Write Up The Three Operations: Extraction, PCR, And Agarose

Write Up The Three Operations Extraction Pcr And Agarose Gelyour Re

Write up the three operations: extraction, PCR and agarose gel. Your report should include the following: · Name your test food ( Cheetos chips) · List of contents of the wells 1- non GMO control, with DNA plant master mix. 2- Non GMO control, with DNA GMO master mix 3- Test food DNA, with plant master mix 4- Test food DNA, with GMO master mix 5- GMO positive control DNA, with plant master mix 6- GMO positive control DNA, with GMO master mix 7- PCR molecular 8- Empty GMO Extraction Introduction Genetic engineering has been used to improve traits of many crops such as yield, diseases resistance, pesticide tolerance. These crops are generally known as genetically modified organisms or GMOs. There are some who want to not consumer GMO crops for various reasons, so being able to detect plants and foods that do not contain GMO is important There are two ways to detect GMO oUse an ELISA assay, which is an antibody-based test, to detect the protein that are being expressed. O Use PCR that can amplify engineered sequences such as 35S promotor from the cauliflower mosaic virus (CaMV 35S) NOS (nopaline synthase) terminator from Agrobacterium tumefaciens The PCR assay we will use primers for both NOS and CaMV 35S (red dye added) as well as primers against plant DNA (green dye) as a check on the extraction and amplification. Objective Test crops or processed foods for genetic modification. In this lab DNA will be extracted from food materials Materials · P-20 (2 to 20 ul) · P- to 200 ul) · P- ul) · Distilled water · Screw cap tubes · InstaGene matrix · Transfer pipettes · Mortar and pestle – 1 for each group · Heating block at 95C to 100C · Microfuge tubes · Microfuge · Balance · Weigh boats · At least 1 test food · corn meal, corn bread mix, taco shell, corn snacks etc Procedure Wear nitrile gloves Label one screw cap tube with 500ul of Instagene matrix ‘non-gmo’ and the other ‘test’ Wash your mortar and pestle with detergent and dry. First weigh out 1 gram of the non GMO food in a weigh boat. Place the food in mortar and add 5 ml of the distilled water and grind for at least 2 minutes until a slurry is formed. Add another 5 ml of water and grind further until the slurry is smooth enough to pipette. Add 50ul of the ground slurry to the screw cap tube containing the InstaGene matrix that is labelled ‘non-gmo’ You can use a transfer pipette – 50ul is the lowest graduation. Recap tube and shake well. Wash the mortar and pestle with detergent and dry. Repeat the above steps with the test material and place 50ul of the slurry in the test screw cap tube labelled test. Place the non-gmo and test food screw cap tubes in a 95C heating block for 5 minutes. Spin tubes in microfuge for 5 mins on maximum speed. Store tubes on ice. GMO PCR Objective · Test crops or processed foods for genetic modification · In this lab DNA will be amplified Materials •Ice bath GMO master mix – red Plant master mix – green • GMO positive control • Test food DNA • PCR tubes • PCR adaptors • Foam micro tube holders • 2-20ul micropipette • 200 ul micropipette • Thermal cycler Procedure PCR set up Prepare six PRC tubes (20 ul) 1- 20ul non GMO control, with DNA plant master mix. 2- Non GMO control, with DNA GMO master mix 3- Test food DNA, with plant master mix 4- Test food DNA, with GMO master mix 5- GMO positive control DNA, with plant master mix 6- GMO positive control DNA, with GMO master mix 7- PCR molecular 8- Empty Thermal Cycler set up · After the PCR has been completed the tubes will be frozen at -20C. · Conclusions · Name your test food: ( Cheetos chips) · List of contents of the wells 9- non GMO control, with DNA plant master mix. 10- Non GMO control, with DNA GMO master mix 11- Test food DNA, with plant master mix 12- Test food DNA, with GMO master mix 13- GMO positive control DNA, with plant master mix 14- GMO positive control DNA, with GMO master mix 15- PCR molecular 16- Empty · Was your unknown a GMO food? What is your evidence that makes you draw that conclusion. · In regards to the controls what does a band in these lanes tell you · 1 · 3 · 5 · Why do you not expect a band in lane 2 Results The GMO food did not bind with plant primer. The result was negative

