November 23

NOVEMBER 16TH and 19TH- LABS

  • NOVEMBER 16TH, 2018
    • OBJECTIVE:
      • To pick a plaque and create serial dilutions, so dilutions can be easily plated next time coming into lab 
    • PROCEDURE:
      • Tables were cleaned and lamps were lit
      • Seven micro-centrifuge tubes were labeled according to the dilution (10^0 through 10^-6)
      • 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 
      • The P4 plate was examined under a light microscope, where a plaque was picked using a micropipette 
      • The pipet tip was then swirled in 100𝝁L of phage buffer
      • 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^-6 was reached 
      • After the plaque was picked 8mL of phage buffer was put onto the P4 plate, where the plate was then left to flood for 48 hours 
    • RESULTS: 
      • None
    • CONCLUSION: 
      • None 
    • NEXT STEPS: 
      • Plate dilutions and filter lysate from plate flooding 
  • NOVEMBER 19TH, 2018
    • OBJECTIVE: 
      • To plate serial dilutions and filter lysate
    • PROCEDURE: 
      • Tables were cleaned and lamps were lit 
      • A large test tube was filled with: 
        • 4mL LB
        • 5mL 2X TA
        • 45𝝁L of CaCl2
      • 4.5mL was then pipetted onto 2 plates, one plate where the serial dilutions will be tested, and the control 
      • After allowing 10 minutes for the plates to solidify, 10𝝁L of each dilution was pipetted onto the designated location on the plate 
      • After allowing 10 minutes to let the dilutions sit on the plate, the plate was placed right side up in the incubator
      • The contents of the flooded plate were poured into a top filter, and filtered out creating a new lysate
    • RESULTS: 
      • The results can be viewed in Figure 25 
    • CONCLUSION: 
      • The area on the plate labeled 10^-3 will be used as the high titer for the lysate 
    • NEXT STEPS: 
      • Calculate how much is needed to make a high titer, then plate the amount calculated 
Category: Lauren Foley | LEAVE A COMMENT
November 23

11/19/18 Prepping TEM Grid and Pelleting

Rationale:

The purpose of today’s lab was to prep a TEM grid for microscopy due to a high titer lysate finally being acquired. In addition to TEM, DNA extraction procedure will continue with the pelleting of the phage particles through centrifuge.

Results from 11/16/18:

  • HTL was successfully prepared for DNA extraction with the addition of the nuclease mix and phage precipitant solution. Also, 100 µL of the HTL had been set aside for TEM and will be used to get phage particles onto the grid.

Materials:

  • High Titer Lysate (HTL)
  • 400 Mesh Copper Grid
  • Grid Box for Storage
  • TEM forceps
  • DI Water
  • Uranyl Acetate

Procedure for TEM:

  1. Plates were lined with parafilm and 15 µL of the HTL was spotted onto the parafilm.
  2. Added two 20 µL drops of DI water onto the plate as well, keeping them all separated.
  3. Then a drop of liquid uranyl acetate was spotted onto the plate as well.
  4. A 400 mesh copper grid was removed from the B6 slot on the grid box with the TEM forceps and was left in the HTL shiny side down for 5 minutes.
  5. Once 5 minutes were up, the grid was transferred to the DI water, where it rested for 2.5 minutes and was repeated with the second DI water spot as well.
  6. Once removed from the second DI water spot, the grid was placed on the uranyl acetate spot for 1 minute to stain.
  7. After a minute had passed, the grid was placed into the corresponding B6 slot on a new grid box that contained prepped TEM grids.
  8. Grid box was then stored until ready for TEM.

Procedure for DNA Extraction:

  1. Since the DNA was already prepared and balanced with another conical vial full of water, all that was left was to place the vials in the centrifuge.
  2. Both conical vials were placed in the centrifuge to spin at 10,400 rcf for about 20 minutes at 4 degrees Celsius.
  3. After the 2o minutes had passed, the pelleted lysate was removed from the centrifuge, decanted of any remaining supernatant, and left to freeze to prevent any degradation.

Results:

  • A fair amount of pellet had formed on the bottom of the conical vial, but unaware if it is considered a “large” pellet or not. Also pellet will be left in the freezer for over a week, and it is typically only supposed to be left for 3 days.
  • Ring on the bottom of the conical vial is the pelleted phage particles.

Conclusions:

The amount of phage pellet could be a result of the titer of lysate present. The titer of the lysate is on the lower end of the spectrum of the high titer category, so that could determine a low pellet count. Also, the TEM grid preparation process could yield some over-dyed grids as the TEM forceps had to be shared amongst 8 students, and some had to leave their grids for longer than recommended.

Next Steps:

The next steps for the experiment are to analyze the phage morphology under TEM using the previously prepared grid and continue with the extraction of DNA from the pelleted phage particles.

