November 30

Project Update 11/30/18

Rationale: There is not enough time to continue to pursue a high titre lysate from Mellissa’s phage. The results for MEL X are shown below.

 

Control for MEL X plaque assay

MEL X plaque assay. The agar did not solidify correctly so it is not smooth, but there is no evidence of plaque.

Next Steps:

Focus on group data for the essay and give up on finding a phage 🙁

Category: Rachel Melone | LEAVE A COMMENT
November 30

Plaque Assay for MEL X Plaque 11/28/18

Rationale: Results for the second plaque assay from MEL 2 did not show any plaques so perform a plaque assay for a new plaque picked from Melissa’s plate labeled MEL X

Process:

  1. washed table and setup aseptic zone
  2. picked plaque and swirled it in 50 µL phage buffer (MEL X plaque lysate)
  3. Made LB agar media for 3 plates
    • 6 mL LB broth
    • 68 µL 1 M CaCl2
    • 7.5 mL 2X TA
  4. added 50 µL lysate (Mel X plaque) to 0.5 mL arthro and let sit for 15 minutes
  5. Poured control plate with ~5mL from the LB agar media
  6. Added arthro lysate mixture to  LB agar media
  7. Poured plate from tube and let sit for ~15 minutes
  8. Incubated for 48 hours at 28 degrees Celsius

Next Steps: Another plaque assay but with alterations to get a higher titer.

Category: Rachel Melone | LEAVE A COMMENT
November 30

Calculating and Flooding (11/28/18)

Rationale: To calculate the titer and determine whether or not a new plate has to be made to achieve a high titer. If not, flood each plate.

Procedure:

  • The titer of each plate with phages present was calculated and determined if the titer was high enough to be considered with pure phage.
  • The plate containing 1X concentration and 2X concentration were flooded with 8 mL of phage buffer than placed on the shaking incubator.

Results and Analysis:

Conclusion:

The titer of 1X concentration and 2X concentration were calculated and flooded.

Future Plans:

DNA Extraction

Category: Sabin Patel | LEAVE A COMMENT
November 30

Serial Dilutions (11/27/18)

Rationale: Due to the negative results of the plaque assays, modifications were made to the procedure to achieve a high titer.

 

Procedure:

  • Three sets of dilutions to the 10^-4 were made by adding 90 microliters to each tube.
  • For one set of dilutions, 7 microliters of the 10^0 lysate were used then diluted.
  • For the second set, 14 microliters of the lysate were diluted to the 10^-4
  • For the third set, 30 microliters were used.
  • Using the 10^-4 dilution from each set, three plaque assays were made.
  • Each dilution was combined with 0.5 mL of Arthrobacter and left alone for 10 minutes for infection.
  • Then, three sets of cultures were made using 2.0 mL of LB Broth and 22.5 microliters of calcium chloride.
  • The infected Arthrobacters of each dilution with a culture then top agar was added and plated.

 

Results and Analysis:

Conclusion:

Three concentrations of an original lysate were made with dilutions to the 10^-4. The first concentration was 7 microliters of lysate then diluted. The 2X concentration consisted of 14 microliters and 3X concentration consisted of 30 microliters. Using the 10^-4 of each concentration, plaque assays were made using the lysate, Arthrobacter, LB Broth, calcium chloride, and top agar.

 

Future Plans:

Calculate the titer of each plate then flood in order to get more lysate for DNA extraction

 

Category: Sabin Patel | LEAVE A COMMENT
November 30

Plaque Assay (11/26/18)

Rationale: In order to move to DNA extraction, 10 mL of lysate of pure DNA is needed. Due to a shortage of lysate, plaque assays were created in order to flood and obtain the lysate of pure DNA.

Procedure:

  • After contamination prevention measures were taken, 125 microliters of lysate was added to 0.5 mL of Arthrobacter and allowed for infection for 10 minutes.
  • In another test tube, 2.0 mL of LB broth and 22.5 microliters of calcium chloride were added together.
  • After the 10 minutes, the lysate and Arthrobacter was added to the LB Broth and calcium chloride.
  • Then, 2.5 mL of 1X Top Agar was added to each test tube then quickly added to the plate. To solidify, the plate was left alone for 15 minutes.

