November 20

Preparing a TEM Grid (11/19/18)

Rationale:

A TEM grid will be prepared, so a TEM of Ferranti can be taken next time.

Procedure:

  1. Placed a slip of parafilm paper inside a petri dish.
  2. Pipetted 15 µL of the HTL, 20 µL of DI water twice, and 20 µL of uranyl acetate onto the parafilm.
  3. Placed the copper mesh TEM disc in the HTL drop for 5 minutes, then in the DI water drop for 2.5 minutes, then in the other DI water drop for 2.5 minutes, then in the uranyl acetate drop for 1 minute and 15 seconds.
  4. Blotted the copper mesh TEM disc with filter paper.
  5. Stored the copper mesh TEM disc in A8 in the TEM grid.

Observations:

  • The disc was placed shiny side down on all the drops.
  • The lysate drop appeared to have a faint crystal goldish color. The DI water drops were clear. The uranyl acetate drop had a faint lime green tint. The picture below shows the drops.

  • When placing the copper mesh TEM disc into the A8 spot, the disc crinkled up on itself.

Next Steps:

The TEM grid will be viewed under a TEM. If the TEM does not turn out well, another copper mesh TEM disc will be prepared to view the lysate. Also, the DNA extraction procedures will be completed.

Category: Kathryn Adkins | LEAVE A COMMENT
November 20

11/19 Webbed Plate and Titer calculations

Rationale: Calculate titer from spot titer experiment. Webbed plates made to yield more lysate so each member who contributed to obtaining a high titer lysate could use 10mL of lysate.

Procedure:  Before the experiment was performed, the workspace was cleaned with both Cidecon and 70% Ethanol. Top agar solution made in a 50mL vial using the formula below (6 plates for experiment and one for control):

  • 2mL LB Broth (x7)
  • 2.5mL 2X TA (x7)
  • 22.5µL CaCl2 (x7)
  • lysate
    • 10µL of Flooded lysate x8

4.5mL of this solution was added to a test tube containing 0.5mL Arthrobacter phage + 10µL of high titer lysate. This solution was quickly poured on to a plate, sat for 15 minutes and quickly placed in the incubator for 24 hours at 27 degrees Celsius.

Observations: Spot titer experiment performed on 11/16 during open lab results were used to help calculate titer. The formula used below to calculate titer below:

(22pfu/10µL) x (1000µL/1µL) x 10^8 = 2.2 x 10^11

Leaf for ML soil B was obtained to characterize tree. Tree classified as Quercus palustris.

Conclusions: High titer obtained, but the lysate was used to perform webbed plates. The experiment performed on 11/19 will be flooded on 11/20 and titer will have to be recalculated on 11/26 since the new obtained flooded lysate x9 has different phage concentrations.

Category: Michael Lum, Uncategorized | LEAVE A COMMENT
November 20

Lab Day 27: Serial Dilutions, Plating, Calculations

Rationale

Due to  possible miscalculations from previous titer and webbing, I had to redo the same serial dilution and plate only 10^-3 through 10^-5. From result from previous lab day, no plaque present. From the lab day before that, phage was present in the 10^4 plate. I chose to redo it because new and correct calculations are needed for a new webbed plate.

Detailed Procedure

  1. 10 µL of “SJ 11/12/18 FS lysate 100” was added with 90 µL of phage buffer to create 10-1 dilution which was vortexed.
  2. 10 µL of 10-1 was added with 90 µL of phage buffer to create 10-2 dilution which was vortexed.
  3. 10 µL of 10-2 was added with 90 µL of phage buffer to create 10-3 dilution which was vortexed.
  4. 10 µL of 10-3 was added with 90 µL of phage buffer to create 10-4 dilution which was vortexed.
  5. 10 µL of 10-4 was added with 90 µL of phage buffer to create 10-5 dilution which was vortexed.
  6. Took 50 µL of 10-3 into 0.5 mL of Arthro. Sat for 15 mins to infect.
  7. Took 50 µL of 10-4 into 0.5 mL of Arthro. Sat for 15 mins to infect.
  8. Took 50 µL of 10-5 into 0.5 mL of Arthro. Sat for 15 mins to infect.
  9. Took 8 mL of LB Broth, 10 mL of 2X TA, and 90 µL of 1M CaCl2 into a 50 mL vial.
  10. Took 4.5 mL of top agar mixture onto control plate. Allowed to solidify for 15 mins.
  11. Took another 4.5 mL of top agar mixture into separate 15 mL vial. Combined lysate and 10-3 mixture into same 15 mL vial. Swirled gently to mix and poured onto 10-3 plate.
  12. Took another 4.5 mL of top agar mixture into separate 15 mL vial. Combined lysate and 10-4 mixture into same 15 mL vial. Swirled gently to mix and poured onto 10-4 plate.
  13. Took remiaing 4.5 mL of top agar mixture and combined lysate and 10-5 mixture into same vial. Swirled gently to mix and poured onto 10-5 plate.
  14. Allowed all plates to solidify for 15 mins.
  15. Inverted all plates into incubator after 15 mins.

