October 26

Gel Electrophoresis (10/22/18)

Rationale:

Run a gel electrophoresis using Soil Sample E’s enriched lysate to test for presence of DNA. 

Procedure: 

  1. Obtained a flask, and added 40 mL of TBE, and 0.8 Agarose.  
  2. Microwaved the flask until the solution was clear.  
  3. The flask was let to be cooled before proceeding.  
  4. Added 2 uL of Ethidium bromide into each flask.  
  5. Poured the solutions into the tray and left to set, and after gel set, removed the combs.  
  6. Filled the tray with TBE until covered a layer over the gel.  
  7. Pipetted 10 uL of primer into each of the wells. , and 5 uL in the well for the ladder. 
  8. Pipetted the ladder in the middle row, and left ends empty.  
  9. Used left end for practice.  
  10. Attached the apparatus and plugged into the power source.  
  11. Imaged soil and obtained results about an hour later.  

Observations/ Results : 

The gel electrophoresis results came back negative, as no phage DNA was traced in Soil Sample E.  

Gel Electrophoresis Before Imaging

 

Gel electrophoresis Results

Conclusions/Next Steps: 

The gel electrophoresis came out negative, and therefore can conclude that there is no phage in this soil sample. Next time I will either re-enrich a previous soil sample, or will collect new soil, enrich the new soil sample, and find the soil metadata to create a PCR and run a gel electrophoresis.

Category: Sona Subramanian | LEAVE A COMMENT
October 26

Soil Collection (10.24.18) and Washing + Enrichment 10/24/18

Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

Samples were collected to expand the data size and provide more insight into whether the presence of Arthrobacter phages in the soil around red or white oaks has a correlation with the health condition of oak trees. Washing helps to isolate the arthrobacter and phages from the soil particles itself and Enrichment amplifies the numbers of phages present by adding arthrobacter hosts for phages to infect.

Soil Washing and Enrichment for sample (10.24.18):

Material:

  •  15 ml Conical Tube
  •  50 ml Conical Tube
  • LB Broth
  • Syringe Filter
  • Vortex machine
  • Centrifuge
  • Micropipette
  • Serological pipette

Procedure:

  1. Set up an Aseptic zone
  2. Placed sample Soil (10.1.18) to the 4 ml mark.
  3. Poured LB Broth to the 12 ml mark.
  4. Vortexed for 15 min to mobilize the soil.
  5. Centrifuged at 3000 G for 10 min.
  6. Transferred the supernatant to a new tube
  7. Added 0.5 ml Arthrobacter into the filtered supernatant and placed on shaker and incubate at 38.1 Celcius.

Observations, Results & Data:

Soil Collecting for sample (10.24.18):

  • White Oak
  • Diameter: 16.9 cm
  • Tree Height: 6.3 m
  • Canopy: 224 cm

Interpretations & Conclusions:

Due to shortages of filters, the sample hadn’t been filtered by with a 0.22 um filter, the results of this change of protocol could have an impact on the results of the experiment.

Next Step:

The next step would be to use chloroform to kill the microbes in the lysate and filter the lysate, completing the enrichment process.

Category: Yang-En Yu | LEAVE A COMMENT
October 26

Electrophoresis on Sample Enrichment Lysate (10.1.18) after PCR 10/21/18

Research Question:

To find out how the presence of bacteriophages in the soil around red or white oak trees has a correlation with the health condition of oak trees.

Rationale:

By implementing Electrophoresis, the image captured on the gel after could help determine if there is any trace of DNA amplified after PCR in the filtered Enrichment Lysate sample.

