November 30

TEM and Making a Precipitate 11/28/18

Rationale

Today we will conduct TEM to image our phage. We will also make a precipitate using our lysate.

Procedure

  • Established an aseptic zone.
  • 10 mL of lysate was poured into a tube to begin making a precipitate. 40 µL of nuclease was added to the tube and it was inverted 10 times. 4 mL of phage precipitant solution was added and the tube was inverted once to mix. It was placed in the incubator for 25 minutes.
  • TEM was performed during the incubation period and a grid was prepared.
  • 1 square of parafilm was prepared by aliquotting 20 µL of lysate onto the square, 20 µL of D.I water, another 20 µL of D.I. water, and 20 µL of uranyl acetate in sequential drops.
  • A TEM grid was placed into the lysate for 5 minutes, each D.I. water drop for 2.5 minutes, and the uranyl acetate for 1 minute.
  • Filter paper was used to wick away the excess liquid from the grid and the grid was imaged.
  • The precipitate preparation in the incubator was removed after 25 minutes and left at room temperature for 40 minutes.

Observations

The last spot titer test led to inconclusive results. The 10^-1, 10^-2, and 10^-3 quadrants had completely lysed, however, all the quadrants following did not produce any plaques. This resulted in the titer of our lysate being unknown. However, due to the titer of the current lysate being 1.2×10^7, it was decided to continue onto TEM.

After imaging, the head of the phage was recorded to be 53 nm in width. The tail was recorded to be 110 nm in length.

The centrifuge swinging bucket was not functioning, resulting in precipitate preparation to not be completed.

Conclusion/Next Steps

The precipitate preparation will be completed and DNA extraction will begin. A spot titer test will also be conducted to determine the titer of the lysate.

Category: Emily Gaw | LEAVE A COMMENT
November 30

11/26/18 TEM and DNA Extraction Day 2

Rationale:

The purpose of today’s lab was to observe the purified phage sample under a TEM, and extract the DNA from the pellet acquired previously.

Results from 11/19/18:

  • A TEM grid was prepared to be ready for viewing.
  • Phage particles were pelleted and extracted from a centrifuge, they were left to freeze for the week to preserve the phage structure.

  • Phage Pellet and Supernatant 11/19/18

Materials:

  • TEM Grid
  • Pellet of Phage Material
  • Sterile H2O
  • DNA Resin at 37 °C
  • 80% Isopropanol
  • Elution Buffer
  • Vacuum Manif0ld

TEM Procedure:

  1. Previously prepped TEM grid was loaded into the microscope.
  2. The sample was then adjusted and analyzed until acceptable phage was found.
  3. Measurements were taken of the head and tail of the phage, and a picture was taken for data.

DNA Extraction Procedure:

  1. Began with re-suspending the phage pellet using 500 µl of sterile H2O.
  2. Once re-suspended, 2 ml of DNA clean-up resin was added to the pellet and gently mixed.
  3. After adding the resin, the mixture was equally separated into 2 microcentrifuge tubes and set into the centrifuge at 12,500 xg for 3 minutes to spin.
  4. Microcentrifuge tubes were then removed and contained a cloud of genetic material at the bottom, with a clear liquid on top. The clear liquid (resin) was removed using various micropipettes, careful not to disturb the DNA cloud.
  5. Once the liquid was removed from both tubes, 1 ml of 80% isopropanol was added to each tube and shaken to combine before adding the tubes back into the centrifuge for 3 minutes at 12,500 xg.
  6. The tubes were removed and step 4 was repeated with the DNA sample
  7. 1 ml of 80% isopropanol was then again added to each tube and combined, and poured the mixture into two separate vacuum columns under the fume hood to filter the DNA and resin out from the mixture.
  8. Once filtered, the DNA and resin were added back to the centrifuge to spin, this time for 5 minutes at 12,000 xg.
  9. After removing the microcentrifuge tubes, 50 µl of elution buffer was added to each of the tubes, and they were spun again for 5 minutes at 12,000 xg.
  10. The tubes were removed, and the extracted DNA was combined into one microcentrifuge tube before being taken to the Nanodrop machine.

