November 30

Phage Precipitation for Sample (Gabe) and TEM Imaging 11/26/18

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:

To sequence the phage DNA in the sample, the phages cultured from the webbed plates in the high titer lysate collected must be extracted from the phage buffer solution. The phage in the lysate will precipitate into a pellet and then it could be stored for DNA Extraction.

Pellet Precipitation for Sample (Gabe):

Materials:

  • High Titer Lysate
  • Nuclease Mix
  • Phage Precipitation Solution

Procedure:

  1. Added 40 ul of Nuclease Mix to 10 ml High Titer Lysate and mix gently
  2. Added 4 ml of Phage Precipitation Solution and gently mix
  3. Incubated at 37 degrees Celcius for 30 min and at room temp for 45 min
  4. Centrifuged at 20 g for 20 min
  5. Removed the supernatant, leaving the pellet and stored in freezer.

Observations, Results & Data:

The liquid after centrifuge had opaque cloud like substances suspended inside, and some were lost during the removal of the supernatant.

The imaging of the phage shows that it has a head about 47 nm and tail about 100 nm.

Interpretations & Conclusions:

The centrifuge was supposed to be set at 20000 g for 20 min according to the protocols yet was mistakingly set at 20 g, the results of this deviation of the protocol has yet to be seen.

The size of the phage raised interesting questions regarding the type of the phage, since that three different lysate from the sample yield different lengths of the tail, and since the tail length in phages are heavily conserved it may be likely that the sample contained multiple species of phages.

Next Step:

The next lab would be to start DNA extraction and calculate a concentration for the DNA collected.

Category: Yang-En Yu | LEAVE A COMMENT
November 30

11-28-18 — High Titer Plaque Assay Second Attempt

Date: Wednesday, November 28th, 2018

Title: High Titer Plaque Assay Second Attempt

Rationale: The purpose of today’s lab is to calculate the titer of the lysate using a previous plaque assay and then to attempt to web a plate using the titer calculations.

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. An aseptic zone was set up.
  2. Plates from last lab were evaluated and found to have negative results.
  3. A new 10^-7 dilution was made using the positive lysate.
  4. 20 μL of 10^-7 diluted lysate was added to a culture tube containing .5 mL arthrobacter and left to infect for 15 minutes.
  5. Top agar solution was made using the following recipe:
    1. 4 mL LB Broth
    2. 5 mL 2x top agar
    3. 45 μL 1M CaCl2
  6. 4.5 mL of TA solution was added to a top agar control plate.
  7. 4.5 mL of TA solution was added to the culture tube and pipetted to mix.
  8. The contents of the culture tube were poured into a plate.
  9. The plates were left to sit for 15 minutes before being inverted and placed in an incubator for the next 48 hours.

Observations: The plates from last lab yielded negative results. This is most likely due to the original 10^-7 dilution losing its infectivity. The newly made dilution should produce results, as it has a high titer.

Results: This experiment yielded a top agar control and a plaque assay that, according to titer calculations, should be a webbed plate.

Next Step: The next step is to evaluate the plates and flood it with phage buffer.

Category: Brandon Reider | LEAVE A COMMENT
November 30

11/27/18 Flooding Webbed Plates

11/27/18 Flooding Webbed Plates

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 flooding the webbed plates previously created during the last lab.

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 Flooding a Plate:

  • Phage buffer
  • Refrigerator
  • Syringe Filter
  • 15 ml conical vial
  • 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

Them the seven webbed plates were flooded.

  1. 8 ml of phage buffer was pipetted onto each agar plate
  2. The plates were placed in the refrigerator to sit for ~22 hours
Results:

The results of this lab will not be visible until Wednesday’s lab, but I can say that flooding the plate seemed to occur without incident.

Update: The plates flooded successfully and the lysate was filtered and further testing was run.

Analysis:

The idea behind the procedures as a whole is to enable us to create larger quantities of high titer lysate. In theory, this can be done after a plaque has been purified by creating webbed plates that can be flooded with phage buffer. The resulting mixture then holds many phage, and the titer can be tested with a plaque assay or spot test. This appeared to be a success which we will confirm with a titer test on Wednesday.

Future:

During our next lab, we will perform serial dilutions to determine the titer of the resulting lysate from this lab and because we are crunched for time we will also likely to TEM and start DNA extractions.

