Finding DNA Concentration and Size in Unknown DNA Samples Using Agarose Gel Electrophoresis
Background and Purpose:
In this experiment, two DNA samples with unknown concentrations and sizes are run through agarose gel electrophoresis to determine their concentration and size relative to a ? HindIII standard. The gel is run in an apparatus capable of creating an electric field that enables the negatively charged DNA to navigate its way through the porous agarose gel. The ? HindIII standard serves as a reference for DNA size and concentration since the bands lengths and percent composition relative to the whole ladder are already known 1, p. 23. The gel bands are imaged under a UV light through a fluorescent dye within the gel called SybrSafe 1, p. 24.
To set up the gel apparatus, dams are placed at each end of the gel tray to shape the gel. A ten-toothed comb is used to shape the wells with the teeth height adjusted such that the wells are deep enough to hold the samples but not so deep that the teeth touch the bottom of the tray. Melted gel is poured into the tray and allowed to solidify. As solidification of the gel occurs, 6X loading buffer and water are added to the DNA samples. Sample A and B will be run three times each at the same volume for a total of 6 samples to be loaded onto the gel with one ladder in a well before and after the 6 samples. By the time the samples are prepared, the gel should be solid. TBE buffer is added to the apparatus until the gel is completely submerged in it. The leads are attached to the apparatus such that the negative electrode is on the side of the wells. The external source is turned on and the gel is run until the DNA has migrated 2/3 to 3/4 through the gel. The SybrSafe containing gel can be imaged under a UV light. The DNA size is determined by how far each band traveled relative to that in the ladder and is expressed in terms of base pairs. The DNA concentration is determined by the intensity of each band relative to that in the ladder and is expressed in ng/uL. The intensity of the bands was quantified using Image Studio Lite, resulting in a standard curve for intensity versus amount of DNA (ng). Similarly, a curve was created using the number of base pairs in each band versus how far it traveled. The distance was quantified using a program called Image J and was in terms of pixels. The resulting equation of the lines of each standard curve was used to determine concentration and size of the known DNA samples.
Based on the standard, the results of the gel indicate that Sample A falls between 2.3 to 4.4 kb and Sample B falls below 2.0 kb as shown in Figure 1. A standard curve was created to more accurately depict the relationship between distance traveled by the DNA in terms of pixels and the size of bands in terms of base pairs using ? HindIII. The equation of the line is y=208844e^-0.003x where the size of the bands is y and the distance traveled is x. The concentration of DNA can be determined by comparing the intensity of each sample band to the intensity of the ladder since the percent composition that each band is to the whole ladder is already pre-determined as shown in Figure 1. A standard curve was made comparing the intensity of the bands with the amount of DNA in ng. The equation of the line is y = 0.0003x + 2.6169. By analyzing the intensities of each band using the ? HindIII bands and the standard equation of the line, the concentration of all 6 samples can be calculated. The mean concentration for Sample A was 37.50845 ng/uL with a standard deviation of 7.327516633 and for Sample B, a mean concentration of 5.00395 ng/uL with a standard deviation of 5.087568452 as shown by the taller bar for Sample A than Sample B in Figure 2. Sample A’s higher concentration is also expressed in Figure 1 as more intense bands.
The SybrSafe in the gel allowed the bands to be seen under a UV light relative to the ? HindIII standard. Well 2 and 9 are the HindII ladder. Wells 2, 3 and 4 are Samples A1, A2 and A3, respectively. Wells 5, 6, and 7 are Samples B1, B2 and B3, respectively.
*in 10uL of 500 ng DNA
**4.4 kb band cannot be used as an accurate measure of mass 1, p. 33
The mean concentration of Sample A is 37.50845 ng/uL with a standard deviation of 7.327516633. The mean concentration of Sample B is 5.00395 ng/uL with a standard deviation of 5.087568452. The means for each sample were derived by averaging the concentration of A1, A2 and A3 for Sample A and averaging the concentration of B1, B2 and B3 for Sample B. These individual concentrations were determined using a standard curve comparing the amount of DNA (ng) with band intensity.
According to the gel image in Figure 1 and the bar graph in Figure 2, Sample A was much larger in size and more concentrated than Sample B since it migrated less than Sample A and expressed a brighter band. The results show that band length is inversely proportional to distance traveled across a gel. Thus, the DNA size holds influence in determining how much it can migrate through a porous agarose gel. The sample concentration is expressed through how brightly a band may be relative to the standard. SybrSafe is a fluorescent dye in the gel that enables bands to be visualized under a UV light. As a result, more concentrated DNA samples express brighter bands. The intensities of Sample B bands were fainter than expected. A possible source of error could have been a faulty gel box and the time it took to run the gel after loading the wells. The metal conductors were not entirely screwed on prior to running the gel. It took a few minutes to realize that though the external source was turned on and the leads were connected that our DNA samples were not migrating. The samples could have sat in their wells long enough to potentially allow for dilution of the samples especially amongst the most recently added B samples. Agarose gel electrophoresis serves as a way to visualize the effects of differently sized and differently concentrated DNA in an agarose gel of a certain concentration.
1 M. Butler et al. Lab 1 in: Recombinant DNA Laboratory Manual. Hayden McNeil. Pp 23-24, 33 (2016)