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Gel Electrophoresis Lab Report

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PCR Forensics using Gel Electrophoresis INTRODUCTION In our experiment, we used gel electrophoresis to observe the PCR products of two suspects’ DNA. In our experiment, we were trying to solve the following crime: “A man was assaulted one night when leaving the automated teller machine at his bank. An eyewitness had clearly seen a man fleeing the scene, and almost identified him from pictures of likely suspects. However, the man identified by the eyewitness has a brother who looks very much like him, although they are not twins. The eyewitness could not tell which man he had seen fleeing the crime scene. However, the victim scratched the attacker in self-defense. Forensics experts recovered samples of skin and blood from underneath the …show more content…

We started our procedure by filling the gel box with 1xtris-borate-EDTA (TBE) buffer until it was level with the gel and barely covered the surface of the gel. Next, we carefully removed the comb out of the gel. We prepared the DNA samples by placing the samples into a centrifuge. Next, we loaded the DNA inside the gel’s wells made by the comb. We recorded the order we were going to put the samples in, then started loading the wells. We were careful to use a different pipet each time we loaded a different DNA sample to avoid contaminating the samples. Finally, we closed the top of the chamber and connected the cords to the power source. We made sure the connectors were on the same channel by placing the red went into the red power supply and the black when into the black power supply. We turned on the machine on high (135 Watts) for 45 minutes. After that we disconnected the cords and removed the lid to be able to carefully get the gel into a staining tray. We put CarolinaBLU stain on the gel and let it set for 20 minutes. Next, we poured out the CarolinaBLU and added distilled water on the gel to rise the dye off the gel. We repeated this process, then added distilled water again and let it sit for 30 minutes. We then poured out the distilled water and was ready to look at our results in the

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After its melted and you don’t see any clumps you pour it into the mold to cast the gel and wait 20-30 minutes for the gel to set. Plug in your electrophoresis system and assemble the platform in the tank. After the gel sets remove it from the casting stand, setting the gel tray inside the tank on the platform making sure the wells are facing the negative side. Measure out 125 ml of diluted SB running buffer and pour slowly into one side of the tank, removing all air bubbles. Turn on the low intensity light and load your DNA samples using 10 μl per well, changing your pipette tip after every sample.

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Before we tested anything we had a sense that the blood had to be type A, B, AB, or O. The way we tested between Sam, our primary suspect and the murderer is by using gel electrophoresis. To make the gel we had to use buffer and agarose and this mixture is poured onto a plastic tray. A plastic comb is used to make loading wells, so we can insert the DNA in there. The gel box when turned on the opposite ends generates opposite charges so the DNA will pull downwards. The bigger pieces of the DNA will go down less while the smaller piece of DNA will go down further since it has an easier time with moving through the gel.

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It is evident that gel electrophoresis makes it possible to separate DNA fragments according to size

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We then ran the electric supply to the gel for about 45 minutes at 230 volts to separate the dye and notice how far the dye travels.

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The Primary stain, Heat, Rinse, and Counter stain. The Primary stain, stains the cells and not the spores with a Malachite Green. Then, in the heat step, you heat the cells and spores over boiling water which allows the spores to expand and allow the Malachite to enter the spore. Now both cells are stained. The third step, rinse with water, allows the Malachite Green to rinse out of the cells but not the spores.

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There are two stages of DNA fingerprinting, the Polymerase Chain Reaction (PCR) and the treatment of restriction enzymes (Polymerase Chain Reaction (PCR) (Theory) : Molecular Biology Virtual Lab II : Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab). PCR can amplify a single or several fragments of DNA which is significant because if the piece of DNA is too long, it is not able to be used in this lab. Restriction enzymes help compare DNA between suspects by cutting its palindromic

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The pellet was washed by slowly adding 500 μl of 70% ethanol before being centrifuged for 2 minutes. The supertanent was removed and the DNA pellet was allowed to dry for 5 minutes. The pellet was resuspended in 200 μl of DNA Resuspension Buff by vortexing. The samples were then placed in ice to

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Separation of various DNA fragments by size will be done using a capillary electrophoresis machine by forensic scientists. By doing this, it is quite easy to calculate the number of repetitive units at each site and the length of each STR region. One’s STR repeats must match with the crime scene sample in order to convict a suspect (Winconsin Magazine, 2013). If they are perfectly matched, it is roughly guaranteed that he has got his culprit. Even though there is a single STR mismatch, it is enough to exonerate a suspect (Eugene Garfield, 1989).

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First, the DNA must be amplified through a process known as Polymerase Chain Reaction. PCR can amplify a small amount of DNA to produce a large amount to work with in just a few hours. The DNA can then be separated through Gel Electrophoresis. The DNA strands must be cut by a restriction enzyme, then loaded into an agarose gel. It is important to know which strand of DNA is whose.

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Then, it is air dried and followed by fixing it with flame from Bunsen burner. After fixing the smear, it must be stained using Gram staining solution, firstly crystal violet solution was flood onto it, and allowed for 1 minute, then wash off with tap water. Then, flood the slide with iodine solution for 1 minute and wash it off with tap water again. The formation of a dye-iodine complex will occur in the cytoplasm. Then, it was flooded with ethanol and washed immediately.

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To the double digest tube was added 12 μL double digest master mix, and 2.5 μL sterile dH2O. To the single digest tube was added 11 μL single digest master mix, and 3.5 μL sterile dH2O. To the undigested tube was added 10 μL undigested master mix, and 4.5 μL sterile dH2O. To prepare the unknown plasmid digest, 6.1 μL 41.1 ng/μL of isolated non-recombinant plasmid DNA was pipetted into three micro centrifuge tubes. To the double digest tube was added 12 μL double digest master mix, and 1.9 μL sterile dH2O. To the single digest tube was added 11 μL single digest master mix, and 2.9 μL sterile dH2O. To the undigested tube was added 10 μL undigested master mix, and 3.9 μL sterile dH2O. Digests were then incubated at 37 °C for one hour and then stored at -20 °C for one week. After which the digestions were examined by gel electrophoresis. The samples were run on a 50 mL 0.9% (w/v) agarose gel in 1X TAE buffer at 100 V until the leading track dye traveled 2/3 the distance of the gel.

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Through enzyme digestion experiments, I am able to understand the role of restriction enzymes in bioengineering. And I can clearly see the length of the fragment when the restriction enzyme cleaves DNA. This experiment also made me gain knowledge about DNA analysis. Through the experiment of Restriction Endonuclease Digestion of DNA, I learnt that when using RedSafe stain and the UV light source, I should be careful because their mutagenic effects. Moreover, this experiment makes me understand the role of gel electrophoresis in DNA preparation and analysis.

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In tropical countries, due to high ambient temperature sample degradation is common problem. Particularly in India, there are fewer diagnostic laboratories so the samples have to be transported over a considerably larger distances. The field personnel are also inadequately trained for collection, preservation and transport of samples. All these factors pinnacle the importance of DNA based forensic methods.

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