Rationale

Today we will run gel electrophoresis on the PCR samples created on 10/17/18.

Procedure

• 40 mL of 1X TBE and 0.8 g of Agrose were combined in a flask and microwaved to dissolve. 2 µL of EtBr was added to the warmed solution.
• The mixture was poured onto an electrophoresis tray and was set aside to cool for 20 minutes.
• After cooling, the gel was submerged into 1X TBE until covered.
• 10 µL of each PCR sample and 5 µL of DNA ladder were pippetted into the wells on the gel. Microcentrifuge tubes labeled 1$, 2$, and 3$ were dispensed into wells 5, 6, and 7 respectively.
• 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 display the results.

Observations

After the gels were run, the color produced resembled a light blue while the gels of the other groups produced a more vivid blue.

Conclusion/Next Steps

The results were negative, indicating that new soil must be tested next in pursuit of phage. Previously tested soil may also be re-tested to affirm past discoveries.

Rationale

Phage presence will be tested on the enriched isolation produced on 10/15/18 with soil sample E using PCR.

Procedure

• Established an aseptic zone.
• 8.5 mL of enriched isolation was aliquotted into a tube and centrifuged at 5000g for 5 minutes.
• 1 mL of the centrifuged enriched isolation was put into a microcentrifuge tube and was heated for 10 minutes.
• 4 µL of primer mix 1, 6.5 µL of D.I. water, and two 1 µLs of “phage DNA” was placed into microcentrifuge tube 1 along with 12.5 µL of TAQ polymerase.
• 4 µL of primer mix 2, 6.5 µL of D.I. water, and two 1 µLs of “phage DNA” was placed into microcentrifuge tube 2 along with 12.5 µL of TAQ polymerase.
• 4 µL of primer mix 3, 6.5 µL of D.I. water, and two 1 µLs of “phage DNA” was placed into microcentrifuge tube 3 along with 12.5 µL of TAQ polymerase.
• 12.5 µL of TAQ polymerase, 1 µL of “phage DNA”, 4 µL of primer mix 1, and 7.5 µL of D.I. water were placed into microcentrifuge tube 4 to make a positive control.
• The 4 tubes were then cycled through PCR.

Observations

D.I. water was used in the tubes rather than D.D.I water, which may have an effect on the results of the experiment.

Conclusions/Next Steps

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

Rationale

Due to contamination that was present in the soil E enriched isolation made on 10/10/18, soil sample E must be washed and enriched again.

Procedure

• Established an aseptic zone.
• 2 mL of soil and 10 mL of LB Broth was added to a tube. The tube was vortexed for 10 minutes and then centrifuged at 5000g for 10 minutes.
• After centrifuging, the supernatant produced was filtered using a syringe filter and 0.5 mL of arthrobacter was added. The enriched isolation was placed into the incubator for 48 hours.
• In order to determine the amount of sand, silt, and clay present in the soil, the falcon tube was removed from the fume hood and the mL of sand, silt, and clay present were recorded.
• In order to determine the percent water in the soil sample, the soil sample was taken from the fume hood and was massed.

Results/Observations

The enriched isolation made on 10/10/18 possessed a slightly pink tint, mimicking a diluted color from the red bacteria that was present on the plates observed on 10/10/18.

Mass of the weigh boat: 5.327 g

Percent of water in the sample: 19.4%

Amount of sand: 4.5 mL

Amount of silt: 1.5 mL

Amount of clay: 1 mL

Percent sand: 60%

Percent silt: 26.6%

Percent clay: 13.3%

Conclusion/Next Steps

Next we will begin a new method of testing bacteriophage using our enriched isolation.

Rationale

Today we will collect soil sample E and metadata on the sample as well.

Procedure

• Established an aspectic zone.
• New soil was collected and 2 mL of it was placed into a vial along with 10 mL of LB broth. The vial was shaken for 10 minutes and then was centrifuged at 5000g for 10 minutes.
• After being centrifuged, the supernatant produced was syringe filtered into another vial and 0.5 mL of Arthrobacter was added. The enriched isolation was placed into the incubator for 48 hours.
• To determine the percent water of the soil, the weigh boat was massed, ~3g of soil was added, and then the weigh boat containing the soil was massed again. It was set under the fume hood for 48 hours to dry.
•  To determine the percent sand/silt/and clay of the soil, 10 mL of soil was placed into a falcon tube and DI water was added to the 30 mL line. 3 drops of soil dispersion liquid were added and the tube was shaken for 45 seconds. The tube was then placed under the fume hood for 48 hours.
• To determine the pH of the soil, 1 mL of soil was added to a pH vial and the rest of the vial was filled with DI water. The vial was shaken for 30 seconds and then was set aside for 10 minutes. The pH was then measured using pH paper.