Category: Uncategorized | LEAVE A COMMENT
November 30

11/26/18 Webbing Plates

11/26/18 Webbing Plates

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 webbing enough plates to have enough high titer lysate to proceed with more testing.

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 Plaque Assay:

  • .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

Then plaque assays to web plates were performed.

  1. Seven agar plates were labeled
  2. 10 µL of lysate 1 at 10^-3 dilution were transferred into each of the four culture tube containing .5 ml of Arthrobacter
  3. The culture tubes were set aside for 15 minutes.

While the lysate and bacteria are allowed to sit in the culture tube the agar was prepared.

  1. The agar was prepared according to the following recipe (makes 7 plates):
  2. 4.5 ml of the agar was transferred to the plate labeled “TA control”
  3. The plate was swirled and set aside
  4. 4.5 ml of the agar was transferred into each culture tube
  5. The resulting mixture was poured into the corresponding plates
  6. The was set aside for 10 minutes to allow agar to solidify.
  7. Plates were left to incubate until nest class
Results:

The results of this procedure will not be immediately clear but everything appeared to go according to plan.

Update: These plates essentially lysed making perfect plates to flood (see image below).

Analysis:

The idea behind the procedures as a whole is to enable us to create larger quantities of high titer lysate. In theory, this can be done after a plaque has been purified by creating webbed plates that can be flooded with phage buffer. The resulting mixture then holds many phage, and the titer can be tested with a plaque assay or spot test. This procedure yielded perfect plates to flood so the resulting lysate should be high titer and there should be a lot of it.

Future:

During our next lab period, we will flood these webbed plates.

Category: Uncategorized | LEAVE A COMMENT
November 30

11/19/18 Webbing Plates (Including Flooding of 11/20/18)

11/19/18 Webbing Plates (Including Flooding of 11/20/18)

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. After the spots test showed that lysate 1 was close to high titer we needed to make more of the lysate and amplify it. This procedure will detail the process of flooding plates to get a large amount of high titer lysate.

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 Plaque Assay:

  • .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

Then plaque assays to web plates were performed.

  1. Five agar plates were labeled
  2. 10 µL of lysate 1 at 10^-4 dilution were transferred into each of the four culture tube containing .5 ml of Arthrobacter
  3. The culture tubes were set aside for 15 minutes.

While the lysate and bacteria are allowed to sit in the culture tube the agar was prepared.

  1. The agar was prepared according to the following recipe (makes 5 plates):
  2. 4.5 ml of the agar was transferred to the plate labeled “TA control”
  3. The plate was swirled and set aside
  4. 4.5 ml of the agar was transferred into each culture tube
  5. The resulting mixture was poured into the corresponding plates
  6. The was set aside for 10 minutes to allow agar to solidify.
  7. Plates were left to incubate until nest class
Results:

Update: These plates failed to web, and flooding them did not create enough lysate to test, so we will need to redo the whole procedure.

Analysis:

The idea behind the procedures as a whole is to enable us to create larger quantities of high titer lysate. In theory, this can be done after a plaque has been purified by creating webbed plates that can be flooded with phage buffer. The resulting mixture then holds many phage, and the titer can be tested with a plaque assay or spot test. This procedure was a failure so it will be redone, but if it had gone according to plan, the resulting lysate should have been a high titer.

Future:

During our next lab period, we will create more webbed plates and flood them in an effort to get enough high titer lysate to move on with TEM and DNA extraction.

Category: Uncategorized | LEAVE A COMMENT
November 30

11/28 Phage Precipitation

Rationale: Prepare DNA extraction/PCR by performinf phage precipitation.

Procedure: Lysate obtained and 10mL of lysate was added into a 50mL vial. In the back of the lab, 40µL of nuclease mix was added into the 50mL vial, and this was not done in an aseptic zone. Inverted the vial 10 times and added 4mL of phage precipitant solution. Placed the vial in the incubator at 37 degrees Celcius for 30 minutes, and then incubated the solution at room temperature for 45 minutes. The lysate was then centrifuged in a swinging bucket at 10,000xg for 20 minutes. The supernatant formed from the centrifuge was poured into the sink, but not letting the pellets flow out of the solution. The pellets were poured onto a paper towel, dried, and placed into a microcentrifuge cap labeled ML DNA 11/12/18.

Observations: Experiment performed 11/26 was used to determine titer. Titer calculated to be at 1 x 10^9. Plate from 11/26 experiment showed contaminations, but the phage particles were countable.

