11/28/18 Spot Titer Test, TEM, and DNA Extraction Part 1

Objective:

The goal of this procedure is to assist Lucy P. in getting a large amount of high titer lysate. This is achieved by webbing and then flooding plates. This procedure will detail the process of calculating the titers of the lysate C (the result of webbing plates with lysate 1), creating a TEM grid, running a TEM, and creating a DNA pellet as part of DNA extraction.

The overarching question this test seeks to address is: Is the presence of phage determined by species of oak tree from which soil was collected?

In other words, are specific oak tree species more likely to have Arthrobacter bacteria phages in the soil surrounding them?

The question specific to my lab table is: Is the difference in the presence of phage between live oaks and red oaks on Baylor’s campus?

As a group, we hope to expand our question to include more species as we gather data so that we can better address our overarching question and we will look at our metadata to examine whether or not there are other factors that may determine phage presence.

Procedures and Protocols:

Materials for an Aseptic zone:

• CiDecon
• 70% Ethanol
• Ethanol Burner

Materials for a Serial Dilution:

• Phage Buffer
• Microcentrifuge Tubes
• Vortex Machine
• Pipette

Materials for Lysate Filtering:

• Topfilter
• 50 ml conical
• Flooded webbed plates

Materials for Phage Precipitation:

• High Titer Lysate
• 50 ml conical vial
• Nuclease mix
• Phage Pericpitate solution

Materials for TEM Grid:

• High titer lysate
• 400 mesh copper grid
• Grid box
• TEM forceps
• DI water
• Uranyl Acetate
• Gloves
• p20 micropipettor
• Parafilm

Materials for a Spot Test:

• .5 ml Arthrobacter
• incubator
• Pipette
• Test tube stand
• 50 ml tubes
• Culture tube
• LB Broth
• 2X TA
• 1M Calcium Chloride
• Agar plate
• Serological pipette

In order to complete the procedure, an aseptic zone was created.

1. CiDecon was applied to the lab table with a squeeze bottle and wiped away with a paper towel
2. 70% Ethanol was also applied with a squeeze bottle, spread with a paper towel, and allow to evaporate
3. An ethanol burner was light in order to use the rising heat from the flame to form the aseptic zone
   

Lysate C was collected and filtered

1. The lysate from the flooded plates was poured into a top filter
2. The lysate was filtered into a 50 ml conical

The Phage Precipitation was performed.

1. 10 ml of lysate C were transferred to a conical vial
2. 40 µL of Nuclease mix were added and the vial was inverted several times to mix
3. 4 ml of phage precipitate were added
4. The tube was placed in the incubator for 30 minutes
5. Then the tube was left at room temperature for ~40 minutes
6. The tube was then centrifuged at 10,000g for 20 minutes *Note: the tubes got locked in the centrifuge machine overnight before being centrifuged for 20 minutes*
7. The supernatant was poured into the sink and the pellet was put in the freezer until next lab

While phage precipitation was performed a TEM grid was created.

1. 20 µL of Lysate, 20 µL of DI water, 20 µL of DI water, and ~20 µL uranyl acetate were placed on a strip of parfilm as shown below:
2. TEM forceps were used to place a copper grid shiny side down in the lysate for 5 minutes
3. Forceps were then used to transfer the grid to each sample of DI water for 2.5 minutes each
4. Finally, the grid was transferred to the uranyl acetate for a minute
5. Excess moisture was wicked away with filtered paper and the grid was loaded into the TEM for imaging

Then serial dilutions were performed on lysate C.

1. Seven levels of dilution were created for each lysate (called lysate 1 and 2): 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7
2. Seven microcentrifuge tubes were filled with 90 µL of phage buffer
3. 10 µL of lysate C (10^0) were transferred to one of the 10^-1 vials
4. The tube was vortexed to mix
5. 10 µL of the solution was taken the 10^-1 tube and transferred to the tube labeled 10^-2
6. The tube was vortexed to mix and this procedure of dilutions was repeated through the 10^-7 dilution for lysate C

Then the spot titer test on the new lysates was performed.

1. One agar plates was labeled as shown:
   
2. Agar was prepared according to the following recipe (makes two plates):
3. 4.5 ml of the agar was transferred to the labeled plate
4. The plate was swirled and set aside to allow the agar to solidify
5. When agar was solidified, 10 µL of phage buffer as well as 10^-3, 10^-4, 10^-5, 10^-6, and 10^-7 dilutions for each lysate were transferred to their appropriate spot on the plate

Results:

Update: As can be seen from the image above, our spot test failed. We know our lysate is a high titer, so it seems likely we inverted the plate too quickly so the lower concentration dilutions didn’t have enough time to absorb onto the agar.

In addition, the results of our TEM imagining can be seen. In our group, 2 distinct types of phage were found, as detailed above.

Analysis:

The idea behind these procedures is to learn more about our phage as a whole. From these procedures were were able to learn about our phage morphology and extract DNA for further testing. This is the final step needed to ensure we sucessful have a phage that we can add to the phage database.

Future:

We will finish DNA extraction next lab and we will archive.