Observations

Mass of the weigh boat: 2.326 g

Mass of the weigh boat and soil: 6.048 g

pH of soil: 6

10 mL of enriched isolation was able to be produced.

The plaque assay produced from 10/8/18 resulted in contamination and the presence of colored bacteria. Despite the contamination, it was concluded that soil sample D did not have phage presence, therefore another soil sample was collected.

Conclusions and Next Steps

We will conduct a plaque assay with our enriched isolation to test for phage presence. The results of the metadata experiments will be observed as well.

Rationale

Today we will conduct a plaque assay and collect soil metadata on soil sample D.

Procedure

• Established an aspectic zone.
• The enriched isolation produced on 10/3/18 was respun for 10 minutes at 5000g. The supernatant produced was filtered using a syringe filter into another vial.
• 10 mLs of LB broth, 112.5 µL of CaCl2, and 12.5 mL of 2X TA was added to a tube.
• 10 µL of the filtered enriched lysate was added to a vial containing 0.5 mL of Arthrobacter.
• 4.5 mLs of the solution from the first tube was added to the second tube and then was mixed. The mixture was poured immediately into a petri dish. It was set aside to solidify for 10 minutes.
• The falcon tube was retrieved from under the fume hood and the % sand, silt, and clay were measured.
• The % H2O in the soil was measured by massing the weigh boat.

Observation

There were no air bubbles produced when pouring the plaque assay into the plate.

Amount of sand: 4.2 mL

Amount of silt: 1.3 mL

Amount of clay: 1 mL

Percent sand: 64.6%

Percent silt: 20%

Percent clay: 15.4%

Percent H2O: 18.3%

Conclusion/Next Steps

The results of the plaque assay will be observed. If the plaque assay is negative, more soil will be collected. If the plaque assay is positive, we will begin purification.

Rationale

Today we will collect soil, wash it, and collect metadata.

Procedure

• Established an aspectic zone.
• With new soil collected, 2 mL of soil sample #4 and 8 mL of LB Broth was added to a tube. The tube was then vortexed for 10 minutes and massed.
• After 10 minutes, the vortexed soil was centrifuged at 5000g for 10 minutes. The supernatant produced was filtered with a syringe filter into another vial and 0.5 mL of Arthrobacter was added. The vial was incubated for 48 hours.
• To determine the percent water of the soil, a weigh boat was massed and then massed again after ~3g of soil was added. It was set under the fume hood for 48 hours to dry.
• To determine the percent sand/silt/and clay of the soil, 10 mL of soil was placed into a Falcon tube and was filled to the 30 mL mark with D.I. water. 3 drops of soil dispersion liquid were added and the tube was vigorously shaken for 45 seconds. It was then set under the fume hood to sit for 48 hours.
• To measure the pH of the soil, 1 mL of soil was placed into a pH vial. It was then filled to the top with D.I. water and was shaken for 30 seconds. The vial was set aside for 10 minutes. After 10 minutes, pH paper was placed in and the pH was measured.

Observations

Mass of the weigh boat: 2.328 g

Mass of the weigh boat and soil: 5.985 g

pH of the soil: 6

After the tube was centrifuged, the supernatant produced was clear and slightly yellow in tint. 9 mL of enriched lysate was able to be produced.

Conclusion/Next Steps

Using the enriched lysate from the soil sample, a plaque assay and a spot test will be conducted to test for phage presence. The results of metadata collection will be observed as well.

Rationale

Today we will conduct another plaque assay with soil sample #3. Lucy’s plaque assay results were positive while Sona’s and I were not, despite the soil originating from the same point in the tree; therefore we will test our soil again to confirm the results.

Procedure

• Established an aseptic zone.
• 8 mL of LB broth and 90 µL of CaCl2 were added to a vial.
• 10 µL of enriched lysate was added to a vial containing 0.5 mL of Arthrobacter. It sat for 10 minutes.
• 10 mL of 2X TA was added to the first tube and 4.5 mL of the solution was added to the vial containing the enriched lysate and 0.5 mL of Arthrobacter. The contents of the mixture were then poured onto a plate and sat for 10 minutes.

Observation

When the contents of the mixture were poured onto the plate, no air bubbles were formed.

Conclusion/Next Steps

The results of the plaque assay will be observed on 10/3/18. If negative, we will collect another soil sample.

Rationale

Today we will reconduct the plaque assay from 9/26/18 due to possible contamination in the Arthrobacter used by the class.

