November 28

Lab Day 30: Flooding, Serial Dilution, Spot Titer

Rationale

5 microliters and 203 microliters plates were flooded and had serial dilutions performed up to 10^-12. A spot titer was performed to prevent making 13 plaque assay plates.

Detailed Procedure

  1. Add 8 mL of PB to  203 microliters  plate from previous lab day.
  2. Add 5 mL of PB to  5 microliters  plate from previous lab day.
  3. The plates was shaken on an incubator for one hour.
  4. Filtered lysate from flooded plate in two separate 50 mL 0.22 µm filtered conical vial. Labeled “SJ 11/28/18 FS 2 lysate 100” and “SJ 11/28/18 FS 3 lysate 100.”
  5. 10 µL of “SJ 11/28/18 FS 2 lysate 100” was added with 90 µL of phage buffer to create 10-1 dilution which was vortexed.
  6. 10 µL of 10-1 was added with 90 µL of phage buffer to create 10-2 dilution which was vortexed.
  7. 10 µL of 10-2 was added with 90 µL of phage buffer to create 10-3 dilution which was vortexed.
  8. 10 µL of 10-3 was added with 90 µL of phage buffer to create 10-4 dilution which was vortexed.
  9. 10 µL of 10-4 was added with 90 µL of phage buffer to create 10-5 dilution which was vortexed.
  10. 10 µL of 10-5 was added with 90 µL of phage buffer to create 10-6 dilution which was vortexed.
  11. 10 µL of 10-6 was added with 90 µL of phage buffer to create 10-7 dilution which was vortexed.
  12. 10 µL of 10-7 was added with 90 µL of phage buffer to create 10-8 dilution which was vortexed.
  13. 10 µL of 10-8 was added with 90 µL of phage buffer to create 10-9 dilution which was vortexed.
  14. 10 µL of 10-9 was added with 90 µL of phage buffer to create 10-10 dilution which was vortexed.
  15. 10 µL of 10-10 was added with 90 µL of phage buffer to create 10-11 dilution which was vortexed.
  16. 10 µL of 10-11 was added with 90 µL of phage buffer to create 10-12 dilution which was vortexed.
  17. 4 mL of LB Broth, 45 µL of CaCl2, and 5 mL of 2X TA were combined into a 15 mL vial.
  18. Transferred and mixed 4.5 mL of the Top Agar (TA) mixture from the conical vial into control plate labeled “SJ Control 11/28/18.”
  19. Added 0.5 mL Arthro into remaining 4.5 mL TA mixture and poured onto plate labeled “SJ Spot 11/28/18” which was sectioned off into 12 parts
  20. Waited 15 mins for both plates to solidify.
  21. Micropipetted 10 µL of 100, 10-1, 10-2, 10-3 ,10-4 , 10-5  , 10-6  , 10-7  , 10-8  , 10-9  , 10-10  , 10-11   , 10-12 in according sections of the spot test plate.
  22. Rested into incubator

Conclusion/Next Steps

203 microliter plate was completely lysed, which gives hope that a high titer will be achieved by next lab day. 5 microliter plate was nearly fully webbed, but not enough, so only 5 mL of PB was poured onto the plate. Each plate, after an hour, were filtered out in separate vials. Only the 203 microliter plate was used for serial dilutions because the plate was completely lysed. By next lab day, a new titer will be calculated and hope to achieve a high titer by then.

results from lab day 29 (below)

10^-1 through 10^-12 spot titer plate

Category: Soo-Un Jeong | LEAVE A COMMENT
November 28

TEM and DNA Extraction 11/28-29/18

Rationale: Now that I have enough lysate to run DNA extraction I can proceed with doing so and now I can view my TEM that I prepared a couple weeks ago.

Procedure:

Wednesday:

  1. Pipetted 10mL of lysate into a 50mL conical vial.
  2. Added 40µL of Nuclease Mix and gently shook the vial for about a minute.
  3. Added 4mL of Phage Precipitant Solution and gently shook once again.
  4. Incubated at 37°C for 30 minutes then took out of incubator and left at room temperature for 40 minutes.
  5. Centrifuged at 10,000g for 20 minutes.
  6. Decanted supernatant and stored pellet in freezer overnight.
  7. Since I already prepped the TEM last week I just had to go in and view my phage under  the microscope.

