November 30

SEA Bears Day 26

26 November 2018 ✷ Pick a Plaque again

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 the picked plaque run 11/19 because the titer was too low.

Procedure

  • The table was cleaned with Cidecon and 70% ethanol and an ethanol lamp was lit.
  • Plaque 1 on the plate from the prior lab session was picked and swirled into 30 µL of  phage buffer. 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 1 plaque assay and a control plate were made with the following concentrations and volumes:
  • component volume final concentration
    LB Broth 4 mL –
    2X Top Agar  5 mL 1X
    1M Calcium Chloride 45 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 until Wednesday at 37 degrees celsius.

Observation, Results, Data

3 plaques were seen on the plate that was picked in today’s lab and each was picked and plated. This will help isolate a single phage type from the sample and purify it so it may be studied.

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
November 30

Lab Day 32: Flooding. Serial Dilutions, Spot Titer

Rationale

10 microliter plate was flooded and had serial dilutions performed up to 10^-8. A spot titer was performed to calculate titer by Monday.

Detailed Procedure

  1. Add 5 mL of PB to  10 microliters  plate from previous lab day.
  2. The plates was shaken on an incubator for one hour.
  3. Filtered lysate from flooded plate in two separate 50 mL 0.22 µm filtered conical vial.
  4. 10 µL of 100 was added with 90 µL of phage buffer to create 10-1 dilution which was vortexed.
  5. 10 µL of 10-1 was added with 90 µL of phage buffer to create 10-2 dilution which was vortexed.
  6. 10 µL of 10-2 was added with 90 µL of phage buffer to create 10-3 dilution which was vortexed.
  7. 10 µL of 10-3 was added with 90 µL of phage buffer to create 10-4 dilution which was vortexed.
  8. 10 µL of 10-4 was added with 90 µL of phage buffer to create 10-5 dilution which was vortexed.
  9. 10 µL of 10-5 was added with 90 µL of phage buffer to create 10-6 dilution which was vortexed.
  10. 10 µL of 10-6 was added with 90 µL of phage buffer to create 10-7 dilution which was vortexed.
  11. 10 µL of 10-7 was added with 90 µL of phage buffer to create 10-8 dilution which was vortexed.
  12. 4 mL of LB Broth, 45 µL of CaCl2, and 5 mL of 2X TA were combined into a 15 mL vial.
  13. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into control plate labeled “SJ Control 11/30/18.”
  14. Added 0.5 mL Arthro into remaining 4.5 mL TA mixture and poured onto plate labeled “SJ Spot 11/30/18” which was sectioned off into 8 parts
  15. Waited 15 mins for both plates to solidify.
  16. Micropipetted 10 µL of 100, 10-1, 10-2, 10-3 ,10-4 , 10-5  , 10-6  , 10-7  , 10-8  in according sections of the spot test plate.
  17. Rested into incubator

Conclusion

10 microliter plate was completely lysed, which gives hope that a high titer will be achieved by Monday. Only 5 mL of PB was on the plate since the graduated pipette didn’t have up to 10 mL. By next lab day, a new titer will be calculated and hope to achieve a high titer by then.

Category: Soo-Un Jeong | LEAVE A COMMENT
November 30

PCR and Making of an Agarose Gel (11/28/18)

Rationale:

To find out to which cluster Ferranti belongs, gel electrophoresis will be performed. In preparation for gel electrophoresis, polymerase chain reaction (PCR) will be performed and a 2% agarose gel will be made.

Procedure:

  1. Once an aseptic zone was established, 12.5 µL of 1X MM, 2 µL of extracted DNA, and 6.5 µL of ddH2O were combined three times into three different microcentrifuge tubes.
  2. 4 µL of primer 1, primer 2, and primer 3 were placed into their correlating microcentrifuge tubes.
  3. Placed microcentrifuge tubes in thermo-cycler and activated program STU.
  4. 40 mL of 1X TAE and 0.8 g of powdered agarose were combined and swirled together in an Erlenmeyer flask.
  5. Heated the Erlenmeyer flask until the mixture was boiling then mixed solution until the bubbles disappeared. Repeated until the solution was consistent.
  6. Allowed the solution to cool until it was cool enough to touch.
  7. Added 2.0 µL of EtBr to the flask to achieve a concentration of 0.5 µg/µL.
  8. Poured mixture into gel apparatus and placed comb.
  9. Once the gel solidified, the comb was removed, and TAE buffer was poured over the gel to keep it from drying out.