Nanodrop Procedure:

  1. First the Nanodrop machine was cleaned with sterile wipes, both the bottom and top sensors.
  2. An elution buffer blank was run first by spotting 2 µl of elution buffer onto the Nanodrop sensor, then the arm was slowly lowered and the blank was run.
  3. After running the blank, the machine was cleaned again, and 2 µl of the extracted DNA was spotted onto the sensor and the machine was run to analyze the DNA.

Results/Data:

  • For TEM, it appeared that the mass majority of the phages found in the sample had their heads separated from the tails.
  • For the intact phage that was found, it had a capsid size of approximately 50 nanometers, with a tail size of 174 nanometers.
  • DNA extracted was a clear liquid, could not see any individual parts of it within the elution buffer.
  • The Nanodrop results for the DNA extracted was 805.8 micrograms per ml of DNA

Conclusions:

It is unclear what exactly is causing the damage to the phages which is separating the heads from their bodies. It could be due to improper storage or poor TEM prep that is damaging the phages before they can be seen under the microscope. Several phage were found with portions of their tails missing, but the head size was consistent throughout. Also, the 805.8 micrograms per ml of DNA suggest their is a significant amount of genetic material extracted, it is unclear if it is phage or bacterial DNA, and PCR and gel electrophoresis is needed.

Next Steps:

The next steps for this experiment are to run a PCR on the extracted DNA and prep a gel for gel electrophoresis.

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November 30

Spot Titer Test 11/26/18

Rationale

Today we will perform a spot titer test on the flooded lysate to determine if our lysate is a high titer.

Procedure

  • Established an aseptic zone.
  • 90 µL of phage buffer was aliquotted into 8 microcentrifuge tubes. 10 µL of flooded lysate was pipetted into the microcentrifuge tube labeled 10^-1. The tube was vortexed for a few seconds to mix.
  • 10 µL of the contents in the microcentrifuge tube labeled 10^-1 were pipetted into the microcentrifuge tube labeled 10^-2. The tube was vortexted for 10 seconds to mix.
  • 10 µL of the contents in the microcentrifuge tube labeled 10^-2 were pipetted into the microcentrifuge tube labeled 10^-3. The tube was vortexted for 10 seconds to mix.
  • 10 µL of the contents in the microcentrifuge tube labeled 10^-3 were pipetted into the microcentrifuge tube labeled 10^-4. The tube was vortexted for 10 seconds to mix.
  • 10 µL of the contents in the microcentrifuge tube labeled 10^-4 were pipetted into the microcentrifuge tube labeled 10^-5. The tube was vortexted for 10 seconds to mix.
  • 10 µL of the contents in the microcentrifuge tube labeled 10^-5 were pipetted into the microcentrifuge tube labeled 10^-6. The tube was vortexted for 10 seconds to mix.
  • 10 µL of the contents in the microcentrifuge tube labeled 10^-6 were pipetted into the microcentrifuge tube labeled 10^-7. The tube was vortexted for 10 seconds to mix.
  • 10 µL of the contents in the microcentrifuge tube labeled 10^-7 were pipetted into the microcentrifuge tube labeled 10^-8. The tube was vortexted for 10 seconds to mix.
  • 4 mL of LB Broth, 5 mL of 2X TA, and 45 µL of CaCl2 were combined in a vial. 4.5 mL of the mixture was combined with 0.5 mL of Arthrobacter and was poured onto a plate. The remaining 4.5 mL of the mixture was combined with another 0.5 mL of Arthrobacter and was poured onto another plate. The plates sat aside for 10 minutes to solidify.
  • The 5 µL of each microcentrifuge tube was spotted onto a designated quadrant. 5 µL of phage buffer was spotted onto a control quadrant.
  • The plates set aside for 10 minutes and then placed into the incubator.

Observations

No air bubbles were observed in the process of making the plates.

Conclusions/Next Steps

The results of the plate will be observed on 11/28/18 and the titer of our lysate will be calculated.

Category: Emily Gaw | LEAVE A COMMENT
November 29

Lab Day 31: Titer and web calculations, Webbed plates

Rationale

New titer and web calculations were made since titer is not high enough. Made more webbed plates for new calculations for titer.

