The overall goal of this procedure is to separate DNA fragments of varying sizes. This is accomplished by first preparing a gel with the appropriate concentration of aros and a fluorescent dye. The second step is to pour the gel into an appropriate mold and allow it to set next.
The samples are loaded into the gel and a current is applied. The final step is to visualize the separated DNA fragments under ultraviolet light. In summary, AGROS gel electrophoresis is used in all situations where the routine separation of DNA fragments is required.
The main advantage of this technique over existing method of the time, like sucrose density gradient centrification, is that it provides visualization of DNA bands, and it also enables us to determine the exact sign of exact size of the DNA fragment when separated concurrently with A DNA marker. This method is essential in life science research, such as in the cloning of genes and the purification and sequencing of DNA molecules. Though this method is generally used to separate DNA fragments between a hundred base pairs and 25 kilo bases, it can also be modified to separate DNA fragments up to 10 megabases in size.
Agros gels are prepared using a weight over volume percentage solution. The concentration of aros and a gel will depend on the sizes of the DNA fragments to be separated. To begin the procedure for making a gel weigh out the appropriate mass of aros into an erlenmeyer flask, add the appropriate volume of running buffer to the flask.
The volume of the buffer should not be greater than one third of the capacity of the flask swirl to mix, melt the aros buffer mixture by heating in a microwave at maximum power. At 32nd intervals, remove the flask and swirl the contents to mix. Well repeat until the agros has completely dissolved.
Next, add Atherium bromide to a concentration of 0.5 micrograms per milliliter. It is important to note that atherium bromide is a carcinogen, so gloves should always be worn when handling gels containing atherium bromide allow the agros to cool by incubation in a 65 degrees Celsius water bath. Failure to do so will warp the gel tray while the aros is cooling.
Prepare the gel mold by placing the gel tray into the casting apparatus. Alternatively, one may tape the open edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells.
Pour the mold and aros into the gel mold. Allow the aros to set at room temperature. Once the aros is set, remove the comb.
If the gel is not going to be used immediately, wrap it in plastic wrap and store at four degrees Celsius until use. If the gel is going to be used immediately, place the gel in the gel box. To begin this procedure, add gel loading dye to the DNA samples to be separated.
Loading dye is typically made at a six x concentration program, the power supply to the desired voltage now add enough running buffer into the gel box to cover the surface of the gel. It is important to use the same running buffer as the one used to prepare the gel, attach the leads of the gel box to the power supply and turn on the power supply. Verify both the gel box and the power supply are working.
The appearance of bubbles at the electrodes indicates that current is passing through. Since our hands tend to shake a little naturally, it may be difficult to get a small pipe tip into a small well. One way to prevent our hand from shaking is to rest it on the other arm or the gel box.
Remove the lid of the gel box slowly and carefully Load the DNA samples into the gel. The loading dye in the sample allows the sample to sink into the gel and will help to track how far the sample has traveled. A DNA size marker should always be loaded along with the experimental samples.
Replace the lid. Double check that the electrodes are plugged into the correct slots in the power supply. Turn on the power, run the gel until the dye has migrated to an appropriate distance.
When electrophoresis has completed, turn off the power supply and remove the lid of the gel box. Remove the gel from the gel box and drain off excess buffer on the surface of the gel. Place the gel tray on paper towels to absorb any remaining running buffer.
To visualize the DNA fragments, remove the gel from the gel tray and expose the gel to ultraviolet light. This is most commonly done using a gel documentation system. DNA bands should show up as orange fluorescent bands.
Take a picture of the gel at the end of the experiment, properly dispose of the gel and running buffer per institution regulations. This figure represents a typical result after aros gel electrophoresis of PCR products. After separation, the resulting DNA fragments are as clearly defined bands.
The DNA standard or ladder should be separated to a degree that allows for the useful determination of the sizes of sample bands. In this example, DNA fragments of 765 base pairs, 880 base pairs and 1022 base pairs are separated on a 1.5%AROS gel with a two log DNA ladder. While attempting the procedure, it's important to remember to double check the placement of the electrodes before applying current.
This is to ensure that the DNA molecules travel in the right direction. After watching this video, you should have a good understanding of how to prepare an agro cell, separate DNA, fragments of bearing sizes, and obtain representative images of your results. Don't forget that working with a three bromide can be extremely hazardous and precautions such as wearing gloves, goggles, and lab coats should always be taken when performing this procedure.
