Electrophoretic Mobility Shift Assay Lab Report

This experiment will be done as the first step in determining an unknown protein. The second part of the protein determination will be done using the Western blot technique. We will use the Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) method to separate proteins by their molecular weight. This method is often used to determine the purity and weight of a protein. Electrophoresis is a method of separating proteins based on their chemical and physical properties. Gel electrophoresis has a gel support mediums that includes polyacrylamide, starch and agarose. The gel is chemically inert, it is easy to handle, and can me made to fit a desirable porosity. This makes it good to use for molecules with high molecular weights. After separating the gel, it will be stained with Coomassie Blue to visualize the protein.

Today with my group lab we had to discuss on how to design an experiment that investigated the two herbicides DCMU and DCPIP. We used Elodea leaf and Elodea is basically a genus of aquatic plants often called the waterweeds and can be found anywhere in a pond or under water. So we started by creating a solution of phenol red, by adding 5 drops of concentrated phenol red to 40mL of water, then one of our group member used a straw to gently blow air into the solution until it reaches a neutral pH. We then transferred the solution into two test tubes evenly and labeled each tube dark and light, meaning the dark one will stay in the dark and the light one will stay under light bulbs. We predicted that dark one will stay the same and the light

The results of our experiment were similar to the actual isoelectric point of BSA with the exception of one data record. The calculated pI for BSA was approximately 4.5 after experimentation in comparison to 4.7, the real pI value of BSA. The isoelectric point was calculated after graphing the averages of each buffer solution and taking the x-intercept of the data. The isoelectric point detailed the pH at which the substance had a net charge of zero. The second strip of pH 6.0 with a value of +5mm, was omitted from calculations to improve the accuracy and precision of data. Buffers with a pH greater than the isoelectric move towards the negative end. Most of the buffer solutions with a pH of 4.7 and greater from the data, migrated to the negative

(…) Researchers can edit genes by ridding the faulty genes and replacing the lettering(coding) with the CRISPR-Cas9.

In this experiment, we tested our hypothesis through the use of a PCR and gel electrophoresis. To begin this experiment, we gathered the materials needed. We gathered a screw cap tube that contained the instagene matrix, a 1.5 ml tube, and a small cup containing 0.9% saline solution, P-20 micropipette, P-1000 micropipette, pipet tips, PCR tubes, cap-less microtubes, Master Mix, and water baths. Next, I rinsed my mouth thoroughly for 30 seconds with the saline solution and spit it back into the cup. I then set my P-1000 micropipette to 1,000 μl and transferred 1 ml of my rinse into the test tube. Then, I used the centrifuge to spin the tube at full speed for 2 minutes. I did not have a large enough pellet, so I centrifuged it again for another two minutes. I poured off the supernatant, but I left a small amount of saline at the

CRISPR-Cas9 is not an artificial construction; bacteria use it as a form of adaptive anti-viral immunity. When infected with a pathogen, bacteria that has this system retain a portion of the infecting virus in their chromosomal DNA. The cell then transcribes those sequences (called CRISPRs, or “clustered regularly interspaced short palindromic repeats”) and processes them into short RNAs called crRNAs. The crRNA guides the Cas9 nuclease to its target (normally, an invading viral nucleic acid) and cuts the foreign nucleic acid, rendering the virus useless in the process.

The purpose of this study was to study the plasma membranes of erythrocytes of the human and determine how the majority of polypeptides work with it and where they are located with respect to it. The purification of the erythrocytes was executed by osmotic hemolysis and were washed to the point of being “ghosts”. The polypeptides were then analyzed on the basis of mass by polyacrylamide gel electrophoresis; sodium dodecyl sulfate (SDS) was used to denature the proteins and separate them from the lipids. Furthermore, the location of the membrane proteins were determined by altering the environment around the membrane and recording how they responded. The major conclusion obtained from this study was that the distribution of proteins along the inside and outside of the plasma membranes of erythrocytes exhibits absolute asymmetry.

