Task, Rationale, and Purpose: Lab week 13 entailed the running of the gels with the PCR products we prepared from lab week 12’s Chelex protocol. The task of the lab was to obtain gel electrophoresis results and design a rough draft of the poster for the future presentation of our results. The purpose or rationale for the lab was to determine the efficacy of the Chelex protocol and compare its results to the previous protocols. This is required to determine the optimal protocol to center the presentation and research paper around.

Gel Electrophoresis Protocol:

1. Remove the rubber ends from the agarose gels made from last week’s lab and place the gels in the gel electrophoresis box containing 1X TAE buffer.
2. Ensure that the gel is covered with the TAE buffer and add 5 µl of ladder to well 1 using a p20 micropipette.
3. Using a new micropipette tip for each well, add 10 µl to each designated well:
   1. Well 2: Group 7 – negative control.
   2. Well 3: Group 7 – positive control.
   3. Well 4: Group 7 – environmental DNA.
   4. Well 5: Leave empty.
   5. Well 6: Group 8 – negative control.
   6. Well 7: Group 8 – positive control.
   7. Well 8: Group 8 – environmental DNA
4. Close the electrophoresis box and connect the power sources ensuring that the red cord corresponds with the red input and output sources. Ensure the black cord corresponds with the black input and output sources.
5. Run the gel electrophoresis at 100 volts for at least 30 minutes.
6. Remove the gel and examine the gel under a UV light.
7. Take a picture of the gel and record the results.

Data:

Group 7 and 8’s Gel:

Wells are arranged from well 1 (most left) to well 8 (most right).

Well 2: no band

Well 3: no band

Well 4: band around 500 bp

Well 6: no band

Well 7: no band

Well 8: no band

Storage: The environmental, positive, and negative control samples were given to the TAs for proper disposal. The gels disposed of in plastic bags.

Conclusion: The results from the gel electrophoresis showed negative results for group 8 and one band for group 7’s eDNA well. The positive control well, however, had no bands. No results from the positive control may indicate an error with the making of the solutions or the presence of matter that interfered with the DNA before or after the running of the gel. Group 8 may have improperly followed the Chelex protocol by failing to add primers or DNA. For future steps, the metabarcoding of the remaining eDNA samples is plausible. Further analysis of the Chelex results may present other errors or barriers to positive results such as the lack of purity in the DNA.