Conclusions: DNA extraction will be performed using the products made from 11/28 experiment.

Category: Michael Lum | LEAVE A COMMENT
November 30

DNA Extraction 11/29/18

Rationale

Today we will conduct DNA extraction on the precipitate previously made.

Procedure

  • Established an aseptic zone.
  • 0.5 µL of sterile water was added to the phage pellet to resuspend.
  • 2 mL of incubated Resin was added to the vial and was shaken to fully coat the DNA.
  • Approximately 1.5 mL of the solution was added to one microcentrifuge tube, and 1.5 mL of the solution was added to another microcentrifuge tube.
  • The two tubes were spun at 12-13k g for 3 minutes.
  • The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
  • The two tubes were spun at 12-13k g for 3 minutes.
  • The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
  • The two tubes were spun at 12-13k g for 3 minutes.
  • The supernatant was removed. 1 mL of 80% isopropanol was added to each tube and the pellet was resuspended.
  • A column syringe was assembled and attached to the vacuum hood. The contents were placed in the column syringe filter and the DNA/resin mixture was trapped in the column filter.
  • The two column filters were placed in 2 microcentrifuge tubes and were centrifuged at 12,000g for 5 minutes. This process was repeated twice.
  • 100 µL of Elution Buffer was added to the column and was set aside for 1 minute. The two microcentrifuge tubes were centrifuged at 12,000 g for 1 minute. The product of the two tubes were combined and were placed into the -20 C freezer as a result.

Observation

Dr. Adair centrifuged the phage precipitate preparation before lab since the centrifuging swinging bucket was not functioning at the previous lab.

Conclusions/Next Step

Next, the DNA will be analyzed using a Nanodrop and PCR will occur.

Category: Emily Gaw | LEAVE A COMMENT
November 30

DNA Extraction and Spot Titer Test (11/29/18)

Rationale:

Continued with DNA Extraction and made serial dilutions to perform a spot titer test to calculate the titer of our plate. 

Procedure: 

  1. The pellet was obtained from the freezer and 500 uL of sterile water was added. 
  2. The solution was flicked, and the solution was evenly mixed using a pipette as well.  
  3. Resin was obtained and 2 mL were added into solution containing the pellet.  
  4. Pipetted the solution to mix thoroughly.  
  5. Two microcentrifuge tubes were obtained, and 1.5 mL of solution was added to each.  
  6. Both tubes were centrifuged at 12.5 xg for 3 minutes.  
  7. Using a bulb pipette, the supernatant was removed, without touching the pellet.  
  8. 1 mL of isopropanol was added to each microcentrifuge tube and pipetted up and down to kix.  
  9. The two tubes were spun for a second time at 12.5xg for 3 minutes.  
  10. The supernatant was removed using a bulb pipette and 1 mL of isopropanol was added and mixed by pipetting up and down or flicking   
  11. The tubes were spun for a third time at 12.5 xg for 3 minutes  
  12. The supernatant was removed, and 1 mL of isopropanol, and mixed by pipetting up and down.  
  13. Pipetted the solution into a column syringes under a vacuum hood, and removed the supernatant in this process.  
  14. The column syringes were removed and placed into two new microcentrifuge tubes and the syringes were removed leaving the columns attached to the top of tube. 
  15.  The two tubes were centrifuged at 12 xg for 5 minutes.  
  16.  The columns were transferred to a new microcentrifuge tube and 100 uL of Elution Buffer was added.  
  17. The solution was let to sit for a minute, then spun at 12xg for one minute.  
  18. The column was removed, and the solution was added to a microcentrifuge tube and stored in the freezer.  
  19. Two plates were created for the spot titer test using 4 mL of LB Broth, 45 uL of CaCl2, and 5 mL of Top Agar.  
  20. 4.5 mL of solution was added to each of the two 0.5 mL of Arthrobacter and poured onto the plates to let to set.  
  21. Serial dilutions for a spot test were created using lysate from webbed plates. 
  22. Eight microcentrifuge tubes were obtained and labeled 10^-1 to 10^-8.  
  23. 90 uL of Phage buffer was added to each of the eight tubes.  
  24. 10 uL of lysate was added to the 10^-1 serial dilution. The tube was vortexed and 10 uL of the 10^-1 serial dilution was added to the 10^-2 serial dilution tube.  
  25. Continued this process until a serial dilution from 10^-1 to 10^-8 was obtained.  
  26. 5 uL from each dilution was pipetted onto the respective portion of the plate.  
  27. The plates were left to set for 10 minutes.  
  28. The plates were place upright into the incubator.  

