October 26

Collection of Soil Sample 5 (10/24/18)

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.

Category: Sabin Patel | LEAVE A COMMENT
October 26

Gel Electrophoresis (10/22/18)

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.

 

Category: Sabin Patel | LEAVE A COMMENT
October 26

10/24 ~ Flooding the plate and Webbing a plate

Rationale: Flooded the 10^0 plate, as well as calculated to web a plate by using more lysate.

 

Procedure:

  • Created an aseptic zone to prevent contamination from other bacterium
  • Obtained by plates from 10/22 and observed the results
  • Flooded the 10^0 plate with 6mL PB and let sit for 48 hours
  • Decided to run three plaque assays
    • 10^0 plate would be with 20μL lysate
    • 10^-1 would be 20μL 10^0 lysate + 80μL PB
    • 10^-2 would be 20μL 10^-1 dilution + 80μL PB
  • Obtained a 50mL conical vial and added in 8mL LB Broth and 90μL CaCl2
  • Added 10μL of 10^-1 and 10^-2 (Respectively) to 0.5mL arthrobacter
  • Added 20μL 10^0 into 0.5mL arthrobacter
    • Let the arthrobacter+lysate sit for 10 minutes
  • Added 10mL 2XTA to the conical vial and immediately pipetted 4.5mL into each arthrobacter+lysate vial and plated
  • Allowed the plates to solidify and moved to incubation

 

Observations:

Contaminated TA control

10^-2 plate with markings on it to count the amount of plaques

10^-1 plate with a few plaques

 

Next Steps/Conclusion: Will be attending open lab on 10/26 to filter flooded plate and observe results of the plaque assays. If the plaque assays are positive, will calculate the titer of the lysate. If negative, will redo a plaque assay next lab.

Category: Justin Yu | LEAVE A COMMENT
October 26

Gel Electrophoresis of Soil F

10/24/18

Rational:

To run the samples from last lab through gel electrophoresis in order to determine whether there are arthrobacter phage present in the soil.

Procedure:

  • Cleaned the lab desk with CiDecon and ethanol
  • Weighted .72 g agarose powder
  • Put 35 mL 1x TBE into a flask
  • Added .72 g agarose powder
  • Heated until the powder was dissolved and then it was cooled
  • Added 1.7 ML EtBr and poured into the gel mold
  • Then covered the gel with 1x TBE
  • Added 5 ML of DNA ladder to a well
  • Added dye to PM1 (primer mix 1)
  • Added PM1, PM2, and PM3 to wells
  • Turned on electrophoresis and waited 45 minutes

Observations:

  • The picture of the gel electrophoresis had a white spot (possibly other DNA) covering the middle of the gel

Conclusion:

The spot in the middle of the image of the gel elctrophoresis prevented the results of the gel electrophoresis from being seen. As a result a new gel electrophoresis will be done next lab. A new soil sample will also be collected and filtered in case the results are negative.

Fig.4.F – This image shows the gel electrophoresis when it started. The dye around the wells is what overflowed out of the well when they were beig filled.

Fig.5.F – This image shows the results from the gel elctrophoresis. The white area seen in the middle may be DNA that leaked from a punctured well or from coming in contact with something else that has DNA on it.

 

Category: Emily Balint | LEAVE A COMMENT
October 26

10/22 ~ Re-confirming the titer strength

Rationale: Re-calculate the titer strength of the lysate due to contamination of control plate and plaque assays

 

Procedure:

  • First created an aseptic zone to prevent contamination of plates
  • Obtained previous plates from incubation and observed contamination
  • Obtained previous lysate from refrigeration as well as 4 petri dishes
  • Created a 10^-1 dilution by adding 10μL 10^0 lysate to 90μL PB
  • Created a 10^-2 dilution by adding μL 10^0 dilution to 90μL PB
  • Obtained a 50mL conical vial and added in 10mL LB Broth and 112.5μL CaCl2 (Recipe was multiplied by 5 because a teammate used the same Lb Broth and TA)
  • Added 10μL of lysate (Respectively) to 0.5mL Arthrobacter and let sit for 10 minutes
  • Added 12.5mL 2XTA to the conical vial and immediately added 4.5mL to each arthrobacter + bacteriophage tube and plated
  • Let the plates sit for 15 and incubated

 

Observations:

  • Contaminated Control Plate

    10^0 plate with no results; contamination prevalent

    Contaminated 10^-1 plate with no results

    Contaminated 10^-2 plate with no results

     

    Next Steps/Conclusion: The next steps would be to see if this current plaque assays turn out positive or not. If they do, calculating the titer of the plaque assay or flooding the plate would be the next course of action to create a high titer. If the plates turn out negative, then repeat the plaque assays.

