resulted in the titer of our lysate being unknown. However, due to the titer of the current lysate being 1.2×10^7, it was decided to continue onto TEM.

After imaging, the head of the phage was recorded to be 53 nm in width. The tail was recorded to be 110 nm in length.

The centrifuge swinging bucket was not functioning, resulting in precipitate preparation to not be completed.

Conclusion/Next Steps

The precipitate preparation will be completed and DNA extraction will begin. A spot titer test will also be conducted to determine the titer of the lysate.

Rationale

Today we will perform a spot titer test on the flooded lysate to determine if our lysate is a high titer.

Procedure

• Established an aseptic zone.
• 90 µL of phage buffer was aliquotted into 8 microcentrifuge tubes. 10 µL of flooded lysate was pipetted into the microcentrifuge tube labeled 10^-1. The tube was vortexed for a few seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-1 were pipetted into the microcentrifuge tube labeled 10^-2. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-2 were pipetted into the microcentrifuge tube labeled 10^-3. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-3 were pipetted into the microcentrifuge tube labeled 10^-4. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-4 were pipetted into the microcentrifuge tube labeled 10^-5. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-5 were pipetted into the microcentrifuge tube labeled 10^-6. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-6 were pipetted into the microcentrifuge tube labeled 10^-7. The tube was vortexted for 10 seconds to mix.
• 10 µL of the contents in the microcentrifuge tube labeled 10^-7 were pipetted into the microcentrifuge tube labeled 10^-8. The tube was vortexted for 10 seconds to mix.
• 4 mL of LB Broth, 5 mL of 2X TA, and 45 µL of CaCl2 were combined in a vial. 4.5 mL of the mixture was combined with 0.5 mL of Arthrobacter and was poured onto a plate. The remaining 4.5 mL of the mixture was combined with another 0.5 mL of Arthrobacter and was poured onto another plate. The plates sat aside for 10 minutes to solidify.
• The 5 µL of each microcentrifuge tube was spotted onto a designated quadrant. 5 µL of phage buffer was spotted onto a control quadrant.
• The plates set aside for 10 minutes and then placed into the incubator.

Observations

No air bubbles were observed in the process of making the plates.

Conclusions/Next Steps

The results of the plate will be observed on 11/28/18 and the titer of our lysate will be calculated.

Rationale

Today we will calculate the titer of our lysate from previously performed spot titer test. After calculating the titer, we will web 5 plates.

Procedure

• Established an aseptic zone.
• The calculated titer was used to determine the amount of lysate needed to web a plate.
• 26.7 µL of 10^-2 lysate was added to 0.5 mL of Arthrobacter. This process was repeated 5 times and each mixture was set aside for 10 minutes.
• 12 mL of LB broth, 135 µL of CaCl2, and 15 mL of TA was added to another vial.
• 4.5 mL of the mixture in the second tube was combined with each vial and was poured onto a plate. Five plates were produced along with a control.
• The plates were set aside for 10 minutes to solidify. After 10 minutes, the plates were placed inside an incubator.

Observations

High titer calculations:

Spot Titer Test:

The 10^-4 lysate dilution was used in calculations due to the 10^-3, 10^-2, 10^-1 lysate dilutions creating webbed or lysed spots.

Conclusions/Next Steps

Next, we will flood our hopefully webbed plates to produce a high titer lysate.

Rationale

Today we will conduct spot titers to calculate the titer of an unknown lysate from Lucy P.

Procedure

• Established an aseptic zone.
• 90 µL of phage buffer was aliquotted into 8 microcentrifuge tubes. 10 µL of flooded lysate was pipetted into tube #1 containing 90 µL of phage buffer. 10 µL of the contents in tube #1 was pipetted into tube #2. 10 µL of the contents in tube #2 was pipetted into tube #3. The process repeated down the line for all 8 tubes.
• 4 mL of LB broth, 5 mL of 2X TA, 1.0 mL of Arthobacter, and 45 µL of CaCl2 was combined into a vial. The 10 mL of solution was divided evenly between two plates and poured. The plates were set aside for 10 minutes to solidify.
• 5 µL of the contents in each tube was spot tested onto their designated quadrants on each plate. Phage buffer was spot tested on the control quadrant. The plate was set aside for 10 minutes and then placed into the incubator.

Observations

When pouring the plate, plate #2 possessed large amounts of air bubbles that could possibly be mistaken for plaques when analyzing.

Conclusions/Next Steps

Results will be assessed on 11/16/18 to determine the titer of the lysate.

Rationale

Today we will flood webbed plates created on 11/7/18 to create more lysate from Lucy’s phage.

Procedure

• Established an aseptic zone.
• 5 mL of phage buffer was poured onto one previously webbed plate. The process was repeated for another plate The plates were placed on a shaker for 1 hour.
• After 1 hour had passed, the flood lysate was filtered through a 0.22 µm syringe filter and was placed in the fridge for later use.

Observations

The plates that were flooded are shown above. One plate fell off the shaker, causing 5 mL of phage buffer to be added again and the 1 hour long waiting period to be restarted.

Conclusions/Next Steps

8.3 mL of lysate was collected from flooding, which will allow for use in later plaque assays and spot tests in pursuit of a high titer.

