Rationale: To prepare a TEM grid in order to view the phages under a transmission electron microscope

Procedures:

• Using a petri dish, a strip of parafilm was set across the dish.
• A drop of lysate (.15 µL) was set on the parafilm.
• The, two drops of water (the same amount as lysate) was added.
• After, one drop of uracil acetate was added onto the strip.
• For five minutes, a copper filter was in the lysate.
• After the five minutes, it was moved to the water for two and a half minutes.
• The previous step was done again in the second drop of water.
• After it has been in the water for its allotted time, the filter was placed in the uracil acetate for one minute.
• The filter was removed and placed into its place.

Results and Analysis:

Conclusion:

To prepare a TEM grid, a drop of lysate was added to the strip of parafilm on the petri dish. Two drops of water and one drop of uracil acetate were then added. For five minutes, the copper filter was placed in the lysate then moved to the two drops of water for two and a half minutes each then moved to the uracil acetate for one minute.

Future Plans:

In order to complete a DNA extraction, 10 mL of lysate is needed. Therefore, a plaque assay will be made then flooded in order to get more lysate.

Rationale: To make a plaque assay with the positive lysate to receive a high titer

Procedure:

• The tables were thoroughly wiped down with CiDecon and ethanol and an aseptic zone was also set up to prevent contamination.
• 125 µL of positive lysate and 0.5 mL Arthrobacter were placed in a new tube and left alone for 10 minutes to allow for infection.
• Next, 22.5 µL CaCl2 was combined with 2.0 mL of LB broth.
• After 10 minutes, lysate and Arthrobacter were added with LB broth and CaCl2.
• Then, 2.5 mL 2X Top Agar was added to the LB broth and CaCl2.
• The mixture was poured over the plate and then left alone to solidify before being placed in the incubator.

Results and Analysis:

Conclusion:

Due to the contamination of the negative control, a plaque assay was done. First, lysate and Arthrobacter were combined and left alone for 10 minutes to infect. Then, LB broth was added along with the calcium chloride. After the allotted time, the lysate and Arthrobacter were added to the LB broth and calcium chloride. 2X Top Agar was then added to the solution and poured onto the agar plate. For 15 minutes, the plates remained near an aseptic zone to solidify. After, the plates were placed in an incubator.

Future Plans:

When a plate is high titered with no contamination, the plate will be flooded with phage buffer and filtered to perform dilutions.

Rationale: After flooding the plate, the lysate will be filtered then used to perform serial dilutions to conduct a spot test

Procedure:

• The lysate from the flooded plate was taken using a syringe then filtered.
• Into eight tubes, 90 µL of phage buffer was added to each.
• From the 10^0 dilution, 10 µL of lysate was added to the tube containing phage buffer.
• From the 10^-1 dilution, 10 µL was taken and added to the 10^-2 tube and so on.
• After completing the dilutions, a spot test was made by combining 2.0 mL of LB Broth, 22.5 µL of calcium chloride, and 0.5 mL of Arthrobacter
• Then, 2.5 mL of 1X Top Agar was added then poured onto the plate.
• After 15 minutes of allowing the top agar to solidify, 0.7 µL of each dilution was added to their respective areas within the plate.
• The plate was left alone to allow for the spot dilutions to set in the agar.

Results and Analysis:

While removing the lysate from the flooded plate, the top agar tore.

Conclusion:

After setting up the correct measures to prevent contamination, eight small tubes were filled with 90 µL each. From the 10^0 lysate, 10 µL was transferred to another tube, creating the 10^-1 dilution. From the 10^-1 dilution,  10 µL was transferred to the 10^-2 tube to create the dilution and so on. Next, the spot test was made by combining LB Broth, 1X Top Agar, calcium chloride, and Arthobacter then poured onto a labeled plate.  After 15 minutes, the lysate was dropped in drops of 7 µL in their designated areas. The plates were left alone for 15 minutes to solidify then placed inverted in the incubator.

Future Plans:

Identify the dilution with the highest titer but not lysed then use that lysate to make a plaque assay to determine the titer of the plate, If it has a high titer, flood the plate.

Rationale: Due to the high titer in the last plate, a plaque assay will be made using more lysate to achieve a higher titer

Procedure:

The tables were thoroughly wiped down with CiDecon and ethanol and an aseptic zone was also set up to prevent contamination. 125 µL of positive lysate and 0.5 mL Arthrobacter were placed in a new tube and left alone for 10 minutes to allow for infection. Next, 90 µL CaCl2 was combined with 2.0 mL of LB broth. After 10 minutes, lysate and Arthrobacter were added with LB broth and CaCl2. Then, 2.5 mL 2X Top Agar was added to the LB broth and CaCl2. The mixture was poured over the plate and then left alone to solidify before being placed in the incubator.

Results and Analysis:

Conclusion:

Due to the contamination of the negative control and negative result on the plaque assay, First, lysate and Arthrobacter were combined and left alone for 10 minutes to infect. Then, LB broth was added along with the calcium chloride. After the allotted time, the lysate and Arthrobacter were added to the LB broth and calcium chloride. 2X Top Agar was then added to the solution and poured onto the agar plate. For 15 minutes, the plates remained near an aseptic zone to solidify. After, the plates were placed in an incubator.