Observations/ Results: 

  • Some of the plates for the spot titer test contained bubbles.  
  • While performing DNA Extraction the pellet appeared cloudy.  

Next Steps/ Conclusions: 

Next class we will use the solution containing DNA to perform PCR. Using the results from the spot titer, the titer of the lysate will be determined.  

Category: Sona Subramanian | LEAVE A COMMENT
November 30

TEM and DNA Extraction (11/28/18)

Rationale:

TEM and DNA Extraction were performed since a known medium titer was used to create a new lysate and enough lysate was obtained to start on DNA Extraction.  

Procedure:  

  1. An aseptic zone was created using 70% ethanol, Cidecon, and an ethanol burner.  
  2. For DNA extraction, 10 mL of lysate were obtained and pipetted into a 50 mL tube.  
  3. 40 uL of Nuclease was added into the 50 mL tube and flipped 10 times.  
  4. 4 mL of PEG was added into the 50 mL tube inverted once   
  5. The 50 mL tube was placed in the incubator for 25 minutes.  
  6. While waiting the 25 minutes, TEM grid was made by adding 20 uL of lysate, two 20 uL of DI water, and about 20 uL of Uranyl acetate on parafilm.  
  7. A small mesh copper grid was obtained and placed shiny side down in the lysate for five minutes.  
  8. The 50 mL tube for DNA extraction was moved to sit at room temperature for 40 minutes.  
  9. After the five minutes in the lysate using forceps removed copper TEM grid from lysate and set in DI water for 2.5 minutes.  
  10. The TEM grid was moved to the next DI water for another 2.5 minutes.  
  11. Then the TEM grid was moved to Uranyl Acetate for a minute.  
  12. The TEM grid was then blotted dry and viewed under the electron microscope.  

Observation/Results: 

  • Spot Titer Test lysed for serial dilutions from 10^-1 to 10^-3. The rest of the serial dilutions did not produce any phage, and therefore could not use this plate to determine the titer of the plate.

  • Spot Test Plate 1

    Spot Test Plate 2

  • TEM and DNA Extraction were performed assuming that a high titer would be produced from a medium titer plate of 10^7 since 10^-1 to 10^-3 serial dilutions were lysed as well.  

    Phage on TEM

  • Since the centrifuge machine had some technical difficulties, Dr. Adair centrifuged the lysate solution and separated the pellet from the supernatant and stored the pellet in the freezer. 

Next Step/ conclusions: 

Next class DNA Extraction will be completed to be used for PCR, and another spot titer test will be performed to calculate the titer of the plate.

Category: Sona Subramanian | LEAVE A COMMENT
November 30

Serial Dilutions and Spot Titer Test (11/26/18) 

Rationale:

Performed serial dilutions and spot titer tests to obtain the new titer of the lysate obtained from the medium titer.  

Procedure: 

  1. Created an aseptic zone using Cidecon and 70% Ethanol, and used a ethanol burner.  
  2. Obtained lysate from webbed and flooded plates of new titer lysate. 
  3. Serial Dilutions were performed by adding 10 uL of the 10 ^ 0 lysate to 90 uL of the phage buffer into a small tube.  
  4. The tube was vortexed and used to create serial dilutions from 10^-1 to 10^-8. 
  5. Divided two plates and labeled –1 to –8 as well as one for control.  
  6. 45 uL of CaCl2, 4 mL of LB Broth, and 1 mL of Arthrobacter were added to a 50 mL tube.  
  7. 5 mL of Top Agar was added to the 50 mL tube.  
  8. 5 mL of solution was pipetted into the two plates.  
  9. Waited 10 minutes until the plates set.  
  10. Added 5 uL of lysate into respective portions of the plate.  
  11. 5 uL of phage buffer was added into the control part of the plate.  
  12. The plates were left to sit for another 15 minutes and placed upright into the incubator.  

Observations/ Results: 

  • Small bubbles formed in the middle of the plates while pouring.   

Next Steps/Conclusion: 

Next class we will calculate the titer of the new lysate. If a high lysate is attained, then we will try to create webbed plates and flood them to obtain lysate. Or we could continue by performing TEM using our high lysate.

Category: Sona Subramanian | LEAVE A COMMENT