Observations:

  • The thermocycler used the program STU which started with 5 minutes of initial denaturation at 98.0ºC. Then, 35 cycles of 30 seconds at 98.0ºC followed by 30 seconds at 55.0ºC followed by 45 seconds at 72.0ºC occurred. After the cycles, a final extension happened at 72.0ºC for 5 minutes.
  • Unfortunately, it was realized later that DNA concentration was not taken into account when making PCR Mix.
  • Also, it was realized later that no controls were made.
  • The gel form had 8 wells and had a hazy white tint to it. The following picture shows the gel created.

Next Steps:

Perform gel electrophoresis with DNA samples on agarose gel.

Category: Kathryn Adkins | LEAVE A COMMENT
November 30

11/28/18 PCR

Previous Results:

  • DNA extraction was recently completed and DNA was obtained from the phage sample

Objective:

  • Set up PCR using the concentrated DNA sample and prepare to run a gel test

Procedure:

  1. Aseptic Zone was created with CiDecon, 70% Ethanol, and an Ethanol burner
  2. The DNA microcentrifuge tube “Pippa” was obtained and 3 microcentrifuge tubes were labeled 1-3
  3. 2 microliters of DNA was added to each tube, along with 12.5 mircoliters 1x Master Mix, and 6.5 microliters ddH2O
  4. Each tube, labeled 1-3, was given 4 microliters of primer. Tube 1 had Primer 1, Tube 2 has Primer 2, and Tube 3 had Primer 3. Each tube had a total of 25 microliters liquid
  5. The tubes were then placed in a thermo-cycler @ 98.0 degrees Celsius for 5 minutes, then completed 35 cycles of 94 degrees Celsius for 30 seconds, 55.1 degrees Celsius for 30 seconds, and 72 degrees Celsius for 45 seconds. The process was completed with 5 minutes in 72 degrees Celsius
  6. The tubes were then placed in the freezer at a temp of 4 degrees Celsius until next lab

Results:

  • PCR and gel were not completed, therefore there are no results to report

Next Steps:

  • During the next lab a gel will be ran on the DNA and results will be obtained
Category: Claire Wentzlaff | LEAVE A COMMENT
November 30

TEM Results, DNA Extraction, and Nanodrop (11/26/18)

TEM Results:

Unfortunately, the pictures were not of high quality since the copper mesh TEM disc was exposed to uranyl acetate for 15 seconds longer than the protocol instructed, and the disc was bent causing it to begin to deteriorate. The following pictures were taken on the TEM.

Rationale:

Finish DNA extraction protocols so the extracted DNA can be used on the Nanodrop to find the concentration of DNA.

Procedure:

  1. Re-suspended phage pellet with 0.5 mL of distilled water.
  2. Added 2 mL of 37ºC DNA Clean Up Resin to phage pellet which then was equally distributed into two microcentrifuge tubes.
  3. Centrifuged microcentrifuge tubes at 12,500xg for 3 minutes and removed supernatant.
  4. Added 1 mL of 80% isopropanol to each microcentrifuge tube and re-suspended pellet.
  5. Repeated steps 3 and 4 twice.
  6. Added 1 mL of 80% isopropanol to each microcentrifuge tube and re-suspended pellet.
  7. Filtered each microcentrifuge tube through one-column syringe.
  8. Centrifuged columns at 12,000xg for 5 minutes.
  9. Added 50 µL of 80ºC Elution Buffer to each column.
  10. Allowed columns to sit for 1 minute before centrifuged at 10,000xg for 1 minute.
  11. Performed a blank on the Nanodrop by placing 2.0 µL of Elution Buffer on the sensor, then used 2.0 µL of exacted DNA.

Observations:

  • The second phage of which the picture was taken had a head length of 55 nanometers and a tail length of 128 nanometers.
  • The pellet that formed after adding the resin and centrifuging was white and cloudy.
  • The purified phage genomic DNA was place into a microcentrifuge tube labeled “KEA Ferranti 11/26/18 888.1.”
  • Ferranti contained 888.1 ng/µL of nucleic acid.
Category: Kathryn Adkins | LEAVE A COMMENT
November 30

11/28 ~ DNA extraction

Rationale: Will be running the procedure of pelleting the lysate to obtain the bacteriophage DNA

 

Procedure:

  • Created an aseptic zone to prevent bacterial contamination
  • Added 10mL of lysate into a 50mL conical tube
  • Pipetted 40μL of nuclease into the tube and inverted the tube around 10 times
  • Added in 4mL of PEG and inverted the tube one to two times
  • Placed the tube into the shaker for 30 minutes
  • Took the tube out of the shaker and allowed to sit in room temperature for 45 minutes
  • After the allotted time, moved the tube into a centrifuge and allowed to centrifuge for 20 minutes at 10,000 G
  • After centrifuging, decanted the supernatant in the tube and collected the pellet(s)

 

Observation:

The spot test to confirm the strength of the titer (Was 1 X 10^9)

 

Conclusion/Next Steps: Will be continuing the next steps of DNA extraction with the hopes of breaking open the capsid of the bacteriophage.