Detailed Procedures

  1. Took 0.5 mL Arthro and added 10 microliters of 10^0. Sat for 15 mins.
  2. Took 4 mL of LB Broth, 5 mL 2X TA, and 45 microliters of 1 M CaCl2 into a 15 mL vial.
  3. Took 4.5 mL of top agar mixture and poured onto a plate labeled as “control.”
  4. Took rest of 4.5 mL top agar mixture and poured into a 15 mL vial labeled as “10 microliters.”
  5. Took mixture from step 1 and added to 15 mL vial labeled as “10 microliters.”
  6. Took “10 microliter” vial and poured onto “10 microliters” plate.
  7. Let both plates solidify for around 15 mins and inverted into incubator.

Results

Lab Day 31 Current titer= 1.1 x 10^8

Lab Day 31 web= 0.2627 microliter of 10^0

Conclusion

Plates from lab day 30 did not have any contamination. Plaque size compare to webbed plates from a while ago seems to get smaller. Since pipettes can’t measure 0.2627 microliters, used 10 microliters instead. Since it is the last day, flooding and serial dilutions cannot be made and continue DNA extraction, TEM, or nanodrop. This phage was from a donor, results will be looked over by the donor and compare results.

plaque size is small, used a microscope and found 11 pfu on 10^-5 spot

 

newly webbed plates and control

Category: Soo-Un Jeong | LEAVE A COMMENT
November 29

DNA Extraction Setup

Title: DNA Extraction Setup

Date: 28 November 2018

Rationale/Past Results: The spot titer yielded a growth of one plaque on the 10^-7 spot. This means that the official titer is 1.0 x 10^9. The lysate will then be archived and prepared for DNA extraction. Enzymes and a precipitant will be added to the mixture and it will be incubated in various conditions as well as centrifuged in order to obtain a pellet of phage DNA. Below is a picture of the spot titer result:

Procedure: 

  • 2.8 mL of high titer lysate was aseptically transferred to a 15 mL conical vial for archiving.
  • 10 mL (x4) was transferred to 4 different 50 mL conical vials in order to prepare the lysate for DNA extraction
  • The following was added to the 10 mL of high titer lysate:
    • 40 uL combined DNase 1 + RNase A
    • 4 mL phage precipitant solution
  • The mixture was then incubated at 37 degrees Celsius for 30 minutes
  • The mixture was then removed from the incubator and left at room temperature for 45 minutes
  • The mixture was then spun in a centrifuge for 20 minutes at 10,000 xg
  • The liquid was decanted and the resulting pellets dried and placed into a micro centrifuge tube to be extracted.

Conclusions: This process produces a pellet that contains phage DNA that will be extracted. THe extracted DNA will then be able to be analyzed and characterized in the future.

Category: Uncategorized | LEAVE A COMMENT
November 29

Spot Titer for New Lysate

Title: Spot Titer for New Lysate

Date: 26 November 2018

Rationale/Past Results: About 43 mL of new lysate was acquired through plate flooding (“Flood Lysate #9”) that will be spot titered and run under a TEM in order to discover the official titer and investigate the individual phage, respectively. If the titer is confirmed high, then the lysate will be archived and prepped for DNA extraction.

Procedure: Under an aseptic zone…

  • One spot plate was run in order to determine titer
  • Dilutions of “Flood Lysate #9” were done through the following method
    • 10 uL of original flood lysate was transferred to a new microcentrifuge tube containing 90 uL Phage Buffer. This mixture was designated as the 10^-1 concentration dilution
    • 10 uL of the 10^-1 concentration was then transferred to another microcentrifuge tube containing 90 uL phage buffer. This mixture was designated as the 10^-2 concentration dilution.
    • This process was repeated until a 10^-9 concentration was made.
  • The plate was divided into 6 regions: a control, 10^-5, 10^-6, 10^-7, 10^-8, 10^-9 and labelled accordingly
  • The following recipe was used for the spot plate:
    • 2 mL LB Broth
    • 2.5 mL 2x Top Agar
    • 22.5 uL CaCl2
    • 0.5 mL Arthrobacter culture

~5 mL of mixture was then plated and cooled. 10 uL of phage buffer was spotted onto the control spot while 10 uL each of the 10^-5 through 10^-9 dilutions were spotted in their respective locations. The plate was placed into the incubator.