 

 

 

Category: Gabriel Andino, Uncategorized | LEAVE A COMMENT
November 23

11/16/18 DNA Extraction

Rationale:

The goal in lab today was to analyze the spot titer to calculate the titer of the lysate. If the titer calculated was high enough, then the beginning procedure for DNA extraction would begin. If not, then another flood and plaque assay would be run to continue amplifying the titer of the lysate.

Results from 11/14/18:

  • Despite being set on the bottom rack of the incubator, which is at a different temperature than the rest of the racks, the spot titer yielded visible plaques up to the 10^-6 dilution. It had completely lysed the first few dilutions, but also contained contamination on the top agar control due to unknown contamination.
  • The 10^-6 dilution had approximately 5 plaque forming units (pfu) present, so that was used to calculate the titer of the lysate. Titer was calculated using the equation below.
  • Titer was 5*10^8 pfu/mL, which was a high titer lysate and DNA extraction could begin.

Materials:

  • High Titer Lysate (HTL)
  • Conical Vials
  • Nuclease Mix
  • Phage Precipitant Solution

Procedure:

  1. Established an aseptic zone
  2. Lysate had to be archived, and began with naming the phage “Kiersten”.
  3. After naming, 2.8 mL of lysate was transferred to a conical vial for archiving.
  4. 100 µL of lysate was aliquoted to a micro centrifuge tube for prepping a TEM grid next lab.
  5. 10 mL of the HTL was then transferred to a separate 50 mL conical vial, and 40 µL of the nuclease mix was added to the HTL. It was then mixed gently to combine.
  6. 4 mL of the phage precipitant solution was added as well, and mixed gently to combine.
  7. The lysate solution was then left to sit and incubate at 37 degrees celsius for 3o minutes.
  8. Vial was then weighed out and a conical vial was weighed with water to balance out in a centrifuge and the lysate solution was stored in the freezer for the next lab.

Results/Data:

  • The lysate was prepped for DNA extraction, but no actual DNA extraction took place. With the high titer of lysate, the increased presence of phage particles will allow for a larger pellet to form, which will aid with DNA extraction.

Conclusions:

The prepping of the HTL allows for the pelleting of phage to occur once run through a centrifuge as the nuclease removes any remaining bacterial DNA floating in the lysate, and the phage precipitant solution pulls water molecules out of the lysate to aid pelleting.

Next Steps:

The next steps for this experiment are to prep a TEM grid for microscopy and pellet the phage particles.

Category: Gabriel Andino | LEAVE A COMMENT
November 23

Prepping TEM Grid for Sample (Gabe) and results of Spot Test for Sample (Gabe) 11/19/18

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:

Individual phage particles are too small to be seen with light microscopes, necessitating the use of transmission electron microscopy (TEM). The Grid is vital to the fixing sample and staining them.

Experimental Procedure for TEM grid preparation for Soil (Gabe):

  • High Titer Lysate
  • 400-mesh copper grid
  • TEM forceps
  • DI water
  • Uranyl Acetate
  • Gloves
  • p20 micropipette
  • Parafilm
  • Microcentrifuge tube
  1. Allocated 15 ul lysate sample, two individual H2O drops to the parafilm.
  2. Placed the copper grid on the lysate sample for 5 min.
  3. Placed the copper grid to the H2O for 2.5 min.
  4. Placed the copper grid to the other H2O for 2.5 min.
  5. Allocated one drop of Uranyl Acetate to the parafilm, and placed the grid onto the Uranyl Acetate for 1 min.
  6.  Flick off redundant liquids on the copper grid and store away.

Observations, Results & Data:

The control plate was contaminated. The spotted regions on the sample plate were all completely lysed and appear to be clear.

Interpretations & Conclusions:

The sample plate was lysed on the spotted regions. Since the most diluted sample 10^-8 was also completely lysed that means the sample lysate 10^0 collected is extremely high titer. Therefore was able to start the TEM preparation.

Next Step:

The TEM preparation will continue, and the exact titer of the lysate sample would be calculated and DNA extraction would start.

 

Category: Yang-En Yu | LEAVE A COMMENT
November 21

Filtering Plates For Lysate (11/20/18)

Rationale: To filter the flooded plates to obtain more lysate.  

Procedure: 

  1. Created an aseptic zone by cleaning the table with Cidecon and 70% Ethanol, and using a ethanol burner.  
  2. Added 5 mL of Phage Buffer into each of the plates, and set on the shaking device.  
  3. The plates were shaken for an hour and five minutes. 
  4. Using a syringe and filter, filtered the lysate from each of the plates into a 50 mL tube.  
  5. The 50 mL tube was stored in the fridge.  

Observations/ Results: 

Control plate was not contaminated and plates were webbed.  

Obtained about 13 mL of lysate from the webbed plates.  

When picking up the plates from shaking device realized that the plates were wet and some of the liquid had possibly spilled, and therefore decreasing the amount of lysate that was obtained.  

Next Step/Conclusion: 

Next class we will make more webbed plates and try to create a higher titer plate of either 10^9 or 10^8.

Category: Sona Subramanian | LEAVE A COMMENT