Conclusion

Current titer= 7.4 x 10^-6

Webbed calculations= 97.635 microliters of 10^-5 to web

Only 10^-3 through 10^-5 plates were made because the results from lab day 24 had plaque shown in the 10^-4 spot titer and made calculations based off from there. Since there might have been a miscalculation from that spot titer, individual plates were made from 10^-3 through 10^-5 to insure a more precise calculation. 10^-4 plate was used to calculate the web and current titer since 10^-5 only had 3 plaque present. In the next lab day,  I will be able to make a new webbed plate for it to be flooded.

contamination of control plate, and no phage present on 10^-4 plate from lab day 24

no contamination on control plate, plaque present in 10^-3, 10^-4, and 10^-5 plates.

 

Category: Soo-Un Jeong | LEAVE A COMMENT
November 19

11-19-2018- Flooded plate extraction and Spot Testd

11/19/2018

Objective:

  • Extract lysate from the flooded plate
  • Do a serial dilution for each lysate
  • Make spot tests

Procedure:

  1. On 11/16/18, three plaque assay plates were flooded with 8 ml of phage buffer each. The plates were then placed in the fridge
  2. The phages buffer with phages from the plate was extracted and filtered from each plate.
  3. 100 μl of the lysate from each plate was transferred to three microcentrifuge tubes.
  4. 10 μl of lysate from each microcentrifuge tube was transferred to another microcentrifuge tube with 90 μl buffer.
  5. 10 μl of lysate from each diluted microcentrifuge tube was transferred to another microcentrifuge tube with 90 μl of phage buffer.
  6. 8 ml of LB broth is transferred to a conical vial.
  7. 90 μl of CaCl2 was added to the vial as well.
  8. 10 ml of 2X TA was added to the conical vial.
  9. 4.5 ml of the Top Agar mixture was added to each of the 3 ,0.5 ml arthrobacter test tubes.
  10. The mixture is then plated on 3 agar plates. 4.5 of the Top Agar mixture was added to another plate for a control.
  11. Divide the three plates with arthrobacter into 4 sections.
  12. After Agar has solidified, spot each plate with the dilutions of the respective lysates and phage buffer.
  13. The plates were allowed to absorb the spots for 10 minutes and were then placed in the incubator.

Analysis And Conclusion:

the plates were flooded so that we can have more lysate. the spot tests will now be used to calculate the titer of the lysate on hand.

Category: Uncategorized | LEAVE A COMMENT
November 19

11.19.18 TEM Preparation and Redo of Titer Calculation

11.19.18 TEM Preparation and Redo of Titer Calculation

Rationale: Since the titer calculation plate from the last lab did not display results due to a slipped top agar, it was found to be necessary to redo this calculation to ensure a high titer lysate was present. However, it was found to be possible to continue with the procedure of TEM as each other individual that used Claire’s base lysate had found a high titer lysate from the last round of amplification. Therefore, since we all did similar procedures with similar results, it was concluded that there was a possibility that I too had a high titer lysate.

