Comet Assay

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

· Explain the process of electrophoresis as it relates to separating and identifying different DNA fragments

To do this, 50 ml of buffer solution was added to 0.4 g of agarose in order to create a 0.8% agarose gel in a flask. The flask was placed inside microwave for intervals of first, 20 seconds then swirled, next 30 seconds then a minute and swirled. Then, while the solution was still hot, ethidium bromide (EtBr) was added in order to stain the gel. By staining the gel with EtBr, it allows the DNA to be visible under UV light once the process of electrophoresis has occurred. This is because the staining agent attaches only to DNA fragments.

Although DNA remains mostly stable, it is constantly subject to chemical modification from a variety of different sources. Numerous forms of DNA damage have been identified resulting from different factors such as alkylating agents, hydrolytic deamination, free radicals, and mismatching base pairs. Alkylating agents cause mutations of legitimate bases, transforming them in to either a non-coding or lethal lesion in the DNA. Hydrolytic deamination changes on base on the DNA double helix into another. (Sinha & Häder, 2002) Oxygen radicals are generated during cellular production of O2 gas is known to either attack the bases of DNA (A, T C, or G) or the Deoxyribose to damage the DNA bases or break strands. (Marnett, 2000) DNA remains under threat by other cellular factors, but it does have certain mechanisms in place to repair certain

- 250 μl of buffer P1 (cell suspension buffer ) was added to the cell pellet. The pellet was resuspended to make a homogeneous suspension. The P1 buffer protects the DNA when it is isolated.

The Ames Test: A Method to Test the Mutagenic Effects of Pond Scum. Destiny Reynolds November 17, 2014 219L- 03 With Kayla Wacaster and Vaughan Dees Introduction The purpose of running the Ames test is to see if there is a mutagenic factor that may cause cancer. Mutagens are chemical or physical agents that change the genetic material, in most cases the DNA.

These techniques are sufficient at measuring the telomere content but insensitive to detect individual chromosome telomere lengths which might be more relevant to pathogenesis due to the fact that the. Single telomere length assay is a PCR based technique that measures chromosome specific telomere length from very small quantities of DNA (as few as 50 cells) with the caveat of low throughput and has been utilized in research laboratories.

Among the many types of DNA damage, one of the most severe is DNA double strand breaks (DSB). DNA DSBs occur when two complementary strands break simultaneously generating two strands which are not being held together by base-pairing and chromatin structure. Few known causes for DSB are ionizing radiation, chemical mutagens, reactive oxygen species and drugs which are used for chemotherapy (Goodsell, 2005). This breakage can cause chromosomal rearrangement such as translocation and inversion, consequentially resulting in detrimental phenotypic affects.

A pure quality DNA should have a thick, compact single band. However the electrophoresis of the purified DNA did not exactly complement the above mentioned (Ab260/Ab280) ratio.As from figure 1 it is evident that only the band of ACHN is a single band whereas for the other two bands of MCF7 and H1650 it shows a smear which indicates that DNA is not intact.The smear DNA bands observed could be a result of degradation by nucleases.Thus to overcome this problem,one can use freshly made electrophoresis buffers or ensure that the pipette tips are nuclease –free.It is also possible that tremendously low voltage during gel electrophoresis may cause diffusion of bands orhigh voltage can cause greater heating and denaturation of

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

Gel electrophoresis is a commonly used laboratory technique employed in biochemistry and molecular biology (ARBL, 2000). The two most conventional types of gels used for DNA electrophoresis are agarose and polyacrylamide (PA). The two substances differ in factors such as resolving power and in the difficulty of setting up and handling them (ARBL, 2000). In comparison to the polyacrylamide, agarose gels are used more commonly as it may also be refrigerated and re-used, and runs horizontally (Reina, 2014). Gel electrophoresis through an agarose channel is used to identify, quantify and purify nucleic acid components (Life Technologies, 2015). The samples of DNA are loaded into wells of agarose gel which is then subjected to an electric current,

Figure 2. A 1% LB Agarose Gel that was ran at 185 volts where lanes 1-8 were conducted by students and lane 9 is a standard to be compared to. This is an identical image as Figure 1, but instead here Lane 1 is being analyzed. Lane 1 contains a DNA substance that was labeled D3 and ran through the gel. Although the label D3 was known the true identity of the DNA substance is unknown. After measuring the distance migrated, we calculated the base pairs for each band by using the equation, log(# of bp)= -0.0112 (distance migrated) + 3.9484. The distance migrated for each DNA fragment is located on the left of the image and located adjacent is the calculated base pairs of the fragment. Finally, please note, that although the distance migrated is

8.62 x 10-6pg of nucleic acid were detected per nucleus in this sample, which is a small amount in comparison to the 1.8 pg found in the 1976 experiment. The spectrophotometer does not accurately estimate the amount of DNA per nucleus because it does not differentiate between chromatin, DNA, and RNA in its measurements. The reading therefore does not provide a true answer for the pure contents of DNA within a cell. Reasons why this experimental findings of DNA per nucleus differs from Bennett’s could be because of human error. Since the pellet of nucleic acids was especially miniscule during the latter steps of the experiment, it is possible part of the pellet could have been accidentally poured out with the supernatant. The buffers and other chemicals used in Bennett’s experiment were also unknown by the scientist; therefore it is possible Bennett used different chemicals which were not as degrading to the DNA as the ones provided in this laboratory. Overall, the low yield of DNA per nucleus in this experiment compared to the DNA per nucleus determined by Bennett’s procedure could be because of a lack of precision and accuracy in the use and transference of

We performed gel electrophoresis on DNA that had been cut by a restiction enzyme, in order to separate the strands based on length. It is a routine procedure that allows the separation analyzis, and purification of samples of DNA.4

For the majority of DNA samples, electrophoretic separation is carried out in agarose gels. This is because most DNA molecules and their fragments that are analysed routinely are considerably larger than proteins and therefore, because most DNA fragments would be unable to enter a polyacrylamide gel, the larger pore size of an agarose gel is required. DNA size is referred to in terms of base-pairs (bp) or kilo base-pairs (kbp) though it has now become the accepted nomenclature to abbreviate kbp to simply kb.