Procedure

• Established an aseptic zone.
• The enriched lysate produced on 9/19/18 had formed a pellet near the bottom of the vial, therefore the lysate was separated into two tubes of equal mass and was spun again for 10 minutes to reproduce a new enriched lysate. The supernatant formed after spinning was syringe filtered through a 22 µm filter.
• 8.4 mL of LB broth and 90 µL of 1M CaCl2 were added to a tube.
• 10 µL of the new enriched lysate was added to a vial containing 0.4 mL of Arthrobacter. It sat for 10 minutes.
• 2X TA was added to the first tube and 4.5 mL of the solution was added to the vial containing the new enriched lysate and 0.4 mL of Arthrobacter. The contents of the mixture were then poured onto a plate that sat for 10 minutes.

Observation

The cause of the pellets formed at the bottom of the enriched isolation from 9/19/18 is unknown. The proper technique was used, however the isolation was still unclear in color on 9/26/18. The TA poured onto the plate did not produce any air bubbles.

Conclusion

The results from the plaque assay will be observed again and a spot test will be conducted after.

Rationale

Today we will conduct a plaque assay and collect soil metadata on the new soil collected.

Procedure

• Established an aseptic zone.
• 10 µL of enriched lysate was micropipetted into a vial containing 0.5 mL of Arthrobacter. It was set aside for 10 minutes.
• 8 mL of LB broth was aliquoted into a tube along with 90 µL of CaCl2 and 2X TA. The solution was mixed by pipetting up and down, and 4.5 mL of the solution was pipetted into each tube containing the 10 µL of enriched lysate and 0.5 mL of Arthrobacter.
• The mixed solution was then poured onto a petri dish and was set aside for 10 minutes.
• The percent water in the soil was observed by massing the weigh boat, which was used to determine the % water in the soil.
• The amount of sand/silt/and clay in the soil was observed by seeing the total amount of soil that was present in our falcon tubes , the amount of sand, the amount of silt, and the amount of clay.
• The area was cleaned.

Observations

Percent water in the soil: ((3.88-3.231)/3.88) x 100= 16.7%

Total amount of soil in the falcon tube: 7 mL

Amount of sand: 5.5 mL

Amount of silt: 1 mL

Amount of clay: 0.5 mL

Percent sand: 78.57%

Percent silt: 14.28%

Percent clay: 7.14%

No air bubbles formed when pouring the top agar into the plate.

Conclusion/Next Steps

We will assess the results of the plaque assay and determine if there is phage presence or not. From there we will conduct a spot test to confirm our results.

Rationale

A spot test will be conducted using the possible plaques obtained from the plaque assay on 9/17/18. More soil and soil metadata will be collected as well.

Procedure

1. Use the aseptic technique to clean the area.
2. 10o µL of phage buffer was pipetted into 3 micro test tubes. The tip of a pipet was then placed into each possible phage from the previous plaque assay and the placed back into each micro test tube. Set aside the tubes.
3. Pipet 2 mL of LB broth into a vial along with 0.5 mL of Arthrobacter, 22.5 µL of CaCl2, and 2 mL of 2X TA. The vial was poured onto a plate and the plate sat for 10 minutes.
4. After 10 minutes had passed, 5 µL of each micro test tube was micro pipetted onto each quadrant of the petri dish, along with 5 µL of phage buffer into a control quadrant. The plate then sat for another 10 minutes.
5. New soil was collected by lab partners during this time.
6. Soil washing then began by placing 2 mL of soil and 9 mL of LB broth into a vial. The vial was shaken for 10 minutes to mix.
7. The vial was then massed and centrifuged for 10 minutes.
8. While the soil was centrifuging, % water calculations began by massing a weigh boat, adding soil, and then massing the weigh boat again. The weigh boat was then placed in the fume hood for 48 hours.
9. The percent sand/silt/clay of the soil was also measured by adding 10 mL of soil to a falcon tube. The tube was filled to the 30 mL mark with DI water and 3 drops of soil dispersion liquid was added. The tube was covered and shaken for 30 seconds. After, it was placed in the fume hood for 48 hours.
10. To measure the pH, 1 mL of soil was placed into a vial and then filled with DI water. The vial was shaken for 30 seconds and then sat for 10 minutes. After 10 minutes passed, pH paper was put in the vial and the pH was measured.
11. After the tube containing soil was centrifuged, the supernatant was filtered using a syringe and syringe filter to produce 6.5 mL of enriched isolation.
12. 0.5 mL of Arthrobacter was added and the tube was incubated.

Observations

The mass of the tube before being centrifuged was 17.18 g. The mass of the weigh boat was 2.32 g and the mass of the soil and weigh boat was 6.20 g. The pH of the soil was 6. This soil contained more debris than the previous soil collected.

Conclusions

The results of the spot test will be observed on Monday. From there, we will see if the previous soil sample can be used or if the new sample will be need to be tested.