Procedure:

  1. Aseptic zone established
  2. 100µL of lysate was added to a microcentrifuge tube
  3. Parafilm was placed on petri dish
  4. 15µL of lysate was added to the parafilm at the center
  5. 15µL of water was added to the parafilm to the right of the previous drop two times
  6. Uranyl Acetate was added to the parafilm at the rightmost point.
  7. Copper mesh was placed on the lysate for 5 minutes
  8. Mesh was transferred to first water drop for 2.5 minutes
  9. Step 8 was repeated with second water drop
  10. Mesh was transferred to uranyl acetate for 1 minute, then dried with filter paper. Placed in B8.
  11. 2mL of LB Broth was added to a tube with 0.5mL of arthrobacter and an empty conical tube
  12. 22.5µL of CaCl2 was added to both tubes
  13. 2.5mL of 2X Top Agar was added to both tubes, then the solutions were swished and plated on respective tubes.
  14. Original lysate was used to create dilutions from 10^0 through 10^-9. These lysates were added to labeled sections on the experimental plate in 5µL drops
  15. Plates were incubated overnight.
  16. Station was cleaned

Results:

  • Plate prepared on Wednesday (11/14) that was intended to show the titer of the lysate had issues with the consistency of the top agar. This was predicted to be due to the temperature of the agar when it was used. The plates created today seemed to confirm that the top agar had been allowed to cool for too long, which likely caused the problems with Wednesday’s plate. The same problem was not encountered today.
  • Lysate titer will be reported as the plate created in this lab session is examined this week.

Observations:

  • Top Agar and LB Broth was very clear and was made fresh. This will help reduce the chances of contamination.
  • Drops did not settle into plate for spot test. Therefore, it was found to be necessary to place the plate in the incubator right side up with instructions to avoid flipping or moving the plate to give the drops time to settle.
  • With the TEM preparation, there seemed to be confusion regarding shiny-side up and shiny-side down with the copper mesh. Any adverse results could have been caused by this confusion.

Conclusions:

  • Since the top agar worked today after being taken fresh from the incubator rather than from the table, it can be concluded that the temperature caused the problems that were seen in the previous top agar. The TEM will allow for bacteriophage visualization, and the plate run today will confirm whether or not a high titer lysate has been used for the TEM.
Category: Uncategorized | LEAVE A COMMENT
November 19

Making Webbed Plates and Setting up for TEM 11/19/18

Rationale: Since I need at least 12.8mL of lysate for DNA extraction and archiving I need to make some webbed plates to flood. I calculated that I need 8µL of lysate diluted to the 10^-5 to  web a plate, so I will plate three  plates with  that amount.

Procedure:

  1. Put parafilm on a plate and pipetted 15µL of lysate onto the parafilm.
  2. Pipetted 20µL of water twice onto the parafilm.
  3. A TA aliquotted uracyl acetate onto the parafilm
  4. Placed a TEM stage from section A10 onto the lysate and let sit for 5 minutes.
  5. Transferred stage to water and let sit for 2.5 minutes.
  6. Repeated for the second drop of water.
  7. Transferred stage to uracyl acetate for 1 minute then took off and placed back into container on the side to be used for TEM.
  8. Pipetted 8µL of 10^-5 lysate into 0.5mL of arthrobacter 3 times and let sit for 30 minutes.
  9. Made top agar with 8mL of LB broth, 90µL of CaCl2, and 10 mL of 2X TA.
  10. Plated control with 4.5mL of solution.
  11. Aliquot 4.5mL of solution into each tube of enriched lysate and arthrobacter.
  12. Mixed by pipetting  up and down then plated each.
  13. After letting solidify inverted and placed into incubator for 24 hours.

Observations:

The plate wasn’t fully lysed because the plaque morphology isn’t uniform. I placed back in the incubator over break in hopes of getting a slightly more webbed plate.

Conclusions and Next Steps: This means that there is more than one type of phage in  my  lysate. However, I do not have time to run another round of purification. This means that I will just have to keep running with my current lysate and acknowledge that there are two phages in my lysate when I extract DNA. From there I can differentiate between the two phages.

Category: Sriram Avirneni | LEAVE A COMMENT
November 19

TEM Grid Preparation 11.19.18

Rationale:

To prep a TEM grid with my high titer lysate in order to image the phage it is composed of.

Procedures:

  1. Transferred 15µL of NMN CEW Lysate 10^0 to a piece of parafilm in a plate.
  2. Transferred 15µL of water onto the same piece of parafilm.
  3. Repeated step two for an addtional dot of water.
  4. Added one drop of Uranyl Acetate onto the same piece of parafilm.
  5. Put a copper mesh TEM disc shiny side down onto the lysate drop for 5 minutes.
  6. Transferred the disc to the first drop of water for 2.5 minutes.
  7. Transferred the disc to the second drop of water for 2.5 minutes.
  8. Transferred the disc to the drop of Uranyl Acetate for 1 minute.
  9. Blotted the disc on filter paper and stored for later use with the microscope.