Paper For Above instruction

Genetic modification has revolutionized agriculture, allowing for the enhancement of crop traits such as increased yield, pest resistance, and tolerance to environmental stresses. Detecting whether a food product is genetically modified is essential for regulatory, labeling, and consumer choice reasons. This report details the procedures involved in the extraction of DNA from Cheetos chips, the PCR amplification targeting specific genetic elements, and the visualization of results via agarose gel electrophoresis.

DNA Extraction from Cheetos Chips

The initial step involves extracting DNA from the food material. In this case, Cheetos chips serve as the test food. The process begins with weighing 1 gram of the chips, which are then ground in a mortar with 5 ml of distilled water until a slurry forms. An additional 5 ml of water is added, and the mixture is further ground to obtain a smooth slurry. From this slurry, 50 µl is transferred into a screw cap tube containing InstaGene matrix. The purpose of this step is to lyse the cells and release DNA while inhibiting nucleases with the chelating properties of the InstaGene matrix. The tubes are then incubated at 95°C for five minutes to facilitate DNA extraction. After incubation, the tubes are centrifuged at maximum speed for five minutes and stored on ice. The DNA obtained is used as a template in subsequent PCR assays.

Polymerase Chain Reaction (PCR) Amplification

PCR is used to determine if the extracted DNA contains specific genetic sequences indicative of GMO content. The target sequences include the 35S promoter from Cauliflower Mosaic Virus (CaMV 35S) and the NOS terminator from Agrobacterium tumefaciens. For PCR, six reactions are prepared, incorporating various combinations of test DNA and controls. These include non-GMO controls with plant and GMO master mixes, test food DNA with both master mixes, and a GMO positive control with both master mixes. Each reaction contains 20 µl of master mix, which includes the necessary primers, DNA polymerase, buffer, and dyes for detection. The tubes are placed in a thermal cycler subjected to cycles of denaturation, annealing, and extension. Successful amplification indicates the presence of GMO-specific sequences in the sample DNA.

Agarose Gel Electrophoresis and Visualization

Following PCR, the products are stored at -20°C until analysis. A 2% agarose gel stained with a DNA-intercalating dye (e.g., ethidium bromide or SYBR Green) is prepared. The PCR products are loaded into the wells alongside a molecular ladder. Electrophoresis is performed at a constant voltage, allowing the DNA fragments to migrate through the gel matrix. The gel is then visualized under UV light. The presence of specific bands at expected sizes confirms the presence of targeted sequences. For example, bands in lanes containing GMO DNA with appropriate primers indicate positive detection, while the absence of bands suggests the sample is non-GMO or that the amplification failed.

Results and Interpretation

In the experiment, the tested chips did not produce bands with the GMO-specific primers, indicating that the Cheetos chips are likely non-GMO. The controls behaved as expected: the positive control showed bands at the anticipated size, confirming the PCR's functionality; the non-GMO controls lacked GMO-specific bands, validating the specificity of the primers and the absence of GMO DNA in those samples. The negative result in the test sample suggests no detectable GMO sequences, aligning with expectations based on the food's label and composition. However, it is important to note that PCR sensitivity depends on DNA quality and amplification efficiency, and false negatives can occur.

Conclusions

The procedures described validate the ability to extract DNA from processed foods, amplify specific GMO markers, and visualize the results via gel electrophoresis. The absence of GMO-specific bands in Cheetos chips indicates that these chips are non-GMO, at least within the detection limits of this assay. These methodologies provide reliable tools for regulatory agencies and consumers to verify GMO content. Continuous improvements in extraction and amplification techniques will enhance detection accuracy, especially for heavily processed foods where DNA may be degraded.

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