Electrophoresis for sample (10.1.18):

Materials:

  • 1x TBE
  • Agarose powder
  • EtBr
  • Electrophoresis apparatus and power supply
  • Heat source
  • DNA loading dye
  • DNA ladder
  • Gel Imager

Procedure:

  1. prepare a solution with 40 ml of 1x TBE and 0.8 g Agarose
  2. microwave until the solution boils and retrieves it for cooling
  3. Add EtBr to the solution and have a final concentration of 0.5 %
  4. While the solution is still warm, pour it into the preassembled gel apparatus
  5. wait for solidification
  6. After the gel had solidified, fill the apparatus with 1x TBE
  7. Load the samples(10 ul), DNA dye(10 ul) and DNA ladder(5 ul) into the wells
  8. Run the gel at 100 volts 45 min

Observations, Results & Data:

The result from imaging shows no significant bands anywhere on the gel, indicating there is no significant amount of DNA in the filtered enrichment sample lysate after PCR.

Interpretations & Conclusions:

The lack of distinct bands on the image shows that there are no phage DNA present in the lysate sample, however, it is notable that the bottom part of the gel has traces of band, it might be the primers replicated themselves during PCR.

Next Step:

Due to the negative results of the PCR Electrophoresis experiment, the new objective id to collect more samples to continue the search for phages.

Category: Yang-En Yu | LEAVE A COMMENT
October 25

PCR and Metadata Results

Title: PCR & Metadata Results

Date: 22 October 2018

Rationale: A PCR (Polymerase Chain Reaction) will be done to attempt to replicate any phage DNA present in the most recently acquired soil sample. The metadata results will also be recorded and interpreted.

Procedure:

  • The enriched lysate from the previous lab session was centrifuged at 3000 G for 5 minutes to separate the Arthrobacter from the phage and the lysate suspension.
  • ~1 mL of the centrifuged lysate was placed into a microcentrifuge tube and boiled to release capsid DNA from any potential phage
  • 3 different primer mixes with different DNA primers were made using the following recipe:
    • 12.5 microliters of Taq Polymerase
    • 4 microliters of primer mix (PM1, PM2, or PM3)
    • 6.5 microliters ddH2O
    • 2 microliters of DNA (1 microliter of boiled lysate + 1 microliter of a partner’s lysate)
  • The mixture is put onto ice and the results will be checked with a gel in the following lab
  • The % water and soil dipsersion results were checked and recorded

Results/Conclusions: The final mass of the soil sample was 9.437 g meaning that the % water composition of the soil was 16.19% H20. As for the soil dispersion, the soil was composed of 42.86% Sand, 28.57% silt, and 28.57% clay. The PCR mix is able to recognize phage DNA and, because of the primer mixes, bind to the DNA strands and replicated any phage DNA that are present. This provides another method to test phage presence as opposed to a plaque assay or a spot test.

 

Category: Uncategorized | LEAVE A COMMENT
October 25

10/24/18 Soil Washing and Enrichment of Soil with Known Phage

10/24/18 Soil Washing and Enrichment of Soil with Known Phage

Objective:

The goal of this procedure is to test the efficacy of PCR and Gel Electrophoresis. The way I will be testing this is by creating a newly enriched lysate using soil that already tested positive for phage in the previous testing. The purpose behind this is to test if different ways of cleaning the enriched lysate will affect the results of PCR. I will do this in future labs by using a syringe filter to filter half of the enriched lysate I create, and using chloroform to clean the other half, and then running PCR and Gel Electrophoresis on each sample.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group, we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

  • CiDecon
  • 70% Ethanol
  • Ethanol Burner

Materials For Soil Washing:

  • Syringe filter
  • .5 ml Arthrobacter
  • 50 ml conical vial
  • 15 ml conical vial
  • LB Broth
  • refrigerator
  • Incubator
  • Centrifuge
  • Pipette
  • Test tube stand

In order to complete the procedure, an aseptic zone was created.