Conclusions: If the titer is high enough (≥1.0 x 10^8), then the lysate can be named and archived, and its phage DNA extracted through pellets. A TEM grid can also be run on the phage in order to investigate more closely its characteristics.

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November 29

11.28.18 Finishing DNA Extraction

11.28.18 Finishing DNA Extraction

Rationale: Since a pellet had been obtained through the first part of DNA extraction, it was possible to finish the process of DNA extraction. Therefore, the next logical step was found to be the second half of the procedure that ended with measuring the sample using the nanodrop.

Procedure:

  1. 0.5mL sterile water added to thawed phage pellet
  2. Pellet was resuspended
  3. 2mL of resin was added to the pellet, then the tube was inverted many times to mix
  4. Resin and re-suspended phage pellet was added to two distinct microcentrifuge tubes (split evenly)
  5. Tubes were centrifuged at 12,500g for 3 minutes
  6. Supernatant was removed with pipette and placed in waste
  7. 1mL of 80% isopropanol was added to each microcentrifuge tube
  8. Steps 5-7 were repeated twice
  9. 1mL of 80% isopropanol was added to the pellet to re-suspend
  10. Contents of tubes were added to a column in a vacuum to move DNA to the filter
  11. Column was added to microcentrifuge tube, then centrifuged for 5 minutes at 12,000g
  12. Column was moved to new microcentrifuge tube
  13. 100µL of Elution Buffer (85 degrees Celsius) was added to column, then centrifuged for 1 minute at 12,000g
  14. DNA quantified on nanodrop
  15. Cleaned station

Observations

  • Pellet was initially very difficult to see, which led to doubt about whether or not DNA was strongly present
  • Nanodrop was very sensitive and difficult to place drop on platform without missing or creating air bubbles
  • Pellets after centrifuging formed more of a cloud shape rather than solid in bottom of tube, which made it more difficult to remove the supernatant clanly.

Results

  • The DNA concentration present was 65.69µg/µL. The 260/280 ratio was 1.89, and 260/230 was 0.16.

Conclusions

  • Since the concentration of DNA was above 50, the sample theoretically has a chance to work using the PCR procedure to cluster the phages. The ideal number would have been closer to 500µg/µL, but the sample should still be able to proceed. Furthermore, the ratios found in the results section illustrate that the sample is relatively pure and that the isolation was done reasonably accurately, as the 260/280 ratio should have been close to 2, and it was. The next step will be to process this sample using PCR clustering to see whether or not the procedure will work with the lower concentration and to determine specifics about the phage’s DNA.
Category: Henry Burns, Uncategorized | LEAVE A COMMENT
November 29

11-28-18- Picked old plaques and made a plaque assay

11-28-18

Objective:

To pick 3 plaques and make a plaque assay from the extracted phages.

Pre-Lab Observation:

The plaque assays prepared on 11-26-18 seemed to have a regrowth in Arthrobacter and can therefore not really be used. it is difficult to state whether plaques had formed. Because all the lysate was used, another plaque assay cannot be made. therefore, in a final attempt, an older plate was acquired and plaques were picked.

Procedure:

  1. The aseptic zone was set up.
  2. 3 plaques were picked from the old plaque  assay and the phages were deposited in a microcentrifuge tube with 30μl of phage buffer.
  3. 30 μl of this phage extract was deposited in 0.5 ml of arthrobacter and was allowed to enrich for 15 minutes.
  4. 1 TA mixture was prepared for three plates.
  5. 6 ml of LB broth was transferred to a conical vial.
  6. 67.5 μl of CaCl2 was added to the conical vial.
  7. After the lysate was allowed to enrich for 15 minutes, 7.5 ml of 2X TA was added to the conical vial.
  8. 4.5 ml of the TA mixture was added to the arthro tube.
  9. The contents of the tube were then poured onto the agar plate.
  10. 4.5 ml of the 2X TA mixture was plated on an agar plate for a control.
  11. the plates were allowed to solidify for 15 minutes.
  12. the plates were then placed in the incubator.

