Global Dna Methylation Study

Four test tubes were labeled “H2O A”, “H2O B”, “Enz A”, and “Enz B”. In the test tubes labeled “Enz A” and “Enz B” 1 mL of catalase was pipetted into each, and in the test tubes labeled “H2O A” and “H2O B” 6mL of deionized water was pipetted into each. Next the two test tubes labeled A were placed into an ice bucket, and the test tubes labeled B were placed in a 50°C water bath. These were left in their respected locations for 5 minutes each, and then the temperature of the test tubes in the ice bucket were recorded. Next the “Enz A” test tube was mixed with the “H2O A” test tube and 100µL of guaiacol and 150µL of .1% H2O2 were added to this mixture using a micropipette. The test tube was then covered with aluminum foil and inverted to mix the solution and then placed in a curvette and into the spectrometer. The initial absorbance was recorded and the curvette was placed back in the ice bucket. Then the contents of the test tubes labeled B were mixed and 100µL of guaiacol and 150µL of .1% H2O2 were added to this mixture using a micropipette. This was then placed in a curvette and into the spectrometer and the initial absorbance was recorded and the curvette placed back in the water bath. Finally after 5 minutes of time had passed each of these were placed in the spectrometer again and their absorbance levels recorded. This process should be a very quick one to ensure that the

10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted.

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

The dark, navy blue colored graph represented the absorbance curve for the S1 sample. The red colored graph represented the absorbance curve for the S2 sample. The green colored graph represented the absorbance curve for the P1 sample. The purple colored graph represented the absorbance curve for the P2 sample. The gaps between the P2 curve was due to the oversaturation that led to the inconclusive spectrophotometer readings. The blue colored graph represented the absorbance curve for the P1 low salt sample. The orange colored graph represented the absorbance curve for the P2 low salt sample. The light blue colored graph represented the absorbance curve for the P1 medium salt sample. The light pink colored graph represented the absorbance curve for the P2 medium salt sample. The light green colored graph represented the absorbance curve for the P1 high salt sample. The light purple colored graph represented the absorbance curve for the P2 high salt

feedlots (2.8 + 0.5%; USDA, 2013) only behind bovine respiratory disease and metabolic/digestive conditions. A survey conducted in Kansas 1970’s by Brower and Kiracofe (1972) indicated that 2% of all feedlot steers in Kansas experienced the buller steer syndrome. In the same manner, Brower and Kiracofe (1978) and Irwin et al. (1979) reported that the annual incidence of buller steers within the feedlot industry fell somewhere between 2 and 4%. A 15-year summary of buller incidence in Midwest feedlots consisting of over 5 million steers reported a buller incidence of 2.45% (Edwards, 1995). Furthermore, a 2-year study by Taylor et al. (1997b) at a western Canada feedlot using 78,445 male (intact) cattle indicated a buller incidence rate ranging from 0.0 to 11.2%, with an average incidence of

vulgaris were prepared by repeatedly rinsing in distilled water to remove surface betalain (Flinders, 2015). A control group and two treatments were taken, the control was at room temperature (25 degrees Celsius), and the treatments were at 50 degrees Celsius and 80 degrees Celsius. Cubes of B. vulgaris were placed into cuvettes with three ml of distilled water, the treatments were started five minutes apart to ensure each treatment was recorded at the correct time. The light absorbency of each treatment was then recorded using a spectrophotometer set at 540nm, the absorbency for each treatment was checked after 0 minutes, 15 minutes and, 30 minutes in the water baths. Before measuring the light absorbency of each treatment a cuvette with 3 millimetres of distilled water was placed in a spectrophotometer to set it to zero. The absorbency in the solution was measured in arbitrary units (AU), the higher the light absorbency, the higher the plasma membrane permeability. Each treatment was gently swirled before being placed into the spectrophotometer to disperse the colour. Each treatment had three replicates to guarantee an accurate average could be recorded. The results were then collated into tables and

The absorbance is measured using a Plate reader and a Standard curve is generated. Also, the different types of pipetting techniques are assessed in this Assay.