Thursday:

  1. Added 500µL of sterile water to pellet and re-suspended by pipetting up and down.
  2. Added 2mL of resin to re-suspended pellet and pipetted up and down to stir.
  3. Split up the solution into two microcentrifuge tubes and spun at 12.5g for 3 minutes.
  4. Decanted the supernatant and added 1mL of 80% isopropanol to each tube. Mixed by flicking tube and gently swirling.
  5. Spun at 12.5g for 3 minutes.
  6. Repeated steps 4 and 5.
  7. Decanted supernatant and added 1mL of 80% isopropanol. Re-suspended pellet.
  8. Poured each tube into separate vacuum filters.
  9. Added each column to a new microcentrifuge tube and spun at 12000g for 5 minutes.
  10. Took column out and placed into another set of new microcentrifuge tubes. Added 100µL of elution buffer to column and let sit for a minute.
  11. Spun at 12000g for 1 minute then placed the solution into one microcentrifuge tube. Stored at -20°C overnight.

Observations:

This image was produced using TEM. My phage has a tail length of 100nm.

Conclusions and Next Steps:
Now I need to find out how much nucleic acid is in my sample using the nanodrop machine. From there I will run PCR and gel electrophoresis to find out what cluster my phage is.

Category: Sriram Avirneni | LEAVE A COMMENT
November 28

Transmission Electron Microscopy

Objectives: Using TEM grid from one week prior, find and photograph novel phages.

Rationale: An important part of classifying the phages is observing their physical characteristics. TEM allows us to observe the phage at a visible level, and record it.

Procedure:

  • Removed the copper TEM grid from Monday, November 19th, from its grid location.
  • Using fine tweezers, placed the copper grid into vacuum tube.
  • Operated the machine to find three separate phages, taking photographs of all three
  • Removed grid from vacuum tube
  • Placed back into used TEM tray

Results:
We now have pictures of our phage.

Interpretations: Its uplifting to finally see the phage, especially in the quantities that we saw it.

Future Plans: DNA extraction next lab period!

Category: Uncategorized | LEAVE A COMMENT
November 27

11/26 Spot titer and TEM

Rationale: Spot titer performed to help calculate titer. The titer is unknown for the newly flooded lysate and high titer is needed to extract DNA from.

Procedure: Lysate was obtained from the refrigerator and was used for 11/26 experiment. TA solution was made, and serial dilutions were performed to perform a spot titer. 100µL of the flooded lysate was used for TEM image. The Formula used to make TA solution:

  • 2mL LB broth
  • 2.5mL 2X TA
  • 22.5µL CaCl2
  • 0.5 Artrhobacter

This TA solution was made in a 15mL vial and quickly poured onto the labeled plates. Plates sat for 15 minutes and then the serial diluted lysate were spotted onto the plates (10^-5 – 10^-9 and one control). The plate was placed in the incubator for 24 hours at 24 degrees Celcius and titer will be counted 11/27.

Observations: 45mL lysate obtained from 11/19 experiment. The lysate would then be used for DNA extraction for each member in group 4, but the titer of the new lysate is unknown. High titer must be obtained in order to extract DNA.

Conclusions: High titer calculations from 11/26 experiment will result in DNA extraction 11/28. If low titer, web plates and obtain newly flooded lysate, which would then need to be spotted to calculate titer.

 

Category: Uncategorized | LEAVE A COMMENT
November 27

Lab Day 29: Titer + web calculations, webbing plate

Rationale

New titer and web calculations were made since plate was not webbed at all. Made more webbed plates for flooding and serial dilutions on next lab day.

Detailed Procedures

  1. Took 0.5 mL Arthro and added 203 microliters of 10^0. Sat for 15 mins.
  2. Took another 0.5 mL Arthro and added 5 microliters of 10^0. Sat for 15 mins.
  3. Took 6 mL of LB Broth, 7.5 mL 2X TA, and 69 microliters of 1 M CaCl2 into a 50 mL vial.
  4. Took 4.5 mL of top agar mixture and poured onto a plate labeled as “control.”
  5. Took another 4.5 mL top agar mixture and poured into a 15 mL vial labeled as “203 microliters.”
  6. Took another 4.5 mL top agar mixture and poured into a different 15 mL vial labeled as “5 microliters.”
  7. Took mixture from step 1 and added to 15 mL vial labeled as “203 microliters.”
  8. Took mixture from step 2 and added to 15 mL vial labeled as “5 microliters.”
  9. Took “203 microliter” vial and poured onto “203 microliters” plate.
  10. Took “5 microliter” vial and poured onto “5 microliters” plate.
  11. Let all three plate solidify for around 15 mins and inverted into incubator.

Results

Current titer= 1.4 x 10^7

web= 2.02 microliter of 10^0

Made a miscalculation and did 203 microliters of 10^0, so made another plate with 5 microliters of 10^0  instead of 2.02 microliters.

