Lab Agarose Gel Electrophoresis

Lab : Agarose Gel Electrophoresis: Introduction: According to NCBI, agarose gel electrophoresis is the most effective way of separating DNA fragments according to varying sizes and charges. The gel itself is porous, and the higher the concentration of the agarose the smaller the pore size and vice versa. The separation of proteins occurs fastest with small proteins and slowest in the larger proteins, however, different forms of DNA move through the gel at different rates and the electrophoretic mobility can be calculated Ze where z is the charge, e is the applied voltage, and f is the using this equation μ= f frictional coefficient. For example, supercoiled plasmid DNA moves through the gel the fastest because it is compact, then linear DNA fragment would move the second fastest, with open circular DNA moving the slowest. In this purification technique, loading dye is used to help track the movement of the DNA through the gel. The negative phosphate backbone fragments will migrate from the cathode or negative end to the anode or the positive end until the fragments reach the end of the gel. The leading model for DNA movement through the agarose gel is known as the biased reptation, this model states that the leading edge moves forward and pulls the rest of the molecule along (Smith et al). From this technique, one should be able to understand to the mechanism by which DNA fragments are separated within a gel matrix, how the conformation of the DNA molecule will determine its mobility through a gel matrix and determine the sizes of separated DNA fragments. Objective: The objectives of this experiment is to use the techniques associated with making an agarose gel from scratch for the electrophoresis, being able to load wells carefully with the samples, performing the electrophoresis experiment, and analyzing the results from the electrophoresis experiment. Procedure: In preparing the casting tray for the gel, the open ends of a clean dry casting tray were taped with laboratory tape. ¾ of an inch-wide tape was then taped over the sides and the bottom edge of the bed, with the extended tape edges folded back onto the sides and the bottom and the contact points pressed firmly in order to form a good seal. A comb was then placed in the set of notches at the top of the casting tray making sure that the comb sits firmly and evenly across the tray. Next, when casting the agarose gel, a flask or beaker was used to prepare enough gel solution of a 1.0% gel solution in order to make a 0.5 cm gel using the casting tray provided at the lab station. The table below was used to determine how much gel was needed for casting the tray. Casting Tray Type/Brand Dimensions (width x length) in cm Fisher Scientific (FB-SB-1320) 13 x 16 Bio-Rad Ready Sub-Cell GT MINI 7 x 10 For the larger tray, it was determined that 1.04 g of agarose powder was needed to cast the tray and for the small tray it was determined that 0.35 g of agarose powder was needed to cast the 1 large tray and both powders were added to 1x TBE buffer to separate flasks. The mixture is then swirled in order to disperse clumps found in the agarose powder and a magnetic stir bar is also added to the flasks in order to stir the solution in order to fully dissolve the agarose. A marker or pen with then used to indicate the level of the solution volume on the outside of the flask and the mixture was then heated in order to dissolve the agarose powder. Two methods of heating could have been used the microwave method to the hot plate method. If the microwave method was used, the flask was covered with paraffin wax in order to minimize evaporation, the mixture was then heated high for 1 minute and the mixture was then swirled with the heat on in bursts of 25 seconds until the agarose was completely dissolved. If the hot plate method of heating was used, the flask was covered with aluminum foil to minimize evaporation. Then the mixture was heated to boiling over a Bunsen burner while swirling occasionally. The mixture was boiled until all the agarose was completely dissolved and the heating will continue until the final solution appears clear without any dissolved particles. The solution was checked carefully, and if crystal particles were still present in the solution, that was an indication that the agarose is not completely dissolved. Once the solution was completely dissolved, the agarose solution was cooled to 55°C with careful swirling in order to promote even dissipation of heat, if detectable evaporation had occurred, distilled water is added to bring the solution to the original volume that was marked in step 3. Another gel was then cooled to 55°C and is placed on the previously prepared casting tray on a level surface and the 55°C agarose solution was then poured into the bed. The gel was then set aside to cool completely and solidify for 20 minutes. Finally, during electrophoresis, the tape was then removed from the ends of the casting tray and the gel was placed into the electrophoresis chamber. The comb was then slowly removed by pulling it straight up, this step was done carefully in order to ensure that the bottoms of the wells do not rip. The gel was then oriented so that the wells were near the negative (black) terminal. The electrophoresis chamber was then filled with 1X TBE, enough to completely submerge the agarose gel. A hole in the foil cover was then made for the sample that was loaded with the pipet tip in order to draw up the sample, the tip was then placed at the top of one of the wells and the tip should be submerged in the buffer. The sample was then pipetted into the well very carefully. A new pipet tip was used for each sample, and each sample was recorded from left to right to make sure that the samples don’t get mixed up when interpreting the data. A cover was then placed back on the electrophoresis and the connections were checked to make sure that the colors match. The electrical leads were then placed on their corresponding ports on the power supply and the area around the electrophoresis chamber was checked to make sure that the area was dry. The power supply was then set to 100v and turned on. Bubbles forming on the electrodes was an indication that the current is flowing properly. After 5 minutes the gel was checked to make sure the sample was migrating through the gel in the correct direction. After 3045 minutes, the power supply was shut off and the results were analyzed and recorded. Results/Discussion: Calculation of small container: Volume=( length )( width)(height) 3 Volume=( 7 )( 10)( 0.5 )=35cm =35mL 1% w/v = 1g/100mL 1g xg =0.35gagarosepowder = 100mL 35mL 2 Calculation of large container: Volume=( length )( width)( height ) 3 Volume=( 13 )(16 )( 0.5 )=104cm =104mL 1% w/v = 1g/100mL 1g xg =1.04gagarose powder = 100mL 104mL A 1. Where any of the colors pure, and did different dye pigments migrate different distances? 3 Answer: Yes, all of the colors were pure except for tubes E and F with E being a mixture of dyes and F being a mixture of blue dyes. Also, all of the different color pigments migrated different distances. 2. What are the main factors that help separate dye pigments? (list 3) Answer: The three main factors that help sperate dye pigments are the strength of the electrical charge, density of the gel, and shape of the molecules. 3. What charge is carried by the dye pigments, and how did you determine this? Answer: A negative charge is carried by the dye pigments, and this was determined because we know that opposite charges attract. Since the dyes are negative, they will migrate towards the positive end of the gel. 4. Which color molecule is most likely the smallest, and how did you determine this? Answer: The color molecule that is most likely the smallest is the orange dye because it is not mixed, and it has migrated the farthest out of all the other gels. 5. Compare the dye pigments in sample E to the pigments from samples A-D, what colors are in sample E? Answer: The colors that are in sample E include orange, purple, red and blue dye number 1. 6. What conclusion can be drawn from sample F? Answer: Sample F was made up of two different types of blue dyes, it can also be concluded that each of the dyes had opposing charges as one dye migrated to the positive end of the gel meaning it had a negative charge and the other dye had a positive charged which migrated to the negative end of the gel. Also, each dye has a different density because per the results, one dye migrated further than the other. Conclusion: In conclusion, agarose gel electrophoresis has been the best method for separating DNA fragments. Since its techniques were adopted in the 1970’s, it has proven most useful in labs all across the nation because it gives scientist a visual representation of the sizes of DNA segments. This technique also allows for scientists to learn the exact order bases in the length of DNA, and its versatility allows for scientist to advance in the field of biological science research.