  1. CiDecon was applied to the lab table
  2. 70% Ethanol was also applied

The soil was washed and enriched according to the following procedure:

  1. Gather previously positive soil
  2. ~2.5 ml of soil was added into a 15 ml conical vial
  3. 11 ml of LB broth was added
  4. The vial was vortexed for 15 minutes
  5. The vial was then centrifuged for 10 minutes
  6. ~ 1.5 ml were pipetted into a fresh 15 ml vial for a direct lysate and placed in the fridge
  7. ~9 ml of lysate were pipetted into a fresh 15 ml vial
  8. .5 ml of Arthrobacter was added to the 15 ml conical and put in the incubator
Results:

The results of this procedure will not be immediately clear because I am not testing for phage presence. After I do the two different procedures to clean the enriched lysate and then run PCR and Gel Electrophoresis I will report on my results. Aside from that, I can say that this appeared to go well and an enriched lysate was created.

Analysis:

PCR works by using polymerases to make many copies of specific strands of DNA  so that it is easier to analyze. Using PCR allows researchers to take small samples and amplify the genetic contents in order to conserve resources and determine what is present before future testing commences.  In this lab, using PCR allows people to determine whether or not there is phage DNA in an enriched lysate. Gel Electrophoresis works by using electricity to separate different strands/fragments of DNA in order to analyze the patterns that ensue.  When electricity is applied, DNA moves toward the oppositely charged end of the gel tray and brings the DNA dye with it. The question of my next few procedures seek to address is whether or not these procedures accurately indicate DNA presence and if they can be affected by different prep methods.

Future:

For my next three lab procedures I will be utilizing two different cleaning methods on my enriched lysate, then running PCR, and then doing Gel Electrophoresis.

Category: Lucy FIsher | LEAVE A COMMENT
October 25

SEA Bears Day 18

24 October 2018 ✷ Soil 6 Enrichment + Metadata

Rationale: Soil sample 6, a sample taken from near the bear habitat was enriched and tests for metadata were conducted in an attempt to isolate phage and gather data on the soil.

Procedure

  • The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
  • 4 mL of soil was vortexed for 15 minutes with 8 mL of LB broth and then centrifuged for 5 min at 3000 g.
  • Percent sand, silt, and clay was tested by combining 4 mL soil with 8 mL DI water and 3 drops soil dispersion fluid and shaking for 45 seconds. That mixture was allowed to sit overnight to settle.
  • Other group members performed the tests for percent water and pH. The tree was not measured because of poor weather conditions, but it will be measured and reported when the rain stops.
  • 1 mL of the supernatant from the centrifuged soil was pipetted into 3 wells in a well plate and heated to 55 degrees Celsius for 6 minutes and then heated to 60 degrees Celsius for 5 minutes in order to kill off any bacteria existing in the mixture before arthro was to be added.
  • 100 microliters of arthrobacter was added to two of the three wells (last one was left empty to serve as a control) and then the shaker thermostat was left to shake and incubate the bacteria overnight (27 degrees Celsius).

 

Observations, results, data

pH: 6.5

The soil was collected during a thunderstorm so the percent water will be much higher because of the rain. It was also very difficult to measure out when attempting the metadata tests, but this shouldn’t change percent sand, silt, clay much. It may have raised the pH because the soil itself was already so watered down from the rain.

The experimental procedure was adapted in this experiment because of a lack of materials, but new filters were recently delivered and the prior trials can be repeated as before.

Interpretations, conclusion, next steps

The arthrobacter/lysate mix will be filtered and prepared for PCR in order to amplify any present phage DNA so that it can be tested for presence of phage DNA.

Category: Lily Goodman | LEAVE A COMMENT
October 25

SEA Bears Day 17

22 October 2018 ✷ Soil 5 gel electrophoresis

Rationale: Soil sample 5, the sample collected from the red oak in front of LL Sam’s Historic Lofts, was tested using gel electrophoresis in order to look for presence of phage DNA.