Category: Justin Yu | LEAVE A COMMENT
November 30

11/26 ~ TEM Imaging

Rationale: Prepared a TEM grid to obtain an image of the bacteriophage in the lysate

 

Procedure:

  • Aseptic zone was set up to prevent bacterial contamination
  • Pipetted 100μL of lysate from the original 50mL conical vial into a micro-centrifuge tube
  • Traveled to the TEM lab
  • Placed a strip of ParaFilm onto/into a agar plate
  • Pipetted 20μL of lysate (in a drop) onto the ParaFilm, as well as two separate drops of 20μL DI water
  • Pipetted one drop of 20μL Uranyl Acetate onto the ParaFilm strip
  • Using precision forceps, placed a copper grid shiny side down into the lysate drop and let sit for five minutes
  • After the allotted time (timed with a phone), transferred the grid into the first DI water drop and allowed to sit for two and a half minutes
  • After the allotted time, transferred the grid into the other DI water drop and allowed to sir for two and a half minutes
  • After the allotted time, transferred the grid into the Uranyl Acetate drop and allowed to sit for one minute
  • After the allotted time, immediately extracted the grid from the Uranyl Acetate and wicked away extra liquid with a paper towel
  • The prepared grid was put into the TEM and then imaged

 

Observations:

Image of the phage with dimensions measured

First image of the bacteriophage

  • The measurement of the head was .055μL
  • The measurement of the tail was .180μL

 

Conclusion/Next Steps: After getting this image, will be moving towards DNA extraction and the procedures following it, such as nano-drop and PCR.

Category: Justin Yu | LEAVE A COMMENT
November 30

11/26/18 DNA Extraction and Nanodrop

Previous Results:

  • The final result of the phage precipitation was a pellet in the bottom of the 50 mL tube. This was the condensed phage that will be used for DNA extraction

Objective:

  • To complete DNA extraction without contamination and obtain enough DNA to continue analyzing it
  • Have enough time to complete the nanodrop process and have positive results

Procedure:

  1. The 50 mL tube containing the phage pellet was brought to room temp
  2. 0.5 ml sterile water was added to tube and mixed to resuspend the pellet
  3. 2 ml of DNA Clean-Up Resin at 37 degrees Celsius was added and mixed
  4. Pellet was separated equally into 2 microcentrifuge tubes and spun at 12.5 x 1000 g for 3 minutes
  5. Supernatant was removed from the microcentrifuge tubes without disturbing the cloudy precipitant (DNA) and add 1 ml 80% Isopropanol to the tubes each
  6. Step 5 was repeated 2 times, then once more but without removing the alcohol
  7. Solution was then transferred to two columns and the vacuum was used to filter the liquid
  8. Columns were centrifuged at 12,000 g for 5 min
  9. 100 microliters of Elution Buffer at 80 degrees Celsius was added to each column. Left to sit for 1 minute
  10. Columns were centrifuged at 12,000 g for 1 minute
  11. Two tubes of DNA sample were combined into 1 microcentrifuge tube and labeled. Phage was named “Pippa”
  12. The nanodrop machine was then used by adding a drop of Elution Buffer to the machine to prepare for DNA drop
  13. DNA drop was added and the machine provided results

Results:

  • The results of the nanodrop showed the sample of DNA was not concentrated enough and had a nucleic acid reading of 43.1 ng/uL

Next Steps:

  • A PCR will be dome on the DNA sample and a gel will be completed
Category: Claire Wentzlaff | LEAVE A COMMENT
November 30