Rationale

Due to the webbed produced from 11/5/18, we will make more webbed plates for potential flooding in the future.

Method

• Established an aseptic zone.
• 10 µL of LIP flooded lysate was added to 0.5 mL of Arthrobacter. This was repeated to produce 2 LIP flooded lysate and Arthrobacter mixes.
• 6 mL of LB Broth, 7.5 mL of 2X TA, and 67.5 µL of CaCl2 was combined into a vial.
• 4.5 mL of the second solution was added to a LIP flooded lysate and Arthrobacter mix and was promptly plated. This was repeated for the second plate.
• Remaining TA mix was poured onto a control plate.
• Plates were set aside for 10 minutes and then placed into the incubator.

Observations

The plate shown was produced on 11/5/18 and indicates what was intended on being replicated. No air bubbles formed when pouring the plate.

Conclusions/Next Steps

Next, we will do serial dilutions on the plate created by LIP and LJF. We will also flood the plates for more lysate.

Rationale

Today we will conduct a plaque assay to help Lucy create a high titer lysate.

Procedure

• Established an aseptic zone.
• 30 µL of phage lysate was added to 0.5 mL of Arthrobacter and was set aside.
• 2 mLs of LB Broth, 22.5 µL of CaCl2, and 2.5 mLs of 2X TA were combined in a separate vial.
• The two tubes were combined and poured onto a petri dish. The dish was set aside for 10 minutes and then incubated for 48 hours.

Observations

No air bubbles were observed when pouring, indicating no possible sources of confusion for plaques when assessing the results at a later date.

Conclusions/Next Steps

Next, we will observe the results of the plaque assay and potentially flood the plate.

Rationale

Today we will run gel electrophoresis on the PCR samples created on 10/31/18.

Procedure

• Established an aseptic zone.
• 40 mL of 1X TBE and 0.8 g of Agarose were added into a flask and microwaved until clear.
• The mixture was set aside to cool for 5 minutes. After cooling, 2 µL of EtBr was added to the flask and was swirled around.
• The contents in the flask were poured into a gel apparatus with a comb inserted and set aside for 20 minutes.
• After the gel had set, the rubber sides of the apparatus were removed as well as the comb. 1X TBE was used to submerge the gel.
• 2.5 µL of dye was added to each PCR tube before pipetting into the wells.
• 10 µL of microcentrifuge tubes 1 •, 2 •, 3 •, positive control 1, positive control 2, and positive control 3 were pipetted into wells 1, 2, 3, 5, 6, and 7 respectively.
• 5 µL of DNA ladder was pipetted into well 4.
• The gel electrophoresis apparatus was plugged into a power source, which was then turned on and set aside for 30 minutes.
• The gels were then imaged to reveal the results.

Observations

After the gel was plugged into the power source, the blue color traveled approximately the same distance across the gel for each sample and positive control.

Conclusions/Next Steps

The results were negative, however, one of the “phage DNA” samples tested was a known positive. This indicates that the gel electrophoresis method for testing for phage presence is flawed.

Rationale

PCR will be conducted on an enriched isolation of soil sample C produced on 10/24/18.

Procedure

• Established an aseptic zone.
• The enriched isolation produced on 10/24/18 was centrifuged at 5000g for 5 minutes.
• 1 mL of the spun enriched isolated was aliquotted into a microcentrifuge tube and was heated for 10 minutes.
• 4 µL of primer mix 1, three 2 µL samples of “phage DNA”, 2.5 µL of DDI water, and 12.5 µL of TAQ polymerase were added to PCR tube 1.
• 4 µL of primer mix 2, three 2 µL samples of “phage DNA”, 2.5 µL of DDI water, and 12.5 µL of TAQ polymerase were added to PCR tube 2.
• 4 µL of primer mix 3, three 2 µL samples of “phage DNA”, 2.5 µL of DDI water, and 12.5 µL of TAQ polymerase were added to PCR tube 3.
• The 3 tubes were then cycled through PCR and stored for later use.

Observations

The mixture in the PCR tubes was colorless rather than the teal color that was observed at the last round of PCR. This is due to a lack of dye that will be added later before conducting gel electrophoresis.

Conclusions and Next Steps

Using the PCR results, gel electrophoresis will be run on Monday to test for phage presence in the sample.

Rationale

Today we will wash and enrich soil sample C for the second time. This soil was previously tested and yielded positive results for one member of the lab group, and negative results for the other two members of the group. Due to the inconsistency, the soil sample C will be tested again.

Procedure

• 2 mL of soil and 10 mL of LB broth were added to a vial. The vial was vortexed for 10 minutes and then centrifuged at 5000g for 10 minutes.
• The supernatant produced was then dispensed into another vial and 0.5 mL of Arthrobacter was added.
• The vial was placed into the incubator where it was shaken for 48 hours.

Observations

Due to the lack of filters, the supernatant was directly poured out of the vial and into another. This allowed for debris to be transferred and the resulting enriched isolation to be cloudy rather than clear.

Conclusion/Next Steps

Next, we will test soil sample C again through PCR and gel electrophoresis in hopes of phage presence.