Category: Justin Yu | LEAVE A COMMENT
October 26

PCR of Soil F

10/22/18

Rational:

To cut and multiply the DNA in the soil F lysate so that it can be tested for arthrobacter phage. Also to record the results for the percent water and soil texture.

Procedure:

  • Cleaned lab desk with CiDecon and ethanol
  • Centrifuged the enriched lysate at 3,000 G for 5 minutes
  • Put the enriched lysate into a microcentrifuge tube
  • Put 4 ML of primer mix 1, 2, and 3 into 3 different tubes
  • Added 6.5 ML of DDI water to the three tubes
  • Added 2 ML of enriched lysate to the tubes
  • Prepare 3 negative control tubes
  • Put 4 ML of primer mix 1, 2, and 3 into 3 different negative control tubes
  • Added 8.5 ML DDI water to the three tubes
  • Put the tubes into the thermocycler

Observations:

  • Sand- 3.5 mL
    • Percent Sand- 35%
  • Silt- 3.5 mL
    • Percent Silt- 35%
  • Clay- 3.0 mL
    • Percent Clay- 30%
  • Soil Type- Clay Loam
  • Dry soil with weigh boat- 9.01 g
  • Dry soil- 6.44 g
  • Percent Water- 18.17%

Conclusion:

The percent water in soil F was similar to all of the other soil sample except for soil C. The soil type which was clay loam was the same as soil Band E. The soil type was also similar to soil C (clay) and soil D (loam). Next lab I will be running a gel electrophoresis with the samples that I ran PCR on.

 

Category: Emily Balint | LEAVE A COMMENT
October 26

10-24-18 — Gel Electrophoresis

Date: Wednesday, October 24th, 2018

Title: Gel Electrophoresis

Rationale: The purpose of today’s lab is to perform gel electrophoresis in order to test samples against a DNA ladder to determine whether or not the sample contains phage DNA.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

  1. 35 mL of TBE buffer was added to a flask.
  2. .72 g agarose was added to the flask with TBE buffer.
  3. The flask was placed in a microwave and cooked in intervals until the solution was clear.
  4. The flask was allowed to sit until it was cool enough to hold with a bare hand comfortably.
  5. 1.7 microliters of Ethidium Bromide (EtBr) was added to the flask.
  6. The solution in the flask was poured into a tray and allowed to harden
  7. A thin layer of TBE was added over the hardened tray so that the comb and dams could be removed without tearing the gel.
  8. TBE buffer was added to the gel electrophoresis tray until it covered the gel section.
  9. Four wells were used:
    1. The first well was filled with 5 microliters of DNA ladder which would serve as a match to possible DNA.
    2. The second well was filled with 10 microliters of the small tube with PM 1. 2.5 microliters of dye was added to the tube before the contents were added to the well in order to weigh down the DNA so it did not float from the well.
    3. The third well was filled with 10 microliters of the small tube with PM 2.
    4. The fourth well was filled with 10 microliters of the small tube with PM 3.
  10. The gel was connected to a power source and allowed to run for 45 minutes. The gel was imaged and yielded negative results.

Observations:

The results from the gel electrophoresis did not match the expected results based on the ladder. This means that the sample in all likelihood did not contain phage DNA.

The compound EtBr is a carcinogen and must be handled carefully. Gloves were used when in contact with EtBr.

Results: This experiment yielded negative results. The PCR and gel electrophoresis did not match the DNA ladder and did not contain phage DNA.

Next Step: The next step is to either collect more soil or adopt from another group.

Category: Brandon Reider | LEAVE A COMMENT
October 26

10-22-18 — PCR Soil Sample C

Date: Monday, October 22nd, 2018

Title: PCR Soil Sample C

Rationale: The purpose of today’s lab is to perform PCR to replicate and search for phage DNA in the soil sample.

Class Question: Is there a difference in bacteriophage presence or type in soil samples taken from live oaks vs those from red oaks?

