In the preparation of agarose gel, agarose powder will be mixed with buffer. Agarose powders need

1. If each individual has such a small amount of DNA, how do the bands on the gel contain enough DNA to be visible?

charged DNA to bind the positively charged minicolumn. 2 ml of column wash is then used to

The idea behind Gel Electrophoresis is that we inject a slab of gel with the DNA we found at the crime scene. We then inject the same gel, next to the crime scene DNA, with suspect 1’s DNA and suspect 2’s DNA. We then send an electric current through the gel and wait for the results. The smaller molecules in the DNA will travel farther than the bigger molecules because the bigger ones will have difficulty making its way through the microscopic beads in the gel. After the separate bands appear in the gel, we stain it with a special chemical called Ethidium Bromide to give it a color under the blue

We placed the gel into the running chamber, and then we completely covered the gel with TAE. 3 microliters of loading dye was added to each tube; this would help distinguish the enzyme from the gel. As before, we tapped the tube on the table to mix. Then we carefully added each of the four samples into their own wells. A total of 33 microliters of each sample was poured into each well. Afterwards, we attached the positive and negative electrodes to their corresponding terminals on the power supply and gel box. We turned on the power to around 80 volts and waited 45-60 minutes for the loading dye to move down the gel approximately 6-8 cm. Finally, we were able to visualize the DNA in the gel and write down the

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

Using the electric current, scientists pass the DNA through gel, and as smaller molecules get through gel quicker than those of bigger sizes, DNA molecules get separated according the sizes of the molecules. We utilize the property that large molecules move slower, and DNA is slightly negatively charged(due to phosphate groups), so it will move to the positive pole of the gel.

it was previously streaked and slide it across on another side of the agar plate. Then using the

Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).

The more agarose that is used and dissolved, the firmer the gel will be. Typical concentrations used are between 0.3% to 2% (Buckingham, 2012). The concentration depends on the type of analysis needed. A higher concentration of agarose, making a stiffer gel, would be used to detect smaller DNA fragments (100-3000 bp) while a lower concentration would be used for larger fragments (5000-60,000 bp). For pieces 50,000 bp and over, pulsed field electrophoresis is used where an alternating current is applied (Buckingham, 2012). A gel comb is placed into one side of the gel mold to create holes (wells) in the gel. The gel will need to cool and solidify, about an hour. The comb is then removed, which leaves the empty wells in the gel.

Agarose is made from agar or polyacrylamide. With gel electrophoresis you can tell how many different things are put together to make one certain item because this process separates all the other ingredients in the mixture. Also, the macromolecules become coated in a negative charge during gel electrophoresis. The other side of the gel chamber is positive and since opposites attract, the macromolecules are pulled to the other side of the gel separating them. An electrical current is applied to the gel which causes the food coloring dye to migrate and separate through the holes In the gel.

In slab gel electrophoresis agarose or polyacrylamide is used. The conducting buffer is contained in the porous gel and the gel is then poured in between glass plates. These plates are separated by spacers. An electric field is applied at the rear after the substance of experimentation is added to the wells at the top. The substance will then move a certain distance towards the positive electrode. This method uses gel unlike the cellulose acetate strips but it still uses a buffer and a electric field to migrate the substances based on their size and charge. Slab gel electrophoresis is usually used in the biological

The Eppendorf tube was then placed within a centrifuge for 10 minutes to separate the insoluble DNA from the rest of the solution. When the 10 minutes were up, a DNA “pellet” appeared at the bottom of the Eppendorf tube and a pipet was used to decant the solution. Once as much liquid was removed from the Eppendorf tube as possible, 1 mL of 70% ethanol was added to the tube, and it was inverted another 5 times. The sample was then placed within the centrifuge for another 5 minutes at maximum speed. The DNA pellet should still be visible within the Eppendorf tube and using a pipet, the ethanol was decanted from the Eppendorf tube.

Each lab bench will make, and run one gel electrophoresis per table. Once the gel is ready to be loaded, load five microliters of PCR DNA ladder into the first well, as a standard. This should be found in a tube in and ice bucket. Next add two microliters of 6x loading dye into the six sample tubes. The dye should be mixed in thoroughly by gently pipetting up and down after adding the dye. Following that you should load fifteen microliters of each sample into the following six wells. Since lane one will have the DNA ladder lane two starts the samples using the orange tube, then the blue, yellow, red, green, and pink tubes go into lanes three, four, five, six, and seven respectively. Once all the samples are loaded turn on the electrophoresis machine, and wait until the bromophenol blue tracking dye has migrated at least half the length of the gel. Lastly using gloves carefully remove the gel and carry it to the UV light box to view, and photograph the gel (Hass C., Woodward D., and Ward A., 2010.).

Electrophoresis is a procedure used to separate molecules by the use of an electric field. Agarose is a gel that comes from seaweed extract. The polysaccharide polymer does not provide uniform pore sizes, but it does well with molecules that exceed a size of 200 kDa. It also is often used for DNA strands that have more than 50 base pairs. So, an agarose gel electrophoresis is used when separating fragments of DNA and RNA by size. The gel acts as a sieve, causing the larger fragments to travel more slowly and less distance than the smaller fragments.

Coarse aggregate passing 12.5mm IS sieve and retained on a10mm. IS sieve and heated at 100 to 110°C for 4 hours and cooled at the room temperature.