During our lab ours today, we continued to improve our PCR skills. Polymerase Chain Reaction, or PCR for short, is the process of replicating DNA exponentially by cycling the DNA product through different temperatures. This replication allows for the ability to run multiple tests using the same strand of DNA without having to extract it from multiple organisms of the same species multiple times. Furthermore, it allows us to generate a usable amount of product to run these reactions.
After PCR, we then analyzed our PCR product in a gel electrophoresis reaction. As I have stated in other blog posts, gel electrophoresis is a useful procedure that allows us to analyze PCR product to determine if we successfully replicated the DNA, among other things. A further explanation can be found on my post all about gel electrophoresis.
Moving Forward
Next week, we should be continuing the analysis of the PCR product in another DNA extraction lab. Eventually, once everyone is sufficiently ready, we will be analyzing the zooplankton samples that Dr. Jewoski gave us at the beginning of the semester. This has the small, but real possibility of us uncovering a new species of zooplankton, which is exciting for everyone in the lab.
Today I have been working on a DNA extraction procedure with our zooplankton practice and actual samples that we obtained during the first few weeks of the course. Dr. Ren completed the first step of the procedure by incubating the samples at 56 degrees centigrade in a buffer and proteinase mixture. I continued the procedure by following a modified procedure that was given to the lab class by Dr. Ren. In short, we added different amounts of different buffers and centrifuged the solutions. We then moved the solution to a spin column to isolate the DNA. After isolating the DNA in the sponge-like surface of the spin column, we then added a buffer that would dissolve the DNA into the buffer, allowing us to obtain the samples after one final centrifuge.
The Next Steps
I will continue this procedure in the afternoon by measuring the concentration of the practice and actual DNA samples we extracted earlier today. This will involve using the NanoDrop machine to obtain the DNA concentration. The extracted DNA will be combined with some amount of water, a master mix, and the same amounts of forward and reverse primers. This will then be cycled in a PCR machine and will be run through a gel electrophoresis reaction next week. I believe that this will be used to determine if the DNA that was extracted belongs to an existing zooplankton species, or if it belongs to an undiscovered species of zooplankton. The primary goal of the research is to test a new DNA sequencing technique.
Today, we have been working on a PCR setup that will be running between class. Polymerase Chain Reaction, or PCR for short, is a series of repeated temperature shifts that allows DNA to be replicated exponentially. Our research has been primarily around species of zooplankton, and today’s lab revolved around replicating the DNA from Daphnia magna, a species of zooplankton, as a practice procedure for later use.
Moving Forward:
After the PCR finishes today, we will be taking the duplicated DNA from D. magna and run them in a gel electrophoresis reaction to assess if we successfully copied the genomic DNA. If the PCR procedure was successful, we will then be able to apply this DNA to other lab procedures. Our next setup will use the D. magna DNA we collected today to do a DNA extraction lab.
In the first few weeks of the class, our instructor, Dr. Ren, gave the class different science topics to research. These topics focused on lab procedures that we would be completing throughout the semester. Myself, along with @rachelatherton12, were assigned to research gel electrophoresis, its mechanisms, and its various applications.
What is Gel Electrophoresis?
Gel electrophoresis is a technique that allows DNA, RNA, and proteins to be separated by their molecular size and charge. The gel mold itself is made from a mixture of agarose powder and a buffer. The amount of agarose that is used in one gel can be changed to accommodate different DNA base pair lengths. A higher amount of agarose powder in the gel mixture will allow for smaller movements. This is useful when analyzing DNA that have very small base pair lengths. A smaller amount of agarose powder in the gel mixture, conversely, will allow for larger movements, which is useful when analyzing DNA that is larger in size.
Why is Gel Electrophoresis Important?
Gel electrophoresis allows scientists to analyze the results of PCR to determine if the cloning was successful. Furthermore, it allows scientists to see visual proof that a setup to a larger experiment (such as developing sample DNA to insert into E. coli bacterium to test for antibiotic resistance in another species) had either succeeded or failed.
What Determines Movement?
Gel electrophoresis banding
There are a few different factors that determine the movement of DNA, RNA, or proteins through the gel. The first factor that determines the movement in gel electrophoresis is the charge of the molecules. The backbone of DNA and RNA is negatively charged. Therefore, these molecules will tend to move toward a positive charge. This is the primary mechanism for how gel electrophoresis is able to occur. The second factor that determines the movement in gel electrophoresis is the size of the molecule itself. Larger molecules will move slower through the agarose gel when compared to smaller molecules. This is the primary way that the gel is analyzed after the electrophoresis experiment has been completed. The final factor that I know of that determines the movement in gel electrophoresis is, as previously stated, the amount of agarose powder in the gel itself. This determines which molecule sizes should be run, with a higher percentage of agarose powder in the gel being better for smaller molecules, and vice versa.
Steps of Gel Electrophoresis
Before discussing the steps to perform a gel electrophoresis procedure, it is worth mentioning that there are different types of machines that can be used to analyze it the same way. Some procedures have a couple extra steps, but most of the set up is universal with some slight tweaks. I will be discussing the procedure to running a gel electrophoresis reaction for the MiniOne electrophoresis system.
MiniOne Electrophoresis system
Prepare the gel. This is done by combining a mixture of agarose powder and TBE Buffer, with the measurements of each depending on the sample size. This mixture is then heated to a boil in a microwave until all of the agarose powder has been dissolved into the buffer. After all of the agarose powder has been dissolved, a DNA stain is added to the mixture. This mixture is then poured into casting trays to solidify over the course of 20 to 30 minutes. These casting trays contain an attachable comb-like piece of plastic that indents wells into the gel. This is how the gel will be loaded with DNA samples later on.
Load the Gel. After the gel has solidified, take one of the gel samples and place it into the electrophoresis chamber. The next thing to do is to fill the chamber with a buffer fluid. This is to prevent the DNA, RNA, and/or protein samples from denaturing. After this, use a 12 microliter micropipette to load the samples into each of the wells.
Run theElectrophoresis Reaction. After covering the electrophoresis chamber, the reaction is ready to be run. I advise turning on the light that is built into the chamber every five minutes to observe the movement of the DNA samples. Once no more movement occurs, stop the reaction, and analyze the gel. The analysis of the bands is already built into this machine. Leave the cover on, turn on the light, and bands should appear.
Rachel inserting a DNA sample into an electrophoresis chamber
Uses of Gel Electrophoresis
Gel electrophoresis separates DNA, RNA, and proteins. This can be used to:
Visualize bands of a molecular marker to genotype individual plants
Verify amplification of Polymerase Chain Reaction (PCR) or sequencing reactions
Check the quality and quantity of genomic DNA after extraction
Separate DNA fragments to clone a specific band
Test plants to determine if they had been genetically modified
OKState DNA Barcoding 