Analysis and Conclusion:

This is the final attempt. If the plate is webbed, it can be flooded and process for DNA extraction can be started.

Category: Aman Patel, Dr. Adair | LEAVE A COMMENT
November 29

DNA Extraction and Nano Drop 11.28.18

Rationale:

To finish extracting the DNA from the pellet frozen on 11.26.18 and to determine the concentration of the DNA via nanodrop.

Procedures:

  1. Defrosted the pellet and added .5mL of sterile water to redissolve the DNA into solution.
  2. Added 2mL of 37˙C resin to the vial and mixed.
  3. Split the contents of the vial into two microcentrifuge tubes.
  4. Centrifuged at 12500g for 3 minutes, removed the supernatant.
  5. Added 1mL of 80% isopropanol alcohol and shook to dissolve the pellet.
  6. Repeated steps 4-5 two more times.
  7. Added 1mL of 80% isopropanol alcohol and shook to dissolve the pellet.
  8. Added the contents of both tubes to separate vacuum columns and vacuumed.
  9. Dried each column filter by centrifuging for 5 minutes at 12,000g in another microcentrifuge tube.
  10. Added 100µL of elution buffer to each filter and transferred to another microcentrifuge tube and centrifuged at 12000g for 1 minute.
  11. Combined the contents of both tubes into one new microcentrifuge tube and labeled “NMN 11.28.18 Pippa”.
  12. Used 2µL of the DNA to conduct a Nanodrop test

Observations/Data:

When redissolving my pellet several chunks remained undissolved. The nanodrop results were 138.92 for nucleic Acid(ng/uL) with an A280/A260 ratio of 1.922.

Conclusions/Next-Steps:

From the nanodrop test, it can be concluded that I have a fairly high concentration of DNA from the phage “Pippa”, which can now be used to run a gel electrophoresis test this week and to sequence the genome in the future.

Category: Nathan Newton | LEAVE A COMMENT
November 28

28 November 2018 Spot Titer Test and TEM – Soil C

Rationale: The purpose of this lab is to collect a high titer lysate and perform a spot titer test to calculate the titer, start the DNA extraction process, as well as perform TEM with the phage.

Description of Procedures:

  1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
  2. The webbed plates were flooded with 8 ml of phage buffer and put in the refrigerator overnight.
  3. Lysate was collected from the webbed plates and filtered using a 0.22 um top filter.
  4. 35 ml was collected and separated into two tubes for two samples.
  5. 10 ml of lysate was poured into a separate tube for DNA extraction. 40 ul of nuclease was added and the tube was inverted 10 times. 4 ml of PEG was then added, the tube was inverted once and then put in the incubator at 37 C for 25 minutes.
  6. The tube was then allowed to sit at room temperature for 40 minutes.
  7. While waiting, electron microscopy was performed. A TEM grid was made with 20 ul of lysate, two 20 ul dots of DI water, and a drop of uranyl acetate. The TEM grid was allowed to sit in the lysate for 5 minutes, and water for 2.5 minutes. It was then allowed to sit in the UA for 1 minute and then dried.
  8. The image of the phage showed it to have an average head size of 58 nm and a tail of 160 nm.
  9. A spot titer test was performed to calculate the titer of the lysate collected, with dilutions out to 10^-7. A 5 ml plate was made and inverted and stored in the incubator until the next lab. It was labeled LIP 11-28-18 STT (Spot Titer Test)
  10. The workstation was cleaned using aseptic technique and materials were properly stored and disposed of.

Observations/Results:

  • Head of phage: 58 nm
  • Tail of Phage: 160 nm
  • 35 ml of lysate collected
  • Bubbles seen on the control plate.

Interpretations/Next Steps:
The procedure was complete for TEM and spot titer test, but the DNA extraction was not complete. The lysate was not spun in the centrifuge due to an error with the machine and will have to be performed in the next lab. The next step will be to finish DNA extraction and possibly run PCR.

 

Flooded Plates and Image of Phage:

 

Category: Lucy Potts | LEAVE A COMMENT