Observations:

There was some questioning regarding if we should have blotted the disc to remove the excess uranyl acetate.

Next Steps:

The next step will be to use the electron microscope to image the phage contained on the copper disc. Additionally, we will also begin the process of prepping the lysate for DNA extraction and analysis.

Category: Nathan Newton | LEAVE A COMMENT
November 19

November 19, 2018 Webbing Plates- Soil C

Rationale: The purpose of this lab is to create webbed plates to create more lysate at a higher titer.

Description of Procedures:

  1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
  2. The titer from the spot titer test was determined to be 7.5 x 10^8.
  3. Top agar was made for 5 plates. 10 ml LB and 112.5 ul of CaCl2 were added to a tube.
  4. 10 ul of the 10^-4 dilution of lysate 1 was added to four tubes of 0.5 ml of arthrobacter and allowed to sit for 10 minutes.
  5. 12.5 ml of 2x TA was added to the top agar solution and pipetted up and down to mix. 4.5 ml of the solution was added to each of the arthrobacter tubes and then poured directly onto plates labeled LIP 11-19-18 PA web. The rest of the solution was poured onto a plate labeled LIP 11-19-18 Control.
  6. The plates were allowed to sit 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:

  • Bubbles were seen on the control plate

Interpretations/Next Steps:
The procedure was complete. The next step will be to flood the plates and collect the lysate.

 

Titer Test and Plates Made:

Category: Lucy Potts | LEAVE A COMMENT
November 16

Dilutions and Titers

Shepard Saabye
November 14th, 2018
SEA PHAGE Lab Journal

Results from the Previous Lab: The control plate was contaminated, but interestingly, it was not contaminated when Dr. Adair looked at it the day prior. I have no way to interpret that information. Another plate had some plaques form but due to contamination, Cooper and Michael’s plates were used for a plaque assay 

Objectives and Rationale: Plate Serial Dilutions in order to determine the titer of the lysate and web a plate

Procedure:

  • Established Aseptic Zone
  • Diluted lysate out to 10^-4 of the original lysate
  • Obtained 3 vials of .5 mL Arthrobacter
  • Added 10 mL LB Broth, 12.5 mL 2X TA and 112.5 uL CaCl to a single 50 mL tube
  • Added 10 uL of 10^-2, 10^-3, and 10^-4 Lysate to their own respective Arthro tubes
  • Added 4.5 mL of Agar mixture to each Arthro tube, and plated
  • Plated remaining 4.5 mL Agar mixture on the control plate
  • Waited 15 min for Top Agar to solidify
  • Left plates in the incubator at 24.5 degrees Celsius

Future Plans: Calculate Titer and continue increasing the concentration of the phage in each lysate

Category: Uncategorized | LEAVE A COMMENT
November 16

Titers and Dilutions

Shepard Saabye
November 12th, 2018
SEA PHAGE Lab Journal

Results from the previous lab: One of the plates was close to webbed. The control came out a little contaminated.

Rationale and Objectives: Today the goal was to generate a new lysate, and have it prepared to calculate titer by the next lab. This is to increase the titer high enough to complete TEM.

  • Procedure:
  • Established Aseptic Zone
  • Flooded webbed plate with 5 mL Phage Buffer
  • Let sit on moving platform for 1 hour
  • Diluted lysate out to 10^-3 of the original lysate
  • Obtained 3 vials of .5 mL Arthrobacter
  • Added 10 mL LB Broth, 12.5 mL 2X TA and 112.5 uL CaCl to a single 50 mL tube
  • Added 50 uL of 10^0 Lysate to the first Arthro tube
  • Added 10 uL 10^-2 Lysate to the second tube
  • Added 10 uL 10^-3 Lysate to the third tube
  • Added 4.5 mL of Agar mixture to each Arthro tube, and plated
  • Plated remaining 4.5 mL Agar mixture on the control plate
  • Waited 15 min for Top Agar to solidify
  • Left plates in the incubator at 24.5 degrees Celsius

Future PLans: use the results from this lab to determine the titer of our lysate, and decide if that is high enough to continue with TEM.

Category: Uncategorized | LEAVE A COMMENT