Conclusion/Next Steps

Plates from lab day 28 did not have any contamination. Since 10^-5 lysate was used, the plaque size was very small in size and quantity, that is why a new titer calculation was made from that. 2.02 microliters of 10^0 was small, so I was advised to used 5 microliters instead. Hopefully a webbed plate will be ready by next lab day so that flooding and serial dilutions will take place and get a high titer by Friday.

results from lab day 28

Category: Soo-Un Jeong | LEAVE A COMMENT
November 27

DNA Extraction and TEM 11.26.18

Rationale:

To extract from DNA from the phage “Pippa” in order to analyze it and to image my TEM grid with “Pippa” on it.

Procedures:

  1. Setup an aseptic zone.
  2. Added 10mL of “NMN CEW Lysate 10^0 11.14.18” to a vial labeled “DNA Extraction”
  3. Added 40µL Nuclease Mix to the vial and mixed.
  4. Added 4mL Phage Precipitant Solution to the same vial.
  5. Incubated on shaker at 37˙C for 30 mins.
  6. Let sit for 40 minutes at room temp.
  7. Centrifuged at 20rpm for 20 minutes.
  8. Centrifuged at 7500rpm for 20 minutes.
  9. Poured off the supernatant from the pellet and froze the pellet.
  10. Imaged the phage “Pippa” via TEM.

Observations:

The vial “DNA Extraction” was erroneously centrifuged at 20rpm for 20 minutes instead of the necessary 7500 rpm for the same amount of time, the proper centrifugation was conducted immediately following the erroneous one. The imaged phage was approximately 26nm  in capsid diameter and 112nm in tail length.

Conclusions/Next Steps:

The next step will be to further prep the pelleted phage for DNA extraction which can then be analyzed for the entire genome of the phage “Pippa”.

Category: Nathan Newton | LEAVE A COMMENT
November 27

11/26/18 – Restart purification proccess

11/26/18

Objective:

  • Make a plaque assay from a picked plaque

Pre-Lab Observation:

The spot test prepared on 11/14/18 resulted in a weak titer or an absolute lack of plaque. Another group member was assigned to extract lysate from the same soil and make a plaque assay. plaques were picked from the plaque assay in this lab in an effort to restart purification.

Procedure:

  1. The aseptic zone was set up.
  2. A plaque was picked from the group members plaque  assay and the phages were deposited in a microcentrifuge tube with 50μl of phage buffer.
  3. 50 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

50 μl of the extract was used to possibly get a webbed plate. the probability of such an outcome is small. due to the lack of time, this would be the last chance for possibly getting to DNA extraction, which seems unlikely at this time.

Category: Aman Patel, Dr. Adair | LEAVE A COMMENT
November 26

11-26-18 — Titer Calculation and Plaque Assay

Date: Monday, November 26th, 2018

Title: Titer Calculation and Plaque Assay

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.
  3. The plaque assay from last lab had 133 plaques present, equivalent to 133 plaque forming units (pfu).
  4. Diameters of the plate and the plaques were measured using a ruler and a microscope.
    1. Plate Diameter: 85 mm
    2. Average Plaque Diameter: 1.5 mm
  5. The area of the plate and the plaques were calculated using A=πr^2
    1. Plate area: A=πr^2 =π(42.5)^2 =5674.5 mm^2
    2. Plaque area: A=πr^2 =π(.75)^2 =1.767 mm^2
  6. The area of the plate was divided by the area of the plaques in order to calculate how many plaques would be necessary to web a plate.
    1. (5674.5 mm^2)/(1.767 mm^2) =3211 pfu
  7. The titer of the lysate was determined using the formula Titer=(pfu x dilution factor x 1000 μL)/(μL lysate used x 1 mL)
    1. Titer =(133 pfu x 10^8 x 1000 μL)/(10 μL x 1 mL) =1.33 x 10^12 pfu/mL
  8. The volume needed to web a plate was determined using the following math:
    1. (3211 pfu x 1 mL x 1000 μL)/(1.33 x 10^12 pfu x 1 mL) =2 x 10^-6 μL lysate needed to web a plate
  9. Since the lysate was diluted by factors of 10, 2 x 10^-6 μL of 10^0 lysate equates to 20 μL of 10^-7 lysate.
  10. A plaque assay and top agar control were set up using the following formula:
    1. 4 mL LB broth
    2. 45 μL 1M CaCl2
    3. 5 mL 2x Top Agar
  11. 20 μL of the 10^-7 lysate was added to a culture tube containing .5 mL arthrobacter and left to infect for 15 minutes.
  12. 4.5 mL of top agar solution was added to a control plate.
  13. 4.5 mL of top agar solution was added to the culture tube solution and pipetted to mix before being transferred to a plate.
  14. The plates were left for 15 minutes to solidify, inverted, and placed in an incubator for the next 48 hours.

