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BIOL 1103L - GMO Results Gel Electrophoresis Assignment (10pts) Last week you isolated DNA from a food sample and performed PCR in order to amplify DNA to find specific genetic markers for GMO (Genetically Modified Organisms). In this lab, you will be analyzing your PCR products using a process called gel electrophoresis. Be sure to write out your responses in complete sentences. Remember how agarose gels and an electric current allow DNA to be separated and then visualized. 1. Syber Safe green nucleic acid stain is a common component of agarose gels, what is its purpose? (1pt) In agarose gel electrophoresis, nucleic acid stains play a crucial role in enabling the detection and measurement of DNA or RNA. These stains make it possible to analyze nucleic acid fragment patterns under UV light and to evaluate the success of electrophoresis. Stains are necessary for procedures like RNA and PCR analysis because they help with findings documentation and concentration estimation. Additionally, they are essential in the purification of particular DNA or RNA bands for use in subsequent processes. 2. What would happen if an agarose gel did not have a nucleic acid stain added? (1pt) It would be difficult to see and analyze the DNA or RNA bands on an agarose gel without a nucleic acid stain since they would be undetectable in normal lighting. Bands are visible thanks to stains, which are made to glow under UV light. This helps with band identification, concentration estimate, and electrophoresis success assessment. The usefulness of agarose gel electrophoresis is reduced when the dye is not used since it makes it more difficult to understand and record results.

3. How will you use the gel to identify if your genetic marker is present? (1pt) After amplifying the DNA region containing the marker using methods like PCR, gel electrophoresis is performed to determine whether a genetic marker is present in an agarose gel. After loading the amplified samples onto the gel, divide the DNA fragments into smaller and larger segments using an electric current. To see bands when the gel is exposed to UV light, stain it with a nucleic acid dye. Examine the resulting pattern in samples lacking the genetic identifier and in samples with a molecular weight marker. The marker's distinctive band will show up if it is present, making identification possible. Quantify with accepted benchmarks. Accurate interpretation is ensured by appropriate documentation and procedures. 4. What should the gel look like if no DNA was amplified from your PCR? (1pt) There shouldn't be any identifiable bands on the agarose gel that match to the target DNA fragment if your PCR failed to properly amp up any DNA. If your samples' DNA was not amplified, the gel lanes would probably show only a light background or be blank. To ensure that the lack of bands is caused by the absence of amplified DNA rather than a technical problem, it is imperative to include a negative control, which is a reaction without template DNA. Frequently, you should check if the predicted product is absent by comparing your results with a molecular weight marker to determine the size of any possible bands. 5. If DNA is present how will you know that it is actually the DNA you meant to amplify using your primers ( think about size )? (1pt) Compare the sizes of the observed bands with the anticipated size of the target fragment to verify the identity of the amplified DNA in the agarose gel. For reference, use a molecular weight marker that contains DNA fragments of known sizes. The successful amplification using the particular primers is further confirmed by the alignment of observed bands with the expected size and a positive control lane containing known target DNA. This size-based comparison guarantees that the DNA in the gel precisely matches the intended amplification using the primers that were designed.

Below is an example of an agarose gel that you need to interpret to answer question 6. PCR was done on 3 plant samples using primers that should result in a 400 bp (base pair) product. The PCR reactions, lanes 2, 3, and 4, were run along with a DNA ladder in lane 1. 6. Did each of the three PCR reactions work? Explain why/how you can determine this. (2pts) Only Lane 2 is properly working and delivers the 400 bps. The PCR in lane 3 is not operational. The PCR mixture can be lacking in some crucial components, which would prevent the gene from being amplified. The lane 4's 1 kbps is higher than the 400 bp that we anticipated. The primer we used is insufficient for our 400 bp product. One kilobases (bp) will be produced when the primer binds to another section of DNA. 7. Briefly describe the results of your experiment. Were the genetic markers of GMOs present? Explain how you interpreted your results. (3pts) For the hint of lime corn chips, the negative bands (5G-, 5P-) were absent because they serve as controls for the experiment. The 5G, 5G-, 5P, and 5P+ all had bands which means that the hint of lime chips contained GMOs.

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