October 24

10.24.28 Gel Electrophoresis Sample E

10.24.28 Gel Electrophoresis Sample E

Rationale: Since sample had been put through the process of PCR, the results of whether or not phage DNA is present can be processed by using gel electrophoresis and matching bands to visualize results. Therefore, it was necessary to use the gel electrophoresis process to determine the presence of phage.

Procedure

  1. Added 35mL 1X TBE to two separate Erlenmeyer flasks.
  2. Added 0.7g Agarose to both flasks, then heated until bubbles formed. Once bubbles formed, tube was shaken until bubbles disappeared. Repeated until solution was created.
  3. Let cool to about 50°C.
  4. Added 1.7µL EtBr to both Erlenmeyer flasks. Swirled to mix
  5. Poured contents of flasks onto two separate gel apparatuses that contained combs to create holes in gel.
  6. Once gel had set, a small amount of TBE buffer was added to the top of both apparatuses. Comb and rubber seals were removed, and gel was set in machine.
  7. 1X TBE was poured over the top of the gel and wells until gel was completely submerged.
  8. 2.5µL dye was added to each sample (1-3 Negative Controls, 1-3 Positive Controls, 1-3 Experimental, and 1-3 Henry Experimental).
  9. 5µL of ladder was added to both gels (well 4 on gel 1 and well 1 on gel 2)
  10. 10µL of each negative control sample was added to the left side of the ladder (wells 1-3) on gel 1
  11.  10µL of each positive control sample was added to the right side of the ladder (wells 5-8) on gel 1
  12. 10µL of each experimental sample was placed in wells 2-4.
  13. 10µL of each Henry experimental sample was placed in wells 6-8.
  14. Power source was attached to apparatus and activated. Let run for 45 minutes.
  15. Used imaging to view results.

Observations:

  • The gel surrounding second negative control well was pierced, causing not all of the sample to be placed into the well. There was enough remaining in the well so the effects should not be overly dramatic.
  • Gel had a blue tint when it was solid, helpful to know when it is discouraged to touch the gel that contains EtBr.

Results:

  • The gels run showed no presence of bands that would indicate phage on both the negative control and experimental wells, but they showed a very present band on one of the positive control wells, a faint band on one of the positive control wells, and no banding on the third positive control.

Conclusions:

  • Since no banding was seen in the experimental wells for the fifth soil sample, it is very likely that there is no phage present. There may be a problem with the PCR procedure, as there have been no positive results since the beginning of its use, but it is overwhelmingly likely if that is not the case that there are no phages in soil sample e. The next steps will be obtaining a new soil sample unless a known positive sample is put through the PCR procedure and results display negative. In that case, the sample would need to be redone with an improved PCR procedure. 
Category: Henry Burns | LEAVE A COMMENT
October 24

Gel Electrophoresis 10.24.18

Rationale:

To conduct a gel electrophoresis test in order to test for the presence of phage in Soil F, by either confirming that phage is absolutely not present or could potentially be present.

Procedures:

  1. Added 35mL TBE, 1.7µL EtBr, and .7g Agarose to an Erlenmeyer flask.
  2. The flask was microwaved to create a solution.
  3. The gel solution was poured into trays and left to solidify, after solidification the dams and comb were removed.
  4. The gel apparatus was filled to about 1cm above the actual gel with TBE.
  5. Repeated steps 1-4 to create another apparatus
  6. Added 5µL of ladder to a well in each apparatus.
  7. Added 2.5µL of 10x DNA gel dye to all PCR tubes from Monday.
  8. Pipetted 5µL of all PCR tubes into different wells utilizing both apparatuses.
  9. Attached a power source and ran the test for 45 minutes.
  10. Analyzed the gel in the gel imager.

Observations/Data:

When pipetting Primer 2 Positive Control into a well the tip went a bit deeper than it should have. The experimental gel came back negative and the control gel performed as expected, however, the lines were relatively faint. The ladder also performed as expected.

Conclusions/Next-Steps:

From the data collected it can be concluded that Soil F is negative for phage, thus the next step will be to collect another soil sample and analyze it via PCR for the presence or absence of phage DNA.

Category: Nathan Newton | LEAVE A COMMENT
October 24

Soil C Washing and Re-Enrichment 10/24/18

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.

Category: Emily Gaw | LEAVE A COMMENT
October 24

Gel Electrophoresis 10/22/18

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.

Category: Emily Gaw | LEAVE A COMMENT
October 24

10/22/18 Gel Electrophoresis of DNA from Soil Sample #3

10/22/18 Gel Electrophoresis of DNA from Soil Sample #3

Objective:

The goal of this procedure is to test the previously created enriched lysate for phage DNA. Gel Electrophoresis will help us determine if there is phage DNA present, which will determine whether or not we do future testing on soil sample #3.