 

Results and Analysis:

First Phage

Head: 52 nm

Tail: 180 nm

 

Second Phage

Head: 47 nm

Tail: 106 nm

 

Conclusion:

Due to the inability to reach the minimum amount of lysate needed for DNA extraction, plaque assays were made to be used for flooding. In order to do this, plaque assays were made using 125 microliters of lysate was added with 5 mL of Arthrobacter then left alone to infect. For the culture, LB broth and calcium chloride were combined together. Then, the infected Arthrobacter was added to the culture. After, the top agar was added then plated.

 

Future Plans:

If phages are present, the titer will be calculated and will be used to determine if it is a high titer. If there are no phages present, plaque assays will be done again.

 

Category: Sabin Patel | LEAVE A COMMENT
November 30

11/28/18 Spot Titer Test, TEM, and DNA Extraction Part 1

 11/28/18 Spot Titer Test, TEM, and DNA Extraction Part 1

Objective:

The goal of this procedure is to assist Lucy P. in getting a large amount of high titer lysate. This is achieved by webbing and then flooding plates. This procedure will detail the process of calculating the titers of the lysate C (the result of webbing plates with lysate 1), creating a TEM grid, running a TEM, and creating a DNA pellet as part of DNA extraction.

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 a Serial Dilution:

  • Phage Buffer
  • Microcentrifuge Tubes
  • Vortex Machine
  • Pipette

Materials for Lysate Filtering:

  • Topfilter
  • 50 ml conical
  • Flooded webbed plates

Materials for Phage Precipitation:

  • High Titer Lysate
  • 50 ml conical vial
  • Nuclease mix
  • Phage Pericpitate solution

Materials for TEM Grid:

  • High titer lysate
  • 400 mesh copper grid
  • Grid box
  • TEM forceps
  • DI water
  • Uranyl Acetate
  • Gloves
  • p20 micropipettor
  • Parafilm

Materials for a Spot Test:

  • .5 ml Arthrobacter
  • incubator
  • Pipette
  • Test tube stand
  • 50 ml tubes
  • Culture tube
  • LB Broth
  • 2X TA
  • 1M Calcium Chloride
  • Agar plate
  • Serological pipette

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

  1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
  2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
  3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone

Lysate C was collected and filtered

  1. The lysate from the flooded plates was poured into a top filter
  2. The lysate was filtered into a 50 ml conical

The Phage Precipitation was performed.

  1. 10 ml of lysate C were transferred to a conical vial
  2. 40 µL of Nuclease mix were added and the vial was inverted several times to mix
  3. 4 ml of phage precipitate were added
  4. The tube was placed in the incubator for 30 minutes
  5. Then the tube was left at room temperature for ~40 minutes
  6. The tube was then centrifuged at 10,000g for 20 minutes *Note: the tubes got locked in the centrifuge machine overnight before being centrifuged for 20 minutes*
  7. The supernatant was poured into the sink and the pellet was put in the freezer until next lab

While phage precipitation was performed a TEM grid was created.

  1. 20 µL of Lysate, 20 µL of DI water, 20 µL of DI water, and ~20 µL uranyl acetate were placed on a strip of parfilm as shown below:
  2. TEM forceps were used to place a copper grid shiny side down in the lysate for 5 minutes
  3. Forceps were then used to transfer the grid to each sample of DI water for 2.5 minutes each
  4. Finally, the grid was transferred to the uranyl acetate for a minute
  5. Excess moisture was wicked away with filtered paper and the grid was loaded into the TEM for imaging

Then serial dilutions were performed on lysate C.

  1. Seven levels of dilution were created for each lysate (called lysate 1 and 2): 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7
  2. Seven microcentrifuge tubes were filled with 90 µL of phage buffer
  3. 10 µL of lysate C (10^0) were transferred to one of the 10^-1 vials
  4. The tube was vortexed to mix
  5. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
  6. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-7 dilution for lysate C

Then the spot titer test on the new lysates was performed.

