Research Introduction

Here, you will find a detailed decription about my research, the outcome, and inferences. The whole process can be divided into four parts: collecting samples, extracting DNA from the samples, performing PCR, and running gel electrophoresis. However, press the button below to see a reflection on the research process and more information about my experience.

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Samples

In total, I collected 8 samples from various sites along the Wolf River and Nonconnah Creek. These locations sites were based off the fact that a) these two waterways feed into the Memphis Aquifer at certain times of the year and b) for the Nonconnah Creek specifically, samples were collected near suspected breach areas. To see a map of my sample sites, use the button below.

I collected approximately 50 milliliters of the sediment below the water at each sample site, focusing on collecting the sediment rather than the water. For protocol, I wore a glove for each sample, and placed the samples in an ice-filled cooler to preserve the contained DNA after collection. To process the samples, I poured off any water inside of the tube, and transferred approximately 1.5 milliliters of each 50 milliliter sample to a smaller tube. While waiting to take the samples to the lab, the samples remained in my home freezer.

Sample Map

DNA Extraction

A FastDNA SPIN Kit for Soil was used to extract the DNA. From the 1.5 milliliter samples, my mentor and I prepared smaller portions to prepare for DNA extraction. Two different bacteria were being observed, so we prepared two 500 milligram (~5 grams) samples from each 1.5 milliliter sample. Next, we carried out the steps for DNA extraction:

1. Prepare the samples

2. Homogenization (facilitates the process)

3. Precipiate proteins

4. Adjust binding conditions

5. Bind the DNA

6. Wash the SPIN filter

7. Dry the SPIN filter

8. Elute the DNA

The steps are based off the manual for the specific DNA extraction kit. To learn more, please press the underlined text to access the detailed version of the protocol. These steps were performed for each sample, and in total, 16 samples were analyzed (2 smaller samples for each of the 8 larger samples).

PCR

PCR, which stands for Polymerase Chain Reaction, is a vital step used for DNA amplification. For this, we used two DNA primers that correspond with a specific microbe they each focus on a targeted gene, and if that gene is present, they amplify, or copy, multiple sequences of the that gene. Each DNA primer went into a different set of 8 extracted DNA samples, and then, they are placed in a thermal cycler, an instrument that executes various temperature change cycles to amplify the DNA. Here are the specific thermal cycler settings for each primer:

Pseudomonas fluorescens primer: (1) 95°C for 15 minutes (2) 40 cycles of thirty seconds at 95°C (3) 51°C for 30 seconds (4) 72°C for 30 seconds (5) 72°C for 5 minutes

Cryptosporidium parvum primer: (1) 30 cycles of thirty seconds at 95°C (2) 50°C for 30 seconds (3) denaturation at 95°C for 5 minutes (4) 72°C for one minute

(5) final extension of 72°C for 10 minutes

Press here to learn more.

Gel Electrophoresis

General: Gel electrophoresis is used to separate DNA fragments based on their size. Its set up entails pipetting your DNA samples into different wells within the gel, and these wells are set up near the negative side of the geI. Then, by running an electrical current, the DNA, which is negatively charged travels towards the positive side. Each fragment travels at a different rate due to its size, and on either far right or left side, there is a comparison to determine the length of the DNA fragment.

For the experiment: If present, the amplified DNA sequences would be present in the samples after PCR. Therefore, if the bacteria are present, our gel to look similar to this:

The bands beyond the first column represent fragmented DNA from a sample. Because we used specific DNA primers, only the targeted genes for each bacteria will be copied, so seeing any bands beyond the comparison indicates that the specific gene and that bacteria are present in the samples.

Results: We did not see any bands present within our gel. There were 16 wells, 8 for each microbe. Consequently, this conveys that neither Pseudomonas fluorescens nor Cryptosporidium parvum were present in any of the soil samples because the corresponding targeted genes were not amplified; amplification can only occur if the genes are present. Therefore, if bands did appear, we could assume the specifc bacteria are present, but sadly, our gel looked similar to this:

The DNA bands that are compared to for determining the length of DNA fragments being anaylzed.

*Both photos are not mine

Inference 1

Because Cryptosporidium parvum is a parasite, the results indicate a positive aspect of the two waterways. From the collected samples, this parasite was not present, so this parasite is not a threat to Memphis's drinking water, which would put the larger city population at risk for diarrheal disease.