Observations: The top agar control from last experiment was free of contamination. The solution in the plate made during this lab session was touched by accident and therefore might have contamination. The arthrobacter may out-compete the contaminants. This will be evaluated next lab. The titer of the lysate is very high, at 1.33 x 10^12. A highly diluted sample will need to be used to web plates.

 

Results: This lab yielded a plaque assay that will be webbed if successful and a top agar control to be evaluated next lab.

Next Step: The next step is to evaluate the plates from this experiment and flood a plate if it is webbed. If the plate is not webbed, a titer calculation might need to be reevaluated and a new plaque assay will be made with a different volume of lysate in order to web one.

Category: Brandon Reider | LEAVE A COMMENT
November 26

11.26.2018 DNA Extraction Initiation

11.26.2018 DNA Extraction Initiation

Rationale: Since results observed by Lathan and sent to me showed that the lysate possessed had a titer of 1e9, the lysate was officially a high titer. Therefore, it is possible to perform the DNA extraction procedure on the lysate to progress with analysis of bacteriophage as part of Claire’s group. Also, since the TEM was available for use, it was found to be beneficial to visualize the bacteriophage using the microscope. The DNA extraction procedure will allow for the genome of the phage to be analyzed if it is selected by the class.

Procedure:

  1. Aseptic zone established
  2. 10mL of lysate labelled “HMB from CEW 10^0 Lysate 11/14/18” was placed into a conical tube.
  3. 40µL of nuclease mix was added to the lysate
  4. 4mL of phage precipitant solution was added to the lysate
  5. Lysate was placed on shaker at 35 degrees Celsius for 30 minutes
  6. Lysate was let sit at room temperature for 45 minutes
  7. Lysate was centrifuged at 7,500g for 20 minutes.
  8. Eliminated supernatant from sample and rinsed sink.
  9. Stored in refrigerator overnight.
  10. Cleaned bench.

Results:

  • These results date back to Monday, November 19 and the procedures performed on this day. As seen, the lysate was strong enough to completely lyse the spots through the 10^-5 dilution, and had plaques form through the 10^-7 dilution. After calculation, the lysate was found to be 1×10^9 pfu/mL. Therefore, a high titer lysate had been obtained! The control also showed no signs of contamination, which confirms that the results could be considered valid.
  • The electron microscope revealed very present bacteriophages. The phages had a head with a diameter of 52nm and a tail of 100nm.

Observations:

  • After the phage precipitant solution was added, the lysate appeared to have smaller particles or granules appeared to be seen. They dissipated more with shaking.
  • Phages appear on the TEM in the shape of a lollipop with a dark head and banded tail.
  • TEM showed phages with a grainy background. Small movements had large effects, and the images moved slightly due to how the electrons interacted with the plastic film.

Conclusions/Next Steps:

  • Since there were plaques present on the 10^-7 dilution, it can be concluded through calculations that there is a high titer lysate present. This is helpful in furthering the processing of the lysate, and allowed the DNA extraction process to begin. The TEM also showed the morphology and size of the bacteriophages from the sample. Since procedures today did not show definitive results, no further conclusions are available to be reported. The next steps in this procedure would be to finish the DNA extraction process, which will be done on Wednesday.

 

Category: Henry Burns | LEAVE A COMMENT
November 26

November 26, 2018 Webbing Plates- Soil C

Rationale: The purpose of this lab is to web plates to obtain more of a higher titer lysate.

Description of Procedures:

  1. The workstation was cleaned using aseptic technique and an ethanol burner was lit.
  2. Lysate 1 was diluted out to 10^-3.
  3. Top agar was made for 7 plates. 14 ml of LB broth and 157.5 ml of CaCl2 were added to a tube.
  4. 10 ul of 10^-3 lysate was added to 6 tubes of 0.5 ml of arthrobacter and was allowed to sit for 10 minutes.
  5. 17.5 ml of 2x TA was added to the tube and mixed. 4.5 ml of the solution was added to each tube of arthrobacter and poured onto 6 plates labeled LIP 11-26-18 webbed plate. The rest of the solution was poured onto a plate labeled LIP 11-26-18 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 on the control plate.

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

Webbed Plates:

Category: Lucy Potts | LEAVE A COMMENT