Procedure

  • The lab table was cleaned with CiDecon and 70% ethanol and an alcohol lamp was lit to promote an aseptic environment.
  • 0.8 g of agarose powder was mixed with 40 mL TBE (buffer) and microwaved. Ethidium Bromide was added and the solution was poured into a gel apparatus and a 8-prong comb was added and the gel was allowed to harden. It was then transferred to an electrophoresis tray and filled with TBE.
  • The first well was used for other group members to practice. The remaining wells were as follows:
    • Well 2: “(: 1” LL Sam’s Oak DNA + Other group’s DNA + primer 1
    • Well 3: “(: 2” LL Sam’s Oak DNA + other group’s DNA + primer 2
    • Well 4: “(: 3” LL Sam’s Oak DNA + other group’s DNA + primer 3
    • Well 5: DNA Ladder
    • Well 6: “★1” DNA from 2 other groups + primer 1
    • Well 7: “★2” DNA from 2 other groups + primer 2
    • Well 8: “★3” DNA from 2 other groups + primer 3
  • The gel electrophoresis was run for roughly 30 minutes at 100 V and analyzed with UV.

observations, results, data

The gel, when analyzed with the UV scanner, was negative for phage DNA. The controls all worked, so the test was effective and the soil tested did not contain phage.

The other two samples collected on the same day, from the basketball hoop and electric pole, were also negative. The use of DI water instead of DD water did not have an adverse effect on the gel.

The gel pictured above “glowed” more than other groups, but this is likely because it ran for less time than the other groups’ gels and there was a higher concentration of dye in one spot instead of being dispersed throughout the entirety of the gel.

 

interpretations, conclusion, next steps

It is possible that the chosen variable for group 6, red oaks, is a negative one, meaning that the scientific question “is there a correlation between the presence of phage in white vs red oaks?” is being answered with, “there are no phages near red oaks” because every sample from red oaks has been negative for phage. It is still somewhat premature to make this conclusion, but the data has not proved this statement incorrect as of yet.

In the coming days, more soil will be collected and enriched. Then, it will be run through PCR and gel electrophoresis.

Category: Lily Goodman | LEAVE A COMMENT
October 25

PA Results and Second Passage PA (10/24/18)

Results:

Many plaques formed on the “KEA 10/22 100-1 PA” plate. The “KEA 10/22 100-0 PA” plate was contaminated. One small plaque appeared on “KEA 10/22 100-2” plate. The shared control plate was contaminated. The pictures below show these plates.

Rationale:

To continue the purification process, a plaque will be chosen from the “KEA 10/22 100-1 PA” plate. Two plaque assays will be performed with this chosen plaque, with one using 100and the other 10-1.

Procedure:

  1. Once an aseptic zone was established, 100 µL of phage buffer was placed into microcentrifuge tube “KEA 10/24 100.”
  2. Used a micropipette tip to touch a plaque from the “KEA 10/22 100-1 PA” plate and then swirled the tip in the “KEA 10/24 100” microcentrifuge tube.
  3. The “KEA 10/24 100” microcentrifuge tube was vortexed.
  4. 10 µL of “KEA 10/24 100” was added with 90 µL of phage buffer to create 10-1dilution which was then vortexed.
  5. 10 µL of 100and 10-1were added to correlated test tubes which already had 0.5 mL of Arthrobacter in them.
  6. 6 mL of LB Broth, 67.5 µL of CaCl2, and 7.5 mL of 2X TA were combined into a conical vial.
  7. Transferred and mixed 4.5 mL of the TA mixture from the conical vial into each test tube.
  8. Each test tube was poured onto their correlating plate.
  9. These plates were placed in the incubator at room temperature.

Observations:

  • The following calculations were performed to determine the amount of LB Broth, 2X TA, and CaCl2needed for 3 plates.

Original Recipe

 X3

2 mL LB Broth

6 mL LB Broth

2.5 mL 2X TA

7.5 mL 2X TA
22.5 μL CaCl2

67.5 μL CaCl2
  • The plaque circled bellowed was picked to make “KEA 10/24 100” plaque, phage buffer mixture. The plaques on this plate were not as distinct as the plaques found on the “KEA 10/12/18 Soil E PA” plate.

