Lab Report Chemistry Lab

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

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

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.

When picking up the gel, the gel broke in half. This could have altered the results because some of our DNA could have broken off. Although it appeared that we saved the DNA, there is a slight chance that some of the DNA could have broken off. Also, my assistants and I had difficulty measuring the agarose powder. We might have poured too much of the powder, which could have made the gel too thick. Next time, we will be more cautious when measuring the powder to have more accurate results.

Purpose: is to determine the unknown bacteria with a variety of biochemical tests. There are many reasons that contribute to why it is so important to test patients for both high and low risks diseases. The most important reason would be to know the identity of microorganism and how it can be treated .This study was performed in microbiology laboratory class by applying the microorganism to the tests that have been performed in the class prior to the identification of the unknown. First, the lab professor handed out a bacteria that was on the unknown streak plate labeled B5 that consisted of an unknown gram positive or gram negative bacteria.

The reason for that is that I believe the TBE buffer in the 1.5% agarose was a little strong which caused the solution mixed into the agarose to spread all over which caused the UV lights to not pick up the concentration of the DNA. As I need more research I came up on the reasoning as to why we use agarose gel electrophoresis, it is because it is more effective with the separation of DNA fragments. That explains the reasoning as to why the separation did not work as well with the 1.5% agarose gel it may have been either a little strong or weak. The gel that came out better was the 3% agarose gel because of the strong concentration of the gel. With troubleshooting agarose gel the important things to know is that if the gel is faint or no bands then there was not enough concentration of DNA and you want to add more DNA.

Once the protein goes through the pores of the gel based on negative charge, if the charge is high, then the faster it moves in an electric field. The shape is important to know based on shape how the protein would travels through it, for example a round shape will make the protein move faster than rod shape due to the rod will bend which this will cause the protein to stop. The size of the is important to determine whether its big or small if its big then the protein are able to have difficulties to get through the pores and move slowly. The protein travel differently than other natural protein in the gel. The mutation change impacts the negatively charge amino acid with hydrophobic amino acid, which cause the protein to change the charge and shape. This process is to either introduce or eliminate a restriction enzyme, which allows it to eliminate the pieces of DNA whether they have it or not. Each of the restriction enzymes is able to identify and eliminate it, which cause the DNA not a normal DNA because of the size of fragments that can be apart from using gel electrophoresis. This causes the DNA fingerprint to change by different pattern on the

Our materials for the extraction of DNA included: the screwcap tubes, capless tubes, pipet tips, disposable transfer pipets, a foam microtube holder, a DI water bottle, a sharpie to label, a hot water bath, Non-GMO food, Test food, and a centrifuge. Our materials for the setup of the PCR reactions included: PCR tubes, screwcap tubes, pipet tips, an aerosol barrier, a foam microtube holder, a PCR tube holder, a 2-20 microliter micropipette, an ice bath, a sharpie to label, a plastic bag, a styrofoam cup with ice water, and a black tray with ice water. Finally, the materials for the electrophoresis of the PCR products included: Agarose gel, PCR sample tubes from previous labs, PCR tube holders, running buffers (300-350 ml), loading dye, a PCR molecular weight

According to data provided in class, the smallest band of Hind III shown on the curve at 2.02 kbp should have traveled 24 cm. As shown in table a, the band with 2.02 kilo base pairs traveled 20.2 cm. III fragment that contained 23.1 kbp traveled only 7.30mm when it should have traveled 11.0 mm. However, the fragment that had 4.36 kbp should have traveled 18mm when it traveled 19mm. This shows the enzymes did not cut right where they were supposed to, resulting from not allowing the fragments to run long enough. An extra half hour or so would have allowed enough time for the fragments to be cut in the right places. After generating a standard curve shown above in example c, the EcoR I fragment sizes were estimated. The largest fragment traveled only 6.79mm, and it was estimated that the fragment was 23.8 kilo base pairs in size. The actual fragment size derived from the provided restriction map was 21.2 kilo base pairs in size. The second smallest fragment traveled 11.6 mm and it was estimated that the fragment was 6.01 kilo base pairs in length. The actual fragment was 5.6kpb in size. The majority of the estimated EcoR I fragments were larger than their actual fragments. This was the result of the Hind III fragments not reaching their end distances in the gel. Since the standard curve was made from the partially traveled Hind III fragments, the estimated numbers of EcoR I were off. The creation of a standard curve was a good way to estimate the DNA fragments sizes, because both the sets of DNA were run at the same time for the same length of time. If one does not have adequate time to travel as far as it should, neither will the other sample being run. EcoR I produced fewer bands than predicted by its restriction map because the smallest band may contain too little DNA to be seen without using a more sensitive dye to illuminate the

The separation of macromolecules for laboratory analysis can involve the use of any of several techniques that isolate macromolecular components on the basis of size, solubility, reactivity, and volatility. Electrophoresis, for example, is a method of macromolecular separation that involves the use of a suspending medium, known as the matrix, and an applied electrical current that separates molecular components based on varying size and reactivity. While a uniform electrical current may activate the movement of molecular components through the matrix, the sponge-like structure of gel matrixes typically used in nucleic acid electrophoresis, such as agarose or polyacrylamide, inhibits the uniform migration of these components through the matrix

To visualize the DNA, the gel is stained with a fluorescent dye that binds to the DNA, and is placed on an ultraviolet trans illuminator that will show the stained DNA as bright bands. The dye can also be mixed with the gel before it is poured. If the gel has run correctly the banding pattern of the DNA marker/size standard will be visible. It is then possible to judge the size of the DNA in the sample by imagining a horizontal line running across from the bands of the DNA marker. The size of the DNA can be estimated in the sample by matching them against the closest band in the

Gel electrophoresis is a laboratory method that allows for the separation of nucleic acids (DNA or RNA) and proteins based on their size. It utilizes a porous gel matrix through which proteins or nucleic acids migrate. Both nucleic acids and proteins can innately and synthetically possess a net-negative electrical charge, a property that is leveraged to facilitate the migration of the desired molecule through the medium. Gel electrophoresis can be conducted in either a horizontal or vertical orientation. Horizontal gels are typically composed of an agarose matrix, while vertical gels are generally composed of an acrylamide matrix.

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.

DNA fingerprinting is used as a forensic skill in criminal investigations to identify an unidentified person or whose identity needs to be confirmed. A tissue sample is obtained and the DNA is extracted. Fragments of different lengths are made by cutting up the DNA. The fragments are split up and visualized in some way and is compared with another sample. In our lab, we practiced a method called Gel electrophoresis a technique commonly used in labs and clinics to separate charged molecules like RNA and DNA according to their size. DNA is negatively charged providing an electric current to the gel, DNA will travel towards the positively charged electrodes. The Short strands of Deoxyribose move quickly through the gel template than longer strands