Future Plans:

If a high titer is achieved, then a plaque will be picked and diluted using phage buffer. If the titer is still low, then another plaque assay will be made but more lysate (150 µL) will be used.

Rationale: Due to contamination, plaque assays were created to get plaques.

Procedure:

The tables were thoroughly wiped down with CiDecon and ethanol and an aseptic zone was also set up to prevent contamination. 10 µL of positive and 0.5 mL Arthrobacter were placed in a new tube and left alone for 10 minutes to allow for infection. Next, 90 µL CaCl2 was combined with 2.0 mL of LB broth. After 10 minutes, lysate and Arthrobacter were added with LB broth and CaCl2. Then, 2.5 mL 2X Top Agar was added to the LB broth and CaCl2. The mixture was poured over the plate and then left alone to solidify before being placed in the incubator.

Results and Analysis:

Contamination from the last plaque assays

Conclusion:

Due to the contamination of the negative control and negative result on the plaque assay, First, lysate and Arthrobacter were combined and left alone for 10 minutes to infect. Then, LB broth was added along with the calcium chloride. After the allotted time, the lysate and Arthrobacter were added to the LB broth and calcium chloride. 2X Top Agar was then added to the solution and poured onto the agar plate. For 15 minutes, the plates remained near an aseptic zone to solidify. After, the plates were placed in an incubator.

Future Plans:

If there is contamination in the control, another plaque assay will be made. If there are plaques in the plate with no contamination in the control, then a plaque will be picked and diluted.

Rationale: To conduct PCR on the lysate

Procedure:

• The lysate was taken out of the shaking incubator.
• One mL of unfiltered lysate was taken out and placed in another tube.
• Then, it was placed in a centrifuge at 10,000 g for 10 minutes.
• After, the supernatant was taken out, carefully avoiding the pellet that formed at the bottom.
• The supernatant was then boiled for 10 minutes,
• After boiling, 2 µL of the enriched lysate ( in each of the three tubes) was added to the 2X Master Mix (12.5 µL of Taq polymerase and dNTP)
• 4 µL of primer was added to their respective tubes.
• 4.5 µL of ddH2O was added to each tube.

Results and Analysis:

Rather than putting 1 µL of enriched lysate solution, 2 µL were put into the primers.

This time, a clear tack was used rather than a dyed one. When doing gel electrophoresis, the dye must be added to the primer.

Conclusion:

Because there were no phages on the plates, another approach to check for phages was taken. The first step to conduct PCR was to boil the enriched lysate was 95 degrees Fahrenheit. Then, 1 µL of the enriched lysate was added to the 2X Master Mix along with 6.5 µL of ddH2O. Primer was added to their respective tube. This was conducted three times, adding each component in equal volumes.

Future Plans:

In the future (10/31/18), gel electrophoresis will be performed using the lysate samples treated with primer.

Rationale: To create a gel for gel electrophoresis which is then used for imaging along with creating a plaque assay with a known, positive lysate

Procedure:

• Agarose powder (0.80 g) was added to 0.40 mL of 1X TBE then stirred.
• The solution was heated in the microwave until it became clear with no visible particles.
• The flask sat on the lab bench until it became warm to the touch then 2 µL of ethidium bromide with a final concentration of 0.5 µg/µL.
• After stirring the solution, the apparatus was set up with the comb set on the negative side on the apparatus.
• The solution was poured in then remained on the bench for 30 minutes to solidify.
• While solidification, 100 µL of a known, positive lysate was taken and mixed with 0.5 mL of Arthrobacter.
• The top agar was set up by combining 22.5 µL of CaCl2 and 2 mL of LB broth.
• After 10-15 minutes, the lysate and Arthrobacter were added to the calcium chloride and LB broth.
• Then, 2.5 mL of 1X Top Agar was added to the plate and poured onto the plate.
• The plates were left alone to solidify then placed in the incubator.

Results and Analysis:

The results from the imaging came back to negative which may be due to the high presence of ions within the soil sample.

Conclusions:

In order to conduct a gel electrophoresis, the gel must first be created. The agarose powder and TBE were combined in a flask and swirled. Then, the solution was heated to reach a homogenous mixture which was then left alone to cool down to a warmer condition. 2 µL of ethidium bromide was added to the solutions then quickly poured onto the gel apparatus with a comb placed on the side. The apparatus was set aside to solidify for 30 minutes. Then, the lysate and Arthrobacter were mixed together to infect. The LB broth and CaCl2 were added together into a vial of which would become the top agar solution. After 1o minutes, the 2X top agar was added along with the lysate and Arthrobacter onto the plate. It was left alone to solidify then put into the incubator. After solidification of the gel, the comb was removed and TBE was poured to cover the surface of the gel. Four sets of phage buffers were placed, two sets on the outside. The next three sets included samples and the fourth was the control. After the samples, the ladder was filled into the slot. The lid was placed on the power source and was turned to 100 V. The power source remained on. After 40 minutes, the gels were examined.