Gel Electrophoresis is a technique used to separate macromolecules fragments like DNA and protein by their size. Also, the proteins can be separated according to their size and their charge of different proteins with the different charges. Therefore, the material being separated and is placed into a gel-like substance called agarose gel. Because each DNA molecule is negatively charged, it can be pulled through the gel by an electric field. Small DNA molecules move more quickly through the gel than larger DNA molecules. (Biotechnology) For example, the DNA are separated to get a DNA fingerprint for forensic purposes, to get a DNA fingerprint for paternity testing, to check a PCR reaction, and to get a DNA fingerprint so that you can look for

Gel Electrophoresis is used to separate macromolecules like DNA and RNA by their size, or proteins by their charge and size. In this experiment, I used the Gel electrophoresis to determine the presence of all dyes in a specific dyes mixture.

The plasma membrane of a cell is like the skin of a human. It is selectively permeable which means it selects what it wants to allow into the cell and blocks out what it doesn't want. The cell membrane is made up of a phospholipid bilayer which contains 75% phospholipids, about 20% cholesterol and 5% glycolipids. Phospholipids are amphipathic which means they contain both polar and non-polar parts, these are their polar heads and non-polar tails.

CRISPR-cas9 allows researchers to edit part of the genome by inserting, deleting, or altering sections of the DNA sequence. The CRISPR-cas9 system consists of two key molecules: Cas9 and single-guide RNA (sgRNA). The enzyme Cas9 is ‘guided’ by sgRNA to the DNA sequences where it cuts the two strands of DNA at the specific location in the genome. Endogenous DNA repair mechanisms try to repair the breaks in both strands which then leads to indels that can cause mutation in the target gene. This process is shown in Figure 1.1. To determine whether CRISPR–Cas9 can be used to understand gene function in human preimplantation development, the POU5F1 gene that encodes the development regulator OCT4 was selected as the target. It was predicted that

We could use CRISPR-Cas 9 complex to determine whether WRKY75 transcription factor directly regulates FLS2 expression. First, we would use two sets of root samples, a control sample with regular a WRKY75 promoter, and an experimental sample that the WRKY74 promoter has been knocked out by CRISPR-Cas 9 complex. Next, we looked up the WRKY75 promoter sequence in the NCBI database and created a guide RNA sequence from the DNA sequence of the particular site we are looking for to cleave. Then, this will allow the Cas9 to come, bind and cleave out the WRKY75 promoter. Once the WRKY75 promoter is being knocked out, transcription factors would not be able to bind and FLS2 gene expression would not be turned on in theory. After that, EMSA could be used to detect whether the WRK75 binds to the FLS2 promoter and

If genetic transformation allows the insertion of a gene into an organism to gain the trait that the inserted gene codes for, then when 250 µl of transformation solution(CaCl2) and 2-4 colonies of bacteria, E. coli, are added to two micro test tubes, labelled +pGLO and –pGLO, with a loopful of pGLO DNA solution in the +pGLO tube, and are incubated for 10 minutes, then transferred to a 42ºC heat bath for exactly 50 seconds, and then incubated in ice again for 2 minutes for a heat shock, and then have 250 µl of LB nutrient broth, and lastly 100 µl of the solutions spread across the surfaces of 4 LB nutrient agar plates, LB/amp, LB/amp/ara, LB/amp, and LB, with +pGLO, +pGLO, –pGLO, and –pGLO used, respectively, the plates with the –pGLO solution will not glow under a UV light after they have

c. The results were analyzed by 3 steps. First of all, specific regions of the genes were PCR amplified and visualized on the gel. Secondy, T7EN1 cleavage assay were used to detect sgRNA:Cas9 mediated on target cleavage of specific genes. Finally, PCR amplified regions were cloned and sequenced to determine the deletions or insertions.

In the cell membrane transport lab, there were many experiments that were done such as osmosis, diffusion in a gel, diffusion in a liquid, diffusion in air, and filtration, A cell membrane transport lab is done to understand the different ways of transport and why they are all important since it relates to the human body.