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 Gel Electrophoresis:

  • Agarose powder
  • 1X TBE
  • Microwave
  • Ethidium Bromide
  • Flask
  • Electrophoresis Tray
  • Electrophoresis apparatus and power supply
  • DNA ladder

The agarose gel was prepared:

  1. The gel was prepared according to the following recipe:
  2. The gel mixture was then microwaved until boiling
  3. The mixture was allowed to cool until warm
  4. 2 μl Ethidium Bromide was pipetted into the mixture
  5. The mixture was poured into an Electrophoresis tray and allowed to solidify

Gel Electrophoresis was run:

  1. The tray was placed into the electrophoresis apparatus and the apparatus was filled with solution to cover the gel
  2. 10 μl of each PCR sample and 5 μl of DNA ladder were pipetted into the gel slots
    • Samples were loaded into the Gel Slots according to the following image (my samples are group 2):
  3. The apparatus was plugged into a power source and turned on
  4. The gel was allowed to run until the DNA had fully traveled across the tray
  5. The results were imaged and recorded
Results:

The results of this Gel Electrophoresis came back negative for all 3 clusters of phage DNA tested. The positive controls however still tested positive, suggesting that there was nothing wrong with the Gel or procedure. These results confirm that there was no arthrobacter phage in my soil sample #3.

Analysis:

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. While there was some concern that using DI water instead of DDI water during PCR would cause odd results, the positive control I created during the last lab still worked, suggesting that I received a true negative result. This means that my previously created PCR tubes did not have phage DNA in them, which means my soil sample #3 likely does not have phage.

Future:

The results of this procedure were disheartening, but also odd because every group received a negative result, and that seems unlikely based on the number of soil samples tested. As a result, I will be testing whether or not cleaning soil with chloroform (as was the case for some of the soil samples tested) effects the results of gel electrophoresis. I will do this by testing soil that has been confirmed to have phage in it. I will wash soil that is known to create phage in order to create an enriched lysate. Then, I will filter half of this lysate with a syringe filter, adn clean half of it with cloroform. I will then run PCR and Gel Electrophoresis on these two different lysates to see if the results are affected. This will likley take me multiple lab periods to complete.

Category: Lucy FIsher | LEAVE A COMMENT
October 24

Lab Day 18: Soil Metadata and Enrichment

Rationale

Begin soil metadata to determine percent water, pH, and type of soil collected. Soil washing and enrichment will be done in order to continue from testing through PCR.

Detailed Procedure: Soil Metadata

  1. Weighed plate and added soil. Recorded both plate and plate +soil
  2. Filled soil up to 10 mL mark in a 50 mL vial and filled rest with D.I water up to 30 mL mark.
  3. Added 3 drops of soil dispenser solution and shook for 30 secs
  4. Left to rest on rack for 48 hours.
  5. Pinched soil into pH vial and filled rest with D.I water and shook for 10 secs
  6. Left to rest for 2 mins
  7. Pinched off 1 inch of pH paper and dipped into pH vial for 45 secs.
  8. Recorded pH of soil

Detailed Procedure: Enrichment

  1. Filled soil in 15 mL vial up to 2 mL mark
  2. Filled LB Broth into vial from Lab Day 14 up to 12 mL mark in aseptic zone
  3. Shook using vortex for 10 mins.
  4. Weighed vials and centrifuged for 5 mins
  5. Took dropper and poured supernatant into 3 separate wells around halfway.
  6. Heated up supernatant at 55 degrees for 5 mins and 60 degrees for 5 mins with shaker-thermostat
  7. Took 100 microliters of Arthro into 2 of the 3 wells.
  8. Incubated it and shook in the machine.

Results metadata

  • pH= 6.5

Conclusion/Next Steps

Group members decided to heat up the supernatant to kill of any bacteria and let it cool down before adding the Arthro. This was a experimental way of making enriched lysate without manually filtering the supernatant. In the next lab day, group members will run PCR.

Category: Soo-Un Jeong | LEAVE A COMMENT
October 24

October 24 2018 Retesting Soil C

Rationale: The purpose of this lab is to re-enrich soil C for further investigation.

Description of Procedures:

  1. The workstation was cleaned using aseptic technique and an aseptic zone was created.
  2. 2 ml of soil was added to a tube with 10 ml of LB Broth.
  3. The soil was shaken for 10 minutes and then spun in the centrifuge for 10 minutes at 5,000 x g.
  4. Supernatant was transferred to a tube and 0.5 ml of arthrobacter was added.
  5. The tube will be shaken at room temperature until the next lab.
  6. The workstation was cleaned using aseptic technique and materials were properly stored or disposed of.

Observations:

  • Filtration process was not done due to no filters.