As of late there has been more of a focus on the Epigenetics component of mental illness.

We turned the Spectrophotometer on and set the value for absorbance to 475nm. We allowed it to properly warm up for at least 15 minutes before we started using it. We prepared 8 clean test tubes to make reaction assay tubes with 4mL of buffer and .5ml of the enzyme in each tube. They were labeled 1 to 8 starting from the lowest concentration to the highest. Tubes 1,3,5 & 7 had .5 ml of buffer added instead to act as the control group.

Incorporation of assay controls included setting up a spectrophotomer and running the chart recorder with a full-scale deflection before the start of the assay. The set recorder had a corresponding value of 1 for the change in the absorbance. Therefore, prior testing was done to observe whether a change occurred in the readings. This helped to indicate that the results were valid, as they could have been affected by a fault during the setting up of the spectrophotometer. On the other hand this was considered as one of the controls for the experiment. Nevertheless, a new cuvette had to be used for each assay.

The penchant for analytical thinking and the scientific system was prevalent in my upbringing, a by-product of being born to two scientists. Therefore, when I began working on epigenetic research on Facioscapulohumeral Muscular Dystrophy (FSHD) at Saint Louis University, I felt comfortable around the slow, systematic approach studded with jargon heavy language. I was excited to experience molecular biology, to work in the same kind of labs I’d visited as a child. When I applied to the job, FSHD was words linked to the epigenetic status of the D4Z4 sequence. In other words an abstract concept. That was the extent of my understanding until I met a 13 year old FSHD patient. If epigenetic changes are the result of environmental factors or

The optimized Rose bengal strip and Bromophenol red strip produced curves with better linearity and better curve fit (R) than previous optimized Bromophenol blue (BPB) indicator strip. R-squared values of these two standard curves were 0.847 and 0.870, respectively. Good linearity of the standard curve is important for providing accurate calculation of a given analyte.

Trying to find the multiple hereditary and environmental causes of TTT, however, is a daunting task; the assumed risk factors include not only epigenetic factors, but also personality traits and social support, and traumatic experiences of the children themselves during their lives. It is difficult to disentangle effects that reflect TEI from early rearing influences and subtle attachment patterns experienced because of having trauma-exposed and/or symptomatic parents. It is precisely because of this complexity that epigenetics makes so much sense as a suitable explanation for

Modification of damaged DNA seems to be an understudied subject, there is much to understand on the restoration of DNA damage, repair and DNA methylation. Genomic DNA can be modified by methylation but much of it is affected on a gene when silenced. When epigenetic modification has been implicated with cancer and aging it causes DNA methylation to also have an impact on the double strand of DNA analysis. Modification as such provoke deteriorating changes like aging found in multicellular organisms and DNA damage may magnify biochemical pathways that regulate a cells growth or control DNA replication with DNA repair. In the article “DNA Damage, Homology-Directed Repair, and DNA Methylation” written by Concetta Cuozzo, Antonio Porcellini, Tiziana Angrisano, et al. they hypothesize how DNA damage and gene silencing may induce a DNA double-strand break within a genome as well as when DNA methylation is induced by homologous recombination that it may somewhat mark its reparation through a DNA segment and protect its cells against any unregulated gene expression that may be followed by DNA damage. The experiments used to demonstration how gene conversion can modify methylation pattern of repaired DNA and when that occurs methylation is able to silence the recombined gene. When exploring the molecular mechanisms that link DNA damage and the silencing gene then there is an induced double strand break that can be found at a specific location or DNA sequence in where the

Whereas the reaction products are not specific the SYBR green dye added to the reaction mixture to quantitatively indicate the amount of dsDNA in the reaction mixture by providing amplification plot and melt curve as well as the melt temperature plot that used in may offers useful information about the sensitivity of the DNA template in the sample (Giglio, Monis and Saint, 2003).