  1. One agar plates was labeled as shown:
  2. Agar was prepared according to the following recipe (makes two plates):
  3. 4.5 ml of the agar was transferred to the labeled plate
  4. The plate was swirled and set aside to allow the agar to solidify
  5. When agar was solidified, 10 µL of phage buffer as well as 10^-3, 10^-4, 10^-5, 10^-6, and 10^-7 dilutions for each lysate were transferred to their appropriate spot on the plate
Results:

Update: As can be seen from the image above, our spot test failed. We know our lysate is a high titer, so it seems likely we inverted the plate too quickly so the lower concentration dilutions didn’t have enough time to absorb onto the agar.

In addition, the results of our TEM imagining can be seen. In our group, 2 distinct types of phage were found, as detailed above.

Analysis:

The idea behind these procedures is to learn more about our phage as a whole. From these procedures were were able to learn about our phage morphology and extract DNA for further testing. This is the final step needed to ensure we sucessful have a phage that we can add to the phage database.

Future:

We will finish DNA extraction next lab and we will archive.

Category: Lucy FIsher | LEAVE A COMMENT
November 30

DNA Extraction and Nanodrop Lysate 8

11/28/18

Rational:

To do DNA extraction on lysate 8 in order to obtain a sample of DNA from the phage found in lysate 8.

Procedure:

  • Cleaned lab desk
  • Added 500 ML of sterile water to phage precipitate
  • Added 2 mL of DNA clean up resin and mixed
  • Put the mixture into two centrifuge tubes and spun at 12,500 G for 3 min
  • Pulled off the supernatent
  • Added 1 mL of 80% isopropenol and spun at 12,500 G for 3 min
  • Pulled off the supernatent
  • Repeated the previous two steps two more times
  • Added 1 mL of 80% isopropenol
  • Put the solution in a column tube and filtered
  • Centrifuged the filtered column at 12,000 G for 5 min
  • Put the remaining solution in a new centrifuge tube and added 100 ML of 80 C elution buffer
  • Centrifuged for 1 min at 12,000 G
  • Ran the nanodrop on the sample

Observations:

  • DNA concentration- 417.74 ng/ML

Conclution:

The nanodrop results showed the the DNA concentration in lysate 8 was 417.74 ng/ML. As there is not enough time nothing else will be done with this sample however, next I would have done PCR and gel electrophoresis on lysate 8 since it has a high titer.

Category: Emily Balint | LEAVE A COMMENT
November 30

TEM ans Phage Precipitation for Lysate 8

11/26/18

Rational:

To get an image of the phage found in lysate 8. Also to start DNA extraction by pelleting the phage in lysate 8 and breaking down the free DNA.

Procedure:

  • Cleaned lab desk
  • Put 10 mL lysate 8 into a tube
  • Added 40 ML nuclease mix
  • Added 4 mL phage precipitant solution
  • Put in shaking incubator for 30 min at 37 C
  • Let sit at room temperature for 40 C
  • Spun down at 10,000 G for 20 min
  • Pulled off teh supernatent
  • Froze the remaining liquid and pellet.

Conclusion:

The TEM showed that thr phage had a 106 Mm tail and a 45 Mm head (diameter). Next lab I will do DNA extraction on the phage pellet formed this lab. Nanodrop will also be done on the phage DNA obtained from the DNA extraction.

Fig.4.8 – This image show one of the phage found in lysate 8.

Category: Emily Balint | LEAVE A COMMENT
November 30

11/28/18 PCR and Gel Prep

Rationale:

With DNA extracted and analyzed, it was time to run a PCR and prep a gel to gain some information on the composition of this phage’s genetic material.

Results from 11/26/18:

  • From the DNA extracted, there is a very good amount of genetic material present. The DNA contains 805.8 micrograms per ml of nucleic acids, but this could be either phage DNA or bacterial, or both.
  • A phage was found intact with a head size of 50 nanometers and a tail size of approximately 174 nanometers.