Procedure:

  1. The enriched lysate from soil sample C was centrifuged at 3000 gs for 10 minutes.
  2. 1 mL of the centrifuged lysate was transferred to a microcentrifuge tube and boiled to release possible phage DNA from capsids.
  3. 6 small tubes were set up. Three were used as a negative control and three used the lysate from soil sample C as well as a partner’s lysate in order to test for phage DNA. The tubes were set up the following way:
    1. 12.5 microliters of Taq Polymerase was added to each of the 6 tubes.
    2. 4 microliters of primer mix 1, 2, and 3 were added to the negative control tubes and the experimental tubes marked with 1, 2, and 3 respectively.
    3. 6.5 microliters of ddH2O were added to each of the 6 tubes.
    4. 2 microliters of lysate (1 microliter from the soil sample C lysate and one microliter from a partner’s lysate) were added to each of the 3 experimental tubes.
    5. An extra 2 microliters of ddH2O were added to each of the 3 negative control tubes.
  4. These tubes were all put into a heat cycle in order to let the primers match with possible phage DNA.

 

 

Observations: The experimental tubes marked for PM (primer mix) 2 and 3 were green while the tube marked for PM 1 was clear. This is because a different material was used in the tube. For gel electrophoresis, a dye will have to be added to the first tube in order to weigh DNA down.

Results: This experiment yielded three experimental tubes that can be tested with gel electrophoresis to search for the presence of phage DNA. This experiment also yielded three negative control tubes that can be tested against to make sure there isn’t contamination or that a procedure was done incorrectly since these tubes should result in strictly negative results.

Next Step: The next step is to use gel electrophoresis in order to check for phage DNA. If this yields negative results, the next step is to find new soil or to adopt phage/soil from another group.

Category: Brandon Reider | LEAVE A COMMENT
October 26

Gel Electrophoresis

Title: Gel Electrophoresis

Date: 24 October 2018

Rationale: After the PCR procedure from the previous lab, a gel will be made and DNA loaded into the gel. An electrical current will be run through the DNA and the gel will show the DNA strands with respect to a primer mix.

Procedure:

  • 35 mL TBE and 0.7g of agarose was mixed into a flask.
  • The mixture was microwaved until the solution was clear
  • Once the solution cooled to a temperature of ~55 degrees Celsius, 1.7 microliters of EtBr was added to the mixture.
  • The mixture was poured onto an electrophoresis plate to set. Once the gel set, the dams and comb was removed and the plate was placed in the electrophoresis apparatus (the “holes” were placed towards the positive end in order to repel the negatively charged DNA)
  • 5 microliters of the protein ladder was loaded into the first lane (bottom lane on picture)
  • 10 microliters of PCR Primer mix 1 + DNA  (from previous lab) was loaded into the second lane (second to bottom lane in picture)
  • 10 microliters of PCR Primer mix 2 + DNA was loaded into the third lane (third from bottom in picture)
  • 10 microliers of Primer mix + DNA 3 was loaded into the fourth lane (fourth from bottom in picture)
  • The power source was attached to the cathode and anode, and 100 V of electricity was run through the gel for ~30 minutes.

Results/Conclusions: The gel appeared to have one thick, yellow strand and one thick, blue strand. The results did not appear to be positive for any DNA Primer mix. New soil will have to be collected or a different procedure will have to be used. Attached is a picture of the gel and computer readout.

Category: Uncategorized | LEAVE A COMMENT
October 26

Soil C Re-enrichment (10/24/18)

Rationale:

Re-enriching Soil Sample C to either confirm or deny the presence of plaque in this soil sample, since one member of group obtained plaque from this soil.  

Procedure: 

  1. Cleaned the table using Cidecon and 70% ethanol.  
  2. Created an aseptic zone by setting up an ethanol burner.   
  3. Added 2 mL of Soil Sample C into a 15 mL tube, and added 10 mL of LB Broth into the same tube, and shook for 10 minutes.  
  4. Obtained the mass of the solution.  
  5. Centrifuged the solution for 10 minutes.  
  6. Obtained the solution from the centrifuge and filtered the supernatant using a pipette into a 50 mL tube. 
  7. Added 0.5 mL of Arthro to the 50 mL of tube containing the enriched lysate.  
  8. Stored the 50 mL tube into shaking incubator until next lab.  

Observations/ Results: 

  • Small particles of soil were not filtered out of our enriched lysate due to the lack of filters. 
  • Mass of solution before centrifuging: 19.74 grams.
  • Enriched Sample amount: 10 mL 

Conclusions/Next Steps: 

Next class, we will prepare a PCR and gel electrophoresis using our newly enriched Soil Sample C in hopes of finding phage.  

Category: Sona Subramanian | LEAVE A COMMENT