Comparing The Non-GMO Sample, Taq DNA Synthesis And

20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.

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.

The purpose of this experiment is to use PCR method to identify genetically modified foods. During the experiment we, as a group, tested a known non- GMO food sample, oatmeal, along with our cornbread mix. We first weighed out 0.77 g of the non- GMO oatmeal. We then proceeded to add 3.85 ml (5.00 ml per 1.00 g) of distilled water (DW) to the oatmeal and ground them together with a pestle. The same was done with 0.99 g of the cornbread mix and 4.95 ml of DW. We pipetted each of these into separate screw-cap tubes consisting of 500 l of an InstaGene solution. These we incubated and centrifuged for approximately 5 minutes each.

To perform each step listed above we needed certain reagents for each process for DNA Extraction we needed a control non-GMO food sample and a testable food sample. Water and a mortar and pestle to grind the sample. Insta gene Matrix was added to two tubes and a water bath. To perform PCR Reactions we needed DNA template that were going to amplify, DNA polymerase, two DNA primers, four DNA base pair subunits and buffers. For electrophoresis we needed PCR product that we collected, molecular weight ruler, and fast blast for staining.

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).

Gel electrophoresis is a procedure used in laboratories to separate DNA, as well as RNA and proteins. A gel slab is placed in a buffer-filled box and an electrical field is applied. The negatively charged DNA will migrate towards the positively charged side, where it can then be recorded and further analyzed.

This is now the solution to load onto the argrose gel. Once completed the first stage of the experiment then continue onto the Gel electrophoresis which is before the isolation of your DNA: 1. Make a 2% agarose gel; add water and add TBE buffer 2. Heat the solution to completely dissolve the solution. (No crystals should be present) 3.

The columns were centrifuged at 15,000 x g for 1 minutes to remove the supernatant. 0.5 mL of Buffer QG was added to each column to remove trace amount of agarose, and 0.75 mL of Buffer PE was added to wash the columns for 5 minutes. Finally, the DNA was eluted with 35 µL of Buffer EB into a clean microcentrifuge tube. The elution buffer was allowed to soak in the membrane for 3 minutes before being centrifuged. Eluted DNA samples were stored at -20

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

Let the agarose powder sit in the buffer for a few minutes. The beaker is covered with plastic wrap an placed in the microwave. Microwave the solution slowly with boiling it. As soon as the solution starts to boil, take it out and carefully mix it with the hot gloves on and continue to heat the solution until it is completely clear. Once the solution is cooled, add 3µL of ethidium bromide stock to the solution and mix it by swirling it. The gel is poured into the prepared mould that is taped on the ends and eliminate any bubbles. Place the comb on the negatively (-) charged side. After the gel solidifies, remove the comb and tape and place the gel into the chamber. Gently pour TAE buffer over the gel as the gel should be completely covered by 2-3 mm of buffer. If air bubbles form, gently displace them with a disposable micropipette tip. Droplets of prepared loading buffer are placed on a piece of parafilm paper. The ladder will consists of only the buffer because it will work as a measuring device to compare the DNA samples. 10 µL from each of the DNA samples will be mixed with the loading buffer using the micropipette tip. The positive control will be consist of 10 µL of water and the droplet of loading buffer. The samples including the ladder and positive control are added to a separate well in the gel. Once all of the samples are loaded into their own

Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)

Agarose Gel Electrophoresis refers to the movement of a charged particle in an electrical field, usually performed for analytical purposes, and is routinely used for the preparation and analysis of proteins, and DNA – often after amplification of DNA. It is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. DNA is negatively charged and when placed in an electrical field, DNA will migrate towards the positive pole (anode). An agarose gel is used to slow the movement of DNA and thus separates the size of molecules based on their rate of movement through the gel under the influence of an electrical field.

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of DNA or proteins in a matrix ofagarose. The proteins may be separated by charge and/or size