Interpretations/Next Steps:
The procedure was complete. The next step will be to filter and run a plaque assay.

Category: Lucy Potts, Uncategorized | LEAVE A COMMENT
October 24

October 24 2018 Picking a Plaque and Purification- Soil C

Rationale: After a negative plaque assay from the flooded lysate, the purpose of this lab is to retest the flooded lysate and pick a plaque for purification. This lab will also retest enriched soil C lysate for further study.

Description of Procedures:

  1. The workstation was cleaned using aseptic technique and an aseptic zone was made with an ethanol burner.
  2. 100 ul of phage buffer was added to a tube. A plaque was picked and mixed in the buffer. The tube was then vortexed to mix.
  3. 10 ul of phage buffer with plaque was added to 0.5 ml of arthrobacter, 100 ul of the flooded lysate was added to 0.5 ml of arthrobacter, and 10 ul of soil C lysate was added to 0.5 ml of arthrobacter, and all were allowed to sit for 10 minutes.
  4. 8 ml of LB Broth and 90 ul of CaCl2 were added to a tube to create the top agar solution. 10 ml of 2x TA were added the tube.
  5. 4.5 ml of the top agar solution was added to 0.5 ml of arthrobacter for each plate, and the poured directly onto plates labeled LIP 10-24-18 PA SC, LIP 10-24-18 PA-P1, and LIP 10-24-18 PA(FL). The rest of the solution was poured directly onto a plate labeled LIP 10-24-18 Control.
  6. The plates were allowed to settle for 10 minutes and then inverted and stored in the incubator until the next lab.
  7. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations:

  • 100 ul of flooded lysate was added instead of the usual 10 ul.
  • Soil C lysate was retested.
  • Some bubbles were seen on the plates.

Interpretations/Next Steps:
The procedure was complete. The next step will be to check for plaques on all the plates, and to flood the plate from the picked plaque.

Category: Lucy Potts | LEAVE A COMMENT
October 24

10/24/18 PCR

Rationale: Set PCR to amplify the phage DNA to prepare for DNA GEL. DNA GEL will determine the presence of phage in the obtained soil sample A.

Procedure: Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. A burner was then placed to set up the aseptic zone. The lysate was obtained and 1mL was added to a microcentrifuge cap. This boiled for 10 minutes to break up the protein ca[sid so the phage DNA could be extracted. In three microcentrifuge caps, Primers 1,2, and 3 were added. The formula below shows the solution concentration added to the caps (x3 for 3 microcentrifuge caps):

  • 12.5 microns Taq
  • 2 microns of phage DNA
  • 6.6 microns of DH2O

After the solutions were added, the three caps were placed in a thermocycler for 52.5 minutes.

Observations: Control and the experiment performed on 10/22 were both negative. No plaques were present on the plaque assay performed on 10/22. No plaques were available to pick, and PCR was done to test whether the soil sample is positive or negative.

Negative Control 10/22

Negative experiment 10/22

Conclusions: Lab experiment performed on 10/22 was negative due to contaminations. Possible contaminations reasons:

  • LB broth and 2X TA are contaminated
  • Opening/Closing of microcentrifuge caps/things not done in an aseptic zone.

The lysate was used to perform PCR in preparation for DNA GEL experiment. DNA GEL will test whether or not the soil sample obtained is positive/negative. If the soil sample is negative, new soil will be needed to be tested via PCR/DNA GEL.

 

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
October 24

Gel Electrophoresis 10/24/18

Rationale: Now that I have replicated phage DNA, if my sample had phage, running a gel electrophoresis will let me see a band showing whether or not there is phage present.

Procedure:

  1. Start by adding 35mL of 1X TBE buffer with 0.7g of agarose powder.
  2. Heat until it starts boiling and swirl to mix. Repeat a couple times to fully mix agarose.
  3. Let mixture cool to about 55ºC then add 1.7μL of Ethidium Bromide and mix.
  4. Pour gel into tray with dams on each side and well comb.
  5. Repeat to make control gel.
  6. Once Solidified adding a bit of TBE buffer to the top to make it easier to remove comb without tearing gel.
  7. Added gel to electrophoresis apparatus and flooded with TBE buffer to fill line.
  8. Ran gel at 100V for 40 minutes.
  9. Imaged using Bio-rad imaging machine.

Observations: On the left is the gel with our samples on it. On the right is the controls. To the left of the ladder are negative controls and to the right are positive controls. As shown none of the samples had phage present.

Interpretations and Next Steps: Yet another soil sample shown to have nothing. This technique also seems to not be working out as it is possible that the sample contained phages of the clusters in primer mix 3 and the band was too faint to see. Next time I’ll bring multiple samples and attempt a multi-well enrichment.

Category: Sriram Avirneni | LEAVE A COMMENT