Materials:

  • 3 Microcentrifuge tubes
  • Extracted DNA
  • Forward Primers and Reverse Primers for clusters (AK, AL, AM, AN, AO, AP, AQ, AR, AS, AT, AU, AV).
  • 2X PCR Mix (dNTP, Taq, Buffer, Mg)
  • ddH2O
  • Thermocycler
  • Agarose powder
  • TAC
  • Microwave
  • Ethidium Bromide (DNA dye)
  • Erlenmeyer flask
  • Electrophoresis apparatus (tray, comb, dams)

Procedure:

  1. Established an aseptic zone.
  2. Labeled each microcentrifuge tube for their respective primer mixes (1-3).
  3. Added 2 µl of extracted DNA to each of the microcentrifuge tubes.
  4. Next, added 12.5 µl of the nuclease master mix to each of the tubes.
  5. After that, 6.5 µl of ddH2O was added to each of the tubes as a final volume of 25 µl is wanted in each of the tubes.
  6. 4 µl of primer mix 1 was added to its respective microcentrifuge tube, then 4 µl of primer mix 2 was added to its respective tube, and the same for primer mix 3 as well.
  7. The micro centrifuge tubes were then put on ice to be transferred to the PCR machine, where the “STU” program was selected to run. This included 35 cycles total of 3o seconds at 98.0 °C, 30 seconds at 55.0 °C, 45 seconds of 72 °C, and a final extension of 5 minutes at 72.0 °C.
  8. While PCR was running, an agarose gel was prepped by measuring out 0.8 g of agarose powder and combined with 40 ml of TAC in an erlenmeyer flask.
  9. The mixture was swirled to combine, and was put in the microwave to boil in one minute increments until it was clear and bubbling. It was then allowed to cool until it was just cool enough to touch without burning ones hand.
  10. Next, 2 µl of ethidium bromide was added to yield a final concentration of 0.5% EtBr.
  11. The gel was then carefully poured into an assembled electrophoresis apparatus with the comb inserted and left to cool. The comb was then removed and the gel was covered with TAC to prevent any drying to store for the rest of the week.

Results/Data:

  • Primer Mix 1 was extremely concentrated when mixed with the DNA sample and could influence the results
  • A positive control was not run in the PCR as well.
  • DNA was not diluted either, which was needed as the DNA has a large amount of nucleic acids per ml.
  • Agarose gel was created with no complications, and the comb was removed without ripping the gel.

Conclusions:

The failure to dilute the DNA or the primer mix will yield interesting potential results. As there is such a high amount of genetic material in the extracted DNA samples, this can cause just a large mass of DNA to be yielded from PCR that will be unable to pass through the gel. PCR should still work theoretically with the highly concentrated primer mix 1, but it is not the most favorable environment for PCR to occur.

Next Steps:

The next steps are to analyze the PCR results and attempt to run a gel with the overly concentrated DNA.

Category: Uncategorized | LEAVE A COMMENT
November 30

SEA Bears Day 27

28 November 2018 ✷ Pick a Different Plaque

Rationale: Members of group 6 adopted phage from Soil 4 from Melissa (collected 10/5/18) in order to help amplify the phage sample. A plaque was picked from Melissa’s original plate because the prior plates run by members of group 6 came up negative.

Procedure

  • The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
  • Plaque “Y” on the plate from the prior lab session was picked and swirled into 50 µL of phage buffer. 30 µL of this mixture was added to 0.5 mL of arthrobacter and allowed to sit for 15 minutes while the plates were made.
  • A plate for 2 plaque assays and a control plate were made with the following concentrations and volumes:
  • component volume final concentration
    LB Broth 6 mL –
    2X Top Agar  7.5 mL 1X
    1M Calcium Chloride  68 µL 4.5 µM
  • The arthro/lysate mix was combined with 5 mL of the above plate mix and the plate was poured, allowed to harden, and then inverted and incubated at 37 degrees celsius. The remaining lysate was refrigerated.

Observation, Results, Data

The originally positive plaque assay was picked and plated but ended up with negative plaque assays that had weak spots that mimicked plaques, but weren’t actually plaques.

 The spots, when looked at under the microscope, weren’t circular nor clear, as true plaques should be. Thus, when Melissa’s initial plate was looked at again under the microscope, the plaques present were more easily identifiable and were able to be picked and plated.

Interpretations, Conclusion, Next Steps

In the following lab periods, the presence of a webbed plate will mean that the lysate is a high titer and can be studied more closely using TEM. The current titer is very low, which may indicate a weaker phage or phage presence.

Category: Lily Goodman | LEAVE A COMMENT