Purpose:

The purpose of this lab was to analyze the existence of DNA in the eDNA, positive control, and negative control samples that were amplified using PCR by running each sample through gel electrophoresis. The presence of a band would determine that the DNA extraction procedure and PCR amplification was successful and that DNA was present and prepared for further analyzation to determine the biodiversity of a sample. In addition, this lab was used as a time to prepare for upcoming poster presentations. Guidelines for posters, critiques and discussion of posters, and the beginnings of poster planning, design, and information needed on posters was discussed among groups.

Procedure:

Gel electrophoresis:

1. Place the agarose gel into the gel electrophoresis apparatus, with the wells closest to the negatively charged side of the box.
2. Pour in the previously prepared and obtained 1x TAE buffer solution onto the gel, filling the box and covering the gel and the wells completely.
3.  Collect your positive control, negative control, and eDNA samples from the front of the room. Carefully micropipette 10 µl of each sample into separate wells in your gel.
4. Micropipette 5 µl of DNA ladder already mixed with loading dye into the well adjacent to your samples.
5. Connect the positive and negative charged wires to their appropriate places on the box and to the power supply.
6. Place the lid onto the apparatus and turn on the power supply to the wires. Run the gel electrophoresis for about 30 minutes at 100 V.
7. Remove the gel after 30 mins and analyze under UV light to examine the bands of DNA present in the gel.

Data:

*Our wells were labeled 1-7, running from the left side of the gel to the right side. Well 1 begins on the left with our DNA ladder. We shared our gel with group 8 to run gel electrophoresis at the same time. Group 8 used wells #2-4 and our group (7) used wells #5-7.

Well #:

1. DNA ladder
2. Group 8 Negative control
3. Group 8 Positive control
4. Group 8 eDNA
5. Group 7 Negative control
6. Group 7 Positive control
7. Group 7 eDNA

None of the samples from our group yielded any DNA bands, however group 8 was able to see very light bands of about 450 bp for both their positive control and eDNA samples. Most of the class were able to see results on their gel electrophoresis and it was determined that the Chelex DNA isolation and extraction method was the most successful of all the methods that we had tested this semester.

Conclusion:

The lab went smoothly and all of the groups were able to complete the procedure for today’s lab with ease. Gel electrophoresis was a skill that we had already practiced and mastered in an earlier lab and we were able to use these skills to complete the procedure smoothly and efficiently in today’s class period. In addition, we were able to have meaningful discussion about our poster regarding its design, layout, and what information would be included on the poster. We were able to decide on a layout and delegated different sections of information on the poster to different group members, so that we could use our time and resources efficiently, while at the same time building each other up and relying on each other’s knowledge. We know what is expected to be completed by next lab time and can now plan accordingly for our next poster workshop day. In addition, when we were able to view the results from our gels toward the end of lab, we saw that our group’s samples yielded no DNA bands or positive results from our gel electrophoresis. These negative results could possibly be due to contamination of DNA that interfered in the PCR amplification process or possible errors of dilution or completion of procedure. There are multiple reasons that could explain why we got negative results, however it is difficult, if not impossible, to pinpoint the exact cause. While we did not see results, a majority of the other groups in our class were able to see results in their gels. Therefore, the Chelex protocol was determined to be the most successful at extracting DNA. When we were finished with lab, our PCR tubes and gel were stored back at the front of the room with the rest of the class and were labeled with the same marking information as referenced in the last log post. The next step for our lab group will be to create a poster presentation that will best describe our research that we have conducted this past semester.