10/29/18 Gel Electrophoresis of DNA from Soil with a Known Phage

Objective:

The goal of this procedure is to test the efficacy of PCR and Gel Electrophoresis. Gel Electrophoresis typically determines if there is phage DNA present, but if the procedure does not work, testing it with a sample that is known to contain phage will demonstrate this.

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 (where s represents syringe filtered samples, c represents chlophorm cleaned samples, and L represents the DNA ladder):
     
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 inconclusive because the DNA ladder didn’t fully travel through the gel. However, it seems as though there might have been issues with the primers as well because primer set three was hard to make out for both the syringe filtered and chloroform cleaned samples.

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. However, in this experiment, something went wrong that prevented this from happening. It is possible that the gel did not have a uniform density, that I added the DNA into the wells wrong, and/or that primer mix 3 wasn’t working. These results prevent me from making a conclusion about whether or not PCR/gel electrophoresis is an effective way to determine phage presence and it prevents me from making conclusions about the differences between syringe filtering and chloroform.

Future:

The results of this procedure were inconclusive so I will likley have to repeat both PCR and gel electrophoresis at some point. I will likley also add more DNA to my PCR tubes when I redo them in order to ensure that phage dna makes it into the tubes and therefore onto the gel.