Gel Electrophoresis 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?

There are three specific steps required to isolate DNA from its cellular contents. The steps used to remove and expose DNA from its cell are: breaking down the food type you are using by crushing it, for example a banana or strawberries, exposing the substance to a sodium chloride (NaCl) solution, subjecting the product to detergent solution (dH2O), filtering the solution and lastly, the addition of ethanol. When beginning with a solid substance, such as a banana, crushing the substance allows for

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

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

Using the electric current, scientists pass the DNA through gel, and as smaller molecules get through gel quicker than those of bigger sizes, DNA molecules get separated according the sizes of the molecules. We utilize the property that large molecules move slower, and DNA is slightly negatively charged(due to phosphate groups), so it will move to the positive pole of the gel.

You can view macromolecules by using a technique called gel electrophoresis. Gel electrophoresis is a process used by scientists to study and view macromolecules. This process separates the different macromolecules and coats the different macromolecules so you can easily see them. A macromolecule is a very large molecule that contains many atoms. Agarose is used in gel electrophoresis to coat the macromolecules.

Throughout analysis of the data, we complete DNA agarose gel electrophoresis in order to determine the size of our plasmid DNA. The electrophoresis

The Material floating at the top of the test-tube after the alcohol was added was all the strands of DNA. The alcohol dissolved the cell walls of the cells and the DNA was free to move about freely.

Gel electrophoresis is a gel technique that separates DNA and proteins based on their mass, by means of an applied electrical field that passes through an agarose or polyacrylamide gel. The concentration of agarose in the gel is commonly 0.8% to 1.0%, since agarose is expensive. The gel is embedded on a buffer, pH of 8.3, which keeps the pH of the solution at equilibrium. Assuming that at typical pH, DNA is negatively charged, denatured protein samples are placed in the wells located on the negative electrode side; hence the positive electrode side is located at the other end of the gel. As the positive and negative electrode sides are connected to a power source, protein sample migrates to the positive side of the gel. Migration of proteins is not necessarily based on their mass or mass to charge ratio; protein migration across the gel is based on their size. In other words smaller molecules will travels further than bigger molecules. Since the SDS gel contains agarose or polyacrilamide, molecules have to be small enough to migrate to the other end of the gel without getting stuck on the way.

Experiment 1 & 2: The DNA concentration was 60 ng/µL (6.00*10^4 ng/mL). The total yield was 0.6 ng (60 ng/µL DNA /100 µL H2O). The DNA sample was divided by 100 µL instead of 25 µL of H2O because the sample was diluted 4 times.

The shorter DNA fragments move faster than the longer fragments. DNA is separated on basis of size. A radioactive material is joined which combines with the DNA fragments to yield a fluorescent image. A photographic copy of the DNA bands is acquired. The pattern of fragment distribution is then examined and

Nucleic acid molecules which are to be analyzed are set upon a viscous medium, thegel, where an electric field induces the nucleic acids to migrate toward the anode, due to the net negative charge of the sugar-phosphate backbone of the nucleic acid chain. The separation of these fragments is accomplished by exploiting the mobilities with which different sized molecules are able to pass through the gel. Longer molecules migrate more slowly because they experience more resistance within the gel. The DNA fragments of different lengths are visualized using a fluorescent dye specific for DNA, such asethidium bromide. The gel shows bands corresponding to different nucleic acid molecules populations with different molecular weight. Fragment size

Let the test tube sit undisturbed for 2 - 5 minutes. You should begin to see air bubbles form at the boundary line between the ethanol and the filtered fruit solution. Bubbles will form near the top, and you will eventually see the DNA float to the top of the ethanol. Gently insert the stir stick into the test tube. Slowly raise and lower the tip several times to spool and collect the DNA. If there is an insufficient amount of DNA available, it may not float to the top of the solution in a form that can be easily spooled or removed from the tube. However, the DNA will still be visible as white/clear clusters by gently stirring the solution and pushing the clusters around the top. Post-Lab Questions What is the texture and consistency of the DNA DNA is viscous and greasy. Why did we use a salt in the extraction solution High salt makes DNA less soluble in water. In order to dissolve, the water needs to interact with the DNA. Since DNA is quite large, it needs to interact with lots of water for this purpose. When you add salt, the water preferentially interacts with the salt (its small, and can move around in solution easier than the DNA can). This makes it so there is less water available to interact with the DNA and it becomes less soluble. Is the DNA soluble in the aqueous solution or alcohol DNA is less soluble in an alcohol such as isopropanol than it is in water. This is because alcohols are non-polar, whereas water is polar. The polar

The polymerase chain reaction or PCR for short can be used to create many copies of DNA. This allows the DNA to then be visualized using a dye like ethidium bromide after gel electrophoresis. The process has been refined over the years, however the basic steps are similar.

Detergents are used to break up the cell, and the nucleus is isolated and treated with chemicals to extract the DNA. Then the DNA sample is multiplied by the polymerase chain reaction. This is artificial DNA replication. The DNA is added to a test-tube and warmed to break the weak hydrogen bonds, separating the two strands of the double helix. Then free DNA nucleotides and DNA polymerase are added to the test-tube. Lastly the solution is cooled allowing the nucleotides to bond to the exposed bases. Next the DNA is subjected to electrophoresis, which separates fragments according to size. Firstly restriction enzymes cut the DNA into fragments. This occurs as the enzymes are able to recognize specific base sequences and cut the DNA at certain points. Next, the fragments are placed on an electrophoretic gel and a charge is applied. Due to the negatively charged phosphate ions in DNA, the fragments are attracted to the positive end of the gel. As they move towards the positive side of the gel, the larger fragments encounter more resistance due to their larger size, and they do not move as far down the gel as the smaller fragments. This creates a pattern unique to that individual’s DNA. To ensure the DNA fingerprint is precise and unique, and reduce the likelihood of falsely identifying an individual, a number of genetic loci are used to generate the DNA fingerprint. This is the ‘fingerprint’ that can then be used for identification purposes or the