NOVEMBER 26TH AND 28TH- Labs

  • NOVEMBER 26TH, 2018
    • OBJECTIVE: 
      • Plate high titer in order to flood plates and get more lysate for DNA extraction 
    • PROCEDURE: 
      • Tables were cleaned and lamps were lit 
      • The dilutions from last lab were used, and the following amounts were placed into a tube with .5mL of Arthrobacter for 15 minutes:
        • 10^-2 45𝝁L  
        • 10^-2 45𝝁L
        • 10^-1 4.5𝝁L 
      • During the 15 minutes a large test tube was filled with: 
        • 8mL LB
        • 90𝝁L  CaCl2 
        • 10mL 2X TA 
      • Then 4.5mL of the 2X TA solution was pipetted into the tube containing lysate and Arthrobacter, then the tube was poured onto a plate
      • The previous step was repeated for all the tubes containing lysate and Arthrobacter 
      • For the control 4.5mL of the 2X TA solution was pipetted onto a plate
      • The plates were then left to solidify for 10 minutes, before being inverted and placed into the incubator 
    • RESULTS: 
      • The Results can be viewed in Figure 26 
    • CONCLUSION: 
      • Minimal to no plaques grew due to how old the lysate was. As the lysate ages, the bacteriophage most likely lost infectivity.
    • NEXT STEPS:
      • Use original lysate to get a high titer plate
  • NOVEMBER 28TH, 2018 
    • OBJECTIVE: 
      • Use lysate from flooded plate (on 12/19) to create serial dilutions and get a high titer
    • PROCEDURE:
      • Tables were cleaned and lamps were lit
      • Six micro-centrifuge tubes were labeled according to the dilution (10^0 through 10^-5)
      • The tube labeled 10^0 was filled with 100𝝁L of phage buffer and tubes 10^-1 through 10^-6 were filled with 90𝝁L of phage buffer 
      • 100𝝁L of lysate was pipetted into the 10^-0 micro centrifuge tube
      • Then 10𝝁L of the 10^0 dilution was pipetted into the 10^-1 tube, then 10𝝁L of solution from the 10^-1 tube was pipetted into the 10^-2 tube, and so on until a dilution of 10^-5 was reached
    • The following amounts of dilutions were placed into a tube with .5mL of Arthrobacter for 15 minutes:
      • 10^-2 45𝝁L  
      • 10^-4 10𝝁L 
      • 10^-1 4.5𝝁L 
      • 10^-2 10𝝁L 
      • 10^-5 10𝝁L 
      • During the 15 minutes a large test tube was filled with: 
        • 14mL LB
        • 157.5𝝁L  CaCl2 
        • 17.5mL 2X TA 
      • Then 4.5mL of the 2X TA solution was pipetted into the tube containing lysate and Arthrobacter, then the tube was poured onto a plate
      • The previous step was repeated for all the tubes containing lysate and Arthrobacter 
      • For the control 4.5mL of the 2X TA solution was pipetted onto a plate
      • The plates were then left to solidify for 10 minutes, before being inverted and placed into the incubator 
    • RESULTS: 
      • Waiting for results 
    • CONCLUSION: 
      • Waiting for results 
    • NEXT STEPS:
      • Wait for results, if high titer was not achieved a TEM will be ran on lysate available, if high titer is achieved plates will be flooded and DNA extraction will be run 
Category: Lauren Foley | LEAVE A COMMENT
November 30

DNA Extraction on Sample (Gabe) 11/28/18

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:

From the pellet collected the DNA could be extracted via DNA extraction protocols, which could help determine the concentration of the DNA and further tests such as PCR and electrophoresis could be run in the future.

Plaque Assay for Sample (Gabe):

Materials:

  • Pellet
  • Sterile Water
  • DNA Clean-Up Resin at 37 degrees Celcius
  • 80% Isoproponal
  • Column
  • Vacuum
  • Elution Buffer at 80 degrees Celcius

Procedure:

  1. Added 0.5 ml sterile water to resuspend the pellet after returning to room temp
  2. Added 2 ml of DNA Clean-Up Resin at 37 degrees Celcius to the resuspended pellet. mixed the resin before adding and the pellet solution after adding the resin
  3. Distribute the pellet to two separate microcentrifuge tubes
  4. Spin at 12.5 x 1000 g for 3 min
  5. Removed the supernatant from the tubes without disturbing the cloudy precipitant and add 1 ml 80% Isoproponal to the tubes each
  6. Repeated step 4 and 5 for 2 times
  7. Removed the supernatant from the tubes without disturbing the cloudy precipitant and add 1 ml 80% Isoproponal to the tubes each
  8. Transfer the solution to two columns and allow the vacuum to pull the liquid through the filter
  9. Centrifuge the columns at 120000 g for 5 min
  10. Added 100 ul of elution Buffer at 80 degrees Celcius to each column and set still for 1 min
  11. Centrifuge the columns at 120000g g for 1 min
  12. Combined the two tubes of DNA sample collected and Quantify the concentration using the nanodrop machine

Observations, Results & Data:

There were large amounts of resin observed through the steps of the experiment

Nucleic Acid(ng/uL) A260/A280 A260/A230 A260 A280 Nucleic Acid Factor Baseline Correction (nm) Baseline Absorbance
417.736 1.996 0.653 8.355 4.185 50 340 0.011

Interpretations & Conclusions:

The low g spin setting while precipitating the pellet seemed to not affect the DNA count on the sample.

Next Step:

The next step would be to run a PCR on the DNA sample and run a gel to test the cluster.

Category: Yang-En Yu | LEAVE A COMMENT