10/26/18 PCR on Lysate from Soil with Known Phage

Objective:

The goal of this procedure is to test the efficacy of PCR and Gel Electrophoresis. The way I will be testing this is by creating a newly enriched lysate using soil that already tested positive for phage in the previous testing. The purpose behind this is to test if different ways of cleaning the enriched lysate will affect the results of PCR. In this lab, I am using a syringe filter to filter one sample of the enriched lysate I created and using chloroform to clean another sample. Then I am running PCR on these two samples and will do Gel Electrophoresis on each sample next lab.

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 PCR:

• 15 ml conical vial
• Syringe Filter
• Chloroform
• PCR Machine
• DI Water
• TAQ Polymerase
• Centrifuge
• microcentrifuge tube
• pipette
• Test tube stand

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

1. CiDecon was applied to the lab table
2. 70% Ethanol was also applied

The syringe filtered sample of enriched lysate was prepped for PCR:

1. ~1 ml of the previously created enriched lysate filtered into a microcentrifuge tube using a syringe filter
2.  The tube was boiled to release phage DNA

The chloroform cleaned sample of enriched lysate was prepped for PCR:

1. ~1 ml of the previously created enriched lysate was aliquotted into a microcentrifuge tube
2. 10 μl of chloroform was added to the tube and allowed to sit for 1 minute
3. The tube was spun in a centrifuge for 5 minutes to pellet the debris
4.  The tube was boiled to release phage DNA

PCR tubes were created:

1. 3 PCR tubes were created according to the following recipe and DNA from the syringe filtered lysate:
2. 3 PCR tubes were created according to the above recipe and DNA from the chloroform cleaned lysate
3. The tubes were placed in the PCR machine until next lab running the following cycle:

Results:

The majority of the information that will be available from these procedures will not be visible until the next lab, so these results will be updated on Monday when the results of PCR will be visible and more testing can be conducted. However,  it seems as though PCR testing went well.

Analysis:

PCR works by using polymerases to make many copies of specific strands of DNA  so that it is easier to analyze. Using PCR allows researchers to take small samples and amplify the genetic contents in order to conserve resources and determine what is present before future testing commences.  In previous labs, PCR was used to determine whether or not there was phage DNA  in an enriched lysate before the time is taken to run spot tests and plaque assays. In this lab, PCR is being run on two samples from the same enriched lysate that is known to be positive for phage. One sample was syringe filtered and the other was cleaned with chloroform, testing both will demonstrate if the PCR/ gel electrophoresis process is working and if there is a difference between the two samples.

Future:

In my next lab, I will be performing gel electrophoresis on the contents of the 6 tubes I created using PCR.

Rationale:

The purpose of today’s lab was to continue the purification process with the phage particles gathered from the previous serial dilutions. If plaque assay results were positive, plaques would have been further picked to purify. If results were negative, plaque assays were to be rerun.

Results from Lab 10/17/18:

• As expected, top agar split and slid, giving no clear indication of phage presence. This was due to the irregular solidification of the top agar during plating the week previous.
• Plaque assays had to be rerun, as the results were inconclusive.
• 

Materials:

• LB Broth
• 2X TA
• Diluted Lysates
• Agar Plates
• Calcium Chloride

Procedure:

1. Established an aseptic zone.
2.  3 separate plaque assays were run with each dilution as well as one top agar control.
3. To begin, 2.0-mL of LB broth was added into 3 separate conical vials and 2.5-mL added to a fourth top agar control vial.
4. Next, 22.5-μL of calcium chloride was added to the each of the conical vials.
5. 10-μL of each of the dilutions were combined with 3 separate 500-μL quantities of Arthrobacter and left to infect for appro.
6. After about 15 minutes, the diluted lysates were combined with their respective labeled LB Broth mixtures and 2.5-mL of 2X TA was added to the all 4 of the broths.
7. Swirled and plated top agars immediately and left to solidify.
8. Once solidified, they were flipped and added to the incubator until the next lab.

Results/Data:

• Top agar and LB Broth used were madd two days prior and contained no evidence of contamination. Also, top agar was not solidified before the top agar pouring process. This lead to a normal solidification of the top agar once plated and should not slide or split.

Conclusions:

• It can be concluded that this previous will yield accurate results in the phage dilution. Top agar did not seem to show any indication of splitting.

Next Steps:

• The next steps are to analyze the top agar results in approximately 48 hours. If the results are plaque positive, new plaques will be picked to get a high titer of phage and continue the purification process. If no plaques are present, then new diluted lysates will be made to rerun plaque assays.