  • Air bubbles formed when pouring the “KEA 10/24 10-1PA” plate.
  • Strange contamination spots formed on the shared control plate as shown below.

Next Steps:

If there is contamination, a plaque assay will be run again with the same dilutions. If there are plaques, a third passage will be performed. If there are no plaques, a different plaque will be chosen.

Category: Kathryn Adkins | LEAVE A COMMENT
October 25

10/24/18- Gel Electrophoresis

10/24/18

Objective:

  • Test for the presence of phage clusters in lysate of soil sample C using PCR and Gel Electrophoresis.

Calculations:

Total Volume= 35 ml

Density of Et Br= 10μg/μl

For 2% agarose = 0.02 X 35= 0.7g

For 0.5% Et Br= 0.005 x 35 x 10= 1.7 μg

Procedure:

  1.  35 ml of TBE water was poured into a flask.
  2. 0.7 g of Agarose powder was added to the flask
  3. The flask was microwaved until the solution bubbled. the flask was taken out  and shaken. Because clumps of powder still remained in the flask, the flask was microwaved and shaken again until all the powder had dissolved.
  4. The solution was allowed to cool until it was warm, at a temperature of about 55°C.
  5. 1.7μl of Et Br was added to the flask.
  6. The solution was then poured onto a electrophoresis apparatus plate (with two dams and combs) and the gel was allowed to solidify.
  7. the comb and dams were carefully removed once the gel solidified.
  8. The plate was then placed into the electrophoresis apparatus.
  9. The apparatus was filled with TBE water until the gel was completely submerged.
  10. 5μl of the DNA ladder was put in one of the wells in the gel.
  11. 10 μl of  the contents of the three green centrifuge tubes prepared on 10/22/18 were placed in three wells in the gel.
  12. The apparatus was connected to the power source and the voltage was set to 100V.
  13. the gel was allowed to rest in this electric field for 45 minutes.
  14. After 45 minutes, the gels were removed from the apparatuses and placed into a machine that emits UV light, analyzes and records the readings on the gel.

Analysis and Conclusion

My Sample ( along with lab partners)control

After analysis of the results from the gel electrophoresis, this soil does not seem to contain any phages and therefore a new soil sample will now be required. There may also be a possibility that adjustment may be required due to varying quality of the result due to use of different Taq polymerases. this may also be further tested. the verity of the PCR will also be tested with the use of lysate samples that contain phages for sure

 

Category: Aman Patel, Dr. Adair | LEAVE A COMMENT
October 25

10/22/18- PCR

10/22/18

Objectives:

  • to test for the presence of bacteriophages in enriched lysate using PCR

Pre-Lab Observations:

the plaque assays and top agar control prepared on 10/17/18 were contaminated. Therefore the results were inconclusive. PCR and gel electrophoresis will be used for testing for the presence of Phages in enriched lysate.

Procedure:

  1. Set up aseptic zone.
  2. transferred 4 ml of enriched lysate for soil sample C into a 15 ml conical vial
  3. centrifuged the conical vial for 5 minutes
  4. transferred 1 ml of centrifuged enriched lysate into a micro-centrifuged tube.
  5. the microcentrifuge tube was then boiled
  6. picked up 3 green vials with 12.5 μl of Taq polymerase.
  7. 4 μl each of primer 1, primer 2 and primer 3 were added to the three green vials and they were labeled 1,2 and 3 according to the primer added.
  8. 6.5 μl of DDI water was added to each of the three green vials.
  9. 1 μl each of the boiled enriched lysate sample from 2 students (one of them being me) was added to each of the three green vials.
  10. the vials were allowed to rest for some time and were then placed in the thermocycler.

Analysis and Conclusion:

judging from the results obtained from the plaque assays, there seem to be no phages in this soil sample. PCR and gel electrophoresis ( the latter will be performed on /10/24/18) will perhaps present a more definitive answer.

Category: Aman Patel, Dr. Adair | LEAVE A COMMENT