What Is Southern Blotting

Based on your procedure and results, how is DNA isolated from plant or animal cells? /4

(PCR), which isolates small fragments of DNA that have a high degree of variability from

This is done through X-ray crystallography and NMR spectroscopy technology and will continue to be an essential method of testing various biological hypotheses3. DNA sequencing and genomic mapping is a newly introduced technology that decodes the individual chromosomes, genes and nucleotide sequences through new technologies, sequencing data, and computerized maps. The quickly

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

The proteins are also added to a Laemmli sample buffer in order to give each protein a negative charge so it is able to get pulled through the polyacrylamide gel. The next step is to put the gel into the electrophoresis module and to run it. It is run until the proteins have almost reached the bottom of the gel. A blue tracking dye is added to the Laemmli sample buffer in order to track the distance in which the proteins travel through the gel. If it is run for too long, the proteins will run off the bottom of the gel and it will mess up your results. Once the protein reach the bottom of the gel, the gel is stained in order to be able to see the individual bands of the different proteins. When the gel is stained, the protein distances will be able to be measured and compared. For a detailed procedure, refer to the Comparative Proteomics Kit I: Protein Profiler Module Lab Manual.

Spin the two tubes in a centrifuge for 5 minutes on opposite side of the centrifuge. The bacterium will collect at the bottom of the tube, so pour out the extraneous supinate. Then, add 250 microliters of buffer. The Ca2+ cation of the buffer neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane allowing the DNA to pass through the cell wall and enter the cells. Place both tubes on ice. Then add 10 microliters of water into one tube and 10 microliters of plasmid DNA into another tube labeling the one with DNA with a + and the one with water -, and place on ice for 10 minutes.

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

Our first step was that we boiled the samples, A&M Std , & Kaleidescope Std. for 5min. in water bath, then we loaded the samples into the wells following the guide given which was as follows: 1- 10 ml of laemmli buffer, 2- 10 ml of molecular weight, 3- 10 ml of salmon, 4- 10 ml of tilapia, 5- 10 ml of catfish, 6- 10 ml of shrimp, 7- 10 ml of actin and myosin, and 8- 10 ml of the laemmli buffer. To load each sample, we used a P-200 (yellow) micropipette tip to withdraw 10 ml of each protein sample from its tube and gently transferred it into the designated well. After loading all samples, on both of the gels we then placed the lid on the tank, and insert the leads into the power supply, matching red to red and black to black. We ran the gel for 45 minutes at a constant voltage of 115V. When the gels were finished running, we discarded the buffer from the inner chamber, we released the cams, and removed the gel cassettes from the assembly. We laid each gel cassette flat on the bench with the short plate facing

Following this action, in tubes 1 and 3 15-µl of Enzyme 1 was dispensed and the same amount was dispensed for tubes 2 and 4 of Enzyme 2. The final volumes of the each of the reaction tubes had a final volume of 40-µl (Table 1). After dispensing what was needed in each of the tubes, one of the team members tapped each tube to mix all the contents together and to be sure that the contents were at the bottom of each tube. Sequentially, after all the contents were mixed and were at the bottom, the tubes were then placed in an incubator at 37°C for 45 minutes (Upadhayaya, 2016). Prior to removing the DNA samples from the incubator, the Gel for Electrophoresis had to be casted.

60uL of each sample transferred to Eppendorf tubes, added with a 30uL sample buffer and heated at 95-degree Celsius for 4 minutes. The electrophoresis apparatus set up and filled with Tri-HEPES SDS running buffer. A 25uL of each sample loaded in this order from right to left: ladder, filtrate, S1, P1, P1 medium salt, P1 high salt, S2, P2, P2 medium salt, P2 high salt. The gel ran for 50 minutes at 110V and analyzed with UVP analyzer.

Next, an ice bath was made by filling up a beaker with ice, them filled it with about two-thirds of tap water. Then, ethanol and the DNA extraction was poured into separate medium-sized test tube and set in the ice bath. Then, six layers of cheesecloth was gathered and cut into square and was fitted into a large funnel. A large test tube was attached to a ring stand and set the funnel and cheesecloth into the top of the large test tube. Then, the strawberries were placed in a clean ziplock bag.

Let the agarose powder sit in the buffer for a few minutes. The beaker is covered with plastic wrap an placed in the microwave. Microwave the solution slowly with boiling it. As soon as the solution starts to boil, take it out and carefully mix it with the hot gloves on and continue to heat the solution until it is completely clear. Once the solution is cooled, add 3µL of ethidium bromide stock to the solution and mix it by swirling it. The gel is poured into the prepared mould that is taped on the ends and eliminate any bubbles. Place the comb on the negatively (-) charged side. After the gel solidifies, remove the comb and tape and place the gel into the chamber. Gently pour TAE buffer over the gel as the gel should be completely covered by 2-3 mm of buffer. If air bubbles form, gently displace them with a disposable micropipette tip. Droplets of prepared loading buffer are placed on a piece of parafilm paper. The ladder will consists of only the buffer because it will work as a measuring device to compare the DNA samples. 10 µL from each of the DNA samples will be mixed with the loading buffer using the micropipette tip. The positive control will be consist of 10 µL of water and the droplet of loading buffer. The samples including the ladder and positive control are added to a separate well in the gel. Once all of the samples are loaded into their own

Southern blotting is a method routinely used in molecular biology to check for the presence of a DNA sequences in a DNA sample. It combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method was invented by the Edwin Southern. In this method, DNA samples are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane can then be probed using a labelled DNA complementary to the sequences of interest. Southern blotting is less commonly used in laboratory science due to the capacity of using PCR to detect specific DNA sequences from DNA samples. However, these blots are still used for some applications, such as measuring transgene copy number in transgenic

Assembly of the Tetra Cell requires following procedures outlined by the manufacturer’s protocol. Once the Tetra cell was assembled, the solution was added to the chambers of the apparatus. The experiment then requires loading 10 l precision plus protein kaleidoscope prestained into well #2, loading wells #3-7 with the 5 l prelabeled fish protein solutions into their own wells, and loading well #8 with 10 l of actin & myosin standard after rising the wells. (See Table 1 in the appendix) The lid was placed on to the vertical electrophoresis apparatus, set the Bio-Rad power supply to 175V, and then the gel was set to run. After 45 minutes, all bands were at the bottom of the gel and the Tetra cell was disassembled. The gel was then placed into a labeled tray with 50 ml of coomassie stain for on hour after being rinsed with distilled water for five minutes. Once done, the gel was wrapped in plastic

The last part of this procedure, antibodies will be used to detect one specific protein from others on the membrane. Incubate the membrane with 10 ml of primary antibody for 10-20 minutes and then place on a rocking platform. Rinse the membrane quickly after with wash buffer on a rocking platform. After three minutes is up, discard the wash and incubate the membrane with 10 ml of secondary antibody for 5-15 minutes on the platform. Rinse the membrane again shortly after and wash the membrane for three minutes. Next, discard the wash and add 10 mL of HRP color detection reagent. Incubation will occur after this for 10-30 minutes. Once it is done, rinse the membrane twice with distilled water and blot dry.