10/22/18 Gel Electrophoresis of DNA from Soil Sample #3

Objective:

The goal of this procedure is to test the previously created enriched lysate for phage DNA. Gel Electrophoresis will help us determine if there is phage DNA present, which will determine whether or not we do future testing on soil sample #3.

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 Gel Electrophoresis:

• Agarose powder
• 1X TBE
• Microwave
• Ethidium Bromide
• Flask
• Electrophoresis Tray
• Electrophoresis apparatus and power supply
• DNA ladder

The agarose gel was prepared:

1. The gel was prepared according to the following recipe:
2. The gel mixture was then microwaved until boiling
3. The mixture was allowed to cool until warm
4. 2 μl Ethidium Bromide was pipetted into the mixture
5. The mixture was poured into an Electrophoresis tray and allowed to solidify

Gel Electrophoresis was run:

1. The tray was placed into the electrophoresis apparatus and the apparatus was filled with solution to cover the gel
2. 10 μl of each PCR sample and 5 μl of DNA ladder were pipetted into the gel slots
   • Samples were loaded into the Gel Slots according to the following image (my samples are group 2):
     
3. The apparatus was plugged into a power source and turned on
4. The gel was allowed to run until the DNA had fully traveled across the tray
5. The results were imaged and recorded

Results:

The results of this Gel Electrophoresis came back negative for all 3 clusters of phage DNA tested. The positive controls however still tested positive, suggesting that there was nothing wrong with the Gel or procedure. These results confirm that there was no arthrobacter phage in my soil sample #3.

Analysis:

Gel Electrophoresis works by using electricity to separate different strands/fragments of DNA in order to analyze the patterns that ensue.  When electricity is applied, DNA moves toward the oppositely charged end of the gel tray and brings the DNA dye with it. While there was some concern that using DI water instead of DDI water during PCR would cause odd results, the positive control I created during the last lab still worked, suggesting that I received a true negative result. This means that my previously created PCR tubes did not have phage DNA in them, which means my soil sample #3 likely does not have phage.

Future:

The results of this procedure were disheartening, but also odd because every group received a negative result, and that seems unlikely based on the number of soil samples tested. As a result, I will be testing whether or not cleaning soil with chloroform (as was the case for some of the soil samples tested) effects the results of gel electrophoresis. I will do this by testing soil that has been confirmed to have phage in it. I will wash soil that is known to create phage in order to create an enriched lysate. Then, I will filter half of this lysate with a syringe filter, adn clean half of it with cloroform. I will then run PCR and Gel Electrophoresis on these two different lysates to see if the results are affected. This will likley take me multiple lab periods to complete.