Future Plans:

If there are plaques on the plaque assay, then two plaques will be picked and diluted for a high titer.

Rationale: Collect and gather data on a new soil sample and to also wash and enrich the sample

Procedure:

• A sample of soil was collected and data was gathered
• 2 mL of the soil sample was taken out and transferred to a new vial.
• 10 mL of LB broth was added to the vial and vortexed for 15 minutes.
• Then, the vial was weighed and placed in the centrifuge for 10 minutes.
• The supernatant was transferred and then enriched with 0.5 Arthrobacter.

Results and Analysis:

• Due to the weather, the soil was of a clay-like texture. The soil also had a different composition an inch from the surface of the soil. While the surface had a darker color, the soil an inch in had a light brown color. The tree was located in the square near the main entrance of Teal Residence Hall of Baylor University.
• The diameter was 53 cm.
• The height was 630 cm.
• The average length of the canopy was 265.183 cm.

Due to the clay-like texture of the soil, half of the soil sample was not incorporated into the LB broth and soil mixture when vortexing.

Conclusion:

Due to weather the day the soil was collected, the soil particles stuck together to form a sort of clay. The composition of the soil changed an inch in the soil. Transfer 2 mL of soil and add 10 mL of LB broth. Vortex for 15 minutes or until most of the soil particles are incorporated into the solution with LB broth. After weighing, place the vial in the centrifuge at 10,000 g for 10 minutes. Transfer the supernatant and add 0.5 mL of Arthrobacter.

Future Plans:

In the future (10/29/18), chloroform the sample then filter using syringe and filters.

Rationale: To prepare the gel and to create a gel electrophoresis to determine whether or not there are phages present within the sample.

Procedure:

• 0.8 grams of agarose powder was collected and poured into an Erlenmeyer flask along with 40 mL of 1X TBE.
• The flask was swirled around to make sure that the components are somewhat mixed together.
• The solution was heated until it becomes a homogenous mixture.
• The flask was left alone to cool to a warmer state.
• 2 µL of ethidium bromide was added to the warm solution.
• The agarose was poured onto the gel apparatus then the comb was inserted on the side.
• The gel was set aside to solidify for 30 minutes.
• Once the plate has solidified, the comb was gently removed and enough TBE was poured to cover the top layer of the gel within the apparatus.
• Then, 10 µL of phage buffer was inserted in the first slot then another 10 µL of phage buffer was added in another slot.
• In the next three slots, the three samples were added in increments of 10 µL.
• After the samples, 0.5 µL of DNA ladder was added.
• In the last two windows, 10 µL of phage buffer was added.
• The lid was laid on top and the power cables attached the apparatus to the power source
• The power source was put to 100 V and 4.5 mA.
• The power source ran for 30-40  minutes to ensure that the DNA had fully expressed itself.
• After the necessary time connected to the power source, the gel was taken out of the apparatus and scanned using the BioRad Gel Doc EZ Imager.

Results and Analysis:

From the BioRad Gel Doc EZ Imager, the results came back to negative. There were some positive results but that was from the phage buffers forming dimers.

Conclusion:

In order to conduct a gel electrophoresis, the gel must first be created. The agarose powder and TBE were combined in a flask and swirled. Then, the solution was heated to reach a homogenous mixture which was then left alone to cool down to a warmer condition. 2 µL of ethidium bromide was added to the solutions then quickly poured onto the gel apparatus with a comb placed on the side. The apparatus was set aside to solidify for 30 minutes. After solidifying, the comb was removed and TBE was poured to cover the surface of the gel. Four sets of phage buffers were placed, two sets on the outside. The next three sets included samples and the fourth was the control. After the samples, the ladder was filled into the slot. The lid was placed on the power source and was turned to 100 V. The power source remained on. After 40 minutes, the gels were examined.

Future Plans:

Due to the negative results from the imaging, collect more soil on 10/24/18 and wash and enrich.

Rationale: To conduct a PCR to determine whether or not there are phages and to amplify the DNA of the phages if they are present within the enriched lysate

Procedure:

• To prevent contamination, wipe the table with CiDecon and ethanol and set up an aseptic zone.
• Due to the presence of no phages, another method was used to determine if there were phages in the sample was taken, PCR.
• First, the sample was boiled.
• 1 µL of the enriched lysate ( in each of the three tubes) was added to the 2X Master Mix (12.5 µL of Taq polymerase and dNTP)
• 4 µL of primer was added to their respective tubes.
• 6.5 µL of ddH2O was added to each tube.
• For the control, 7.5 µL of ddH2O and 4 µL of primer 3 were combined.

Results and Analysis:

On 10^-2, the clear spaces are due to the top agar being torn

Conclusion:

Because there were no phages on the plates, another approach to check for phages was taken. The first step to conduct PCR was to boil the enriched lysate was 95 degrees Fahrenheit. Then, 1 µL of the enriched lysate was added to the 2X Master Mix along with 6.5 µL of ddH2O. Primer was added to their respective tube. This was conducted three times, adding each component in equal volumes.

Future Plans:

If there are phages present in the sample, the DNA will be amplified and will be used for a plaque assay to pick a plaque.