Lab #14- Gel Electrophoresis 11/22/16
Will Mullen
Introduction:
- This lab is the culmination of all the work that has been being done throughout the second half of the semester. It will provide results on whether or not DNA was present in the soil samples. The DNA has now been amplified by PCR and is ready to be run through Gel Electrophoresis. This experiment will match the base pairs present in the DNA with the base pairs that are present in the DNA ladder that will also be run. Ethidium bromide is used in the gel so gloves were worn at all times.
Method:
- In order to make the gel to run the DNA, 20 mL of stock TAE (Tris acetate EDTA) was added to 0.6 g of agarose
- The solution was then microwaved twice for thirty seconds and stirred after being microwaved each time in order to ensure that the solution was mixed properly
- The gel was then poured into the electrophoresis plate
- The comb that makes the wells for the DNA samples was then placed into the plate
- 5 microliters of the DNA ladder was added to provide a good comparison of base pairs for the PCR products
- 10 microliters of the PCR products were added to the slots
- After the PCR was added, the power supply was connected to the plate and turned onto 120 volts
- The gel was run for 30 min
- The DNA was run towards the cathode because it is negatively charged
- The gel was then imaged with a special UV light and produced the image below
- After the gel was imaged, it was disposed of properly and not just thrown away because of the carcinogen that was in the gel
Conclusion:
- Starting at the tall column in the middle and moving left, the first I1 contained Euk primer and the DNA. The next well contained the Cox1 primer and the DNA. The next well was the Euk control. The last well was the Cox1 control. This picture shows that the primers were not able to bind with the ciliate DNA that was present. This could be for various reasons including the fact that the DNA present was not the type of DNA that the primers were looking to amplify. Though the results are not positive, they are still results that were achieved through steps that will help us students to better understand not only the scientific process but also teach us to respect research as a difficult but fascinating field.
Further Research:https:
This video was very beneficial to me when I found out that we were going to be doing gel electrophoresis as a lab experiment. I wanted to find something that would simplify the process as well as giving all the information necessary to understand the steps that I would be taking in the experiment and this video really did the job.
I also found this website to just be fascinating because it shows just how many things can be done with ciliates and their DNA. The website is for the Ciliate Genome Consortium which is a program that has undergraduate students looking into the genomes of Tetrahymena thermophila. The consortium is based out of Claremont, California and encourages students from Claremont McKenna, Pitzer, and Scripps Colleges to join the program. The curriculum includes topics like bioinformatics, gene expression profiling, and coding sequence determination.
http://faculty.jsd.claremont.edu/ewiley/project.php