Analysis The experiment performed was conceptually simple, which was to determine the concentration and purity of several

DNA samples through spectrophotometric processes and the Beer-Lambert law, relating light absorption to the material which absorbs it. The first phase was to determine the absorbance of the DNA samples at 260 nm. Prior to this, however, the plasmid DNA from the previous experiment (which was kept at low temperatures to prevent DNA degradation) was diluted to 0.5% in TE buffer. The purpose of this was to solubilize the sample as well as prevent the DNA in the sample from degrading, which may have caused different absorbance readings and thus, incorrect purity and concentration results later. Before running the sample through the spectrophotometer, a 100% TE buffer blank was used to standardize the machine at 260 nm. This is the maximal absorption wavelength of DNA. This is so because of the nitrogenous bases DNA contains, purine (adenine, guanine) and pyrimidine (cytosine, thymine). These nitrogenous bases have maximal absorbance at the Ultra Violet range due to their aromatic ring structures. The samples were then run through and their absorbance values at 260 nm were recorded. The entire process was repeated for a 280 nm absorbance with the same samples used in the previous part. Values were recorded, along with the calculated (Abs260/Abs280) ratio, DNA concentration and purity. (See Table 1.)

Sample 1 2 3 4 5 6 7

Abs260 0.170 0.095 0.093 0.036 0.032 0.209 0.125

Abs280 0.082 0.049 0.041 0.022 0.032 0.105 0.07

(Abs260/Abs280) 2.07 1.94 2.27 1.64 1.0 1.99 1.79

Purity RNA Contamination RNA Contamination RNA Contamination Protein Contamination Protein Contamination RNA Contamination Pure

[DNA] (g/mL) 1700 950 930 360 320 2090 1250

Table 1: Absorbance values of samples, Purity and DNA concentration The purity of the sample can be determined by dividing the absorbance of the sample at 260 nm by the absorbance of the same sample at 280 nm. As mentioned, the maximal absorbance of nucleic acid is at a 260 nm wavelength. Protein also absorbs Ultra Violet light, but its peak light absorption is at 280 nm. DNA isolation from protein is relatively difficult since protein is tightly bound to it, thus when a sample is run through a spectrophotometer, both the DNA and the proteins absorb the UV light. A theoretically pure sample, containing no protein,

would yield a low 280 absorbance value and thus the 260:280 ratio will be high. Conversely, a contaminated sample will have a lower 260:280 ratio indicating protein contamination.
As seen in Table 1, samples 1, 2, 3 and 6 have 260:280 ratio values beyond 1.9. This indicates RNA contamination. A pure RNA sample has a 260:280 ratio value at 1.8 2.0. Samples 4 and 5 have an absorbance ratio below 1.7 which indicates protein contamination. The absorbance of samples at 280 nm had most likely yielded high values and thus lowered the 260:280 ratio. Sample 7 had an absorbance ratio of 1.79 which falls within range of what is considered to be a pure DNA sample. Many factors could have affected the sample purity. Besides the quantity of protein contaminants, things like the oils left on the quartz cuvette from human fingers could have affected spectrophotometer absorbance. Contamination during TE buffer dilution could also have an effect. Although, improper aspiration of the supernatants or disturbance of DNA suspension, during extraction (previous experiment) would probably be the most probable cause. Anything that may affect the absorbance at either 260 nm or 280 nm will affect results. In relation to the agarose profile, there was no observable parallelism to the smudging and purity values. Since plasmids are double-stranded, the concentration unit used to calculate for DNA

concentration was 50 g/ml.

Conclusion Based from the experiment, the findings show that the group sample (sample 7) is pure from any contamination of protein or RNA with the absorbance ratio of 0.79, which falls between the interval of pure sample. The results indeed shows that in identifying the purity of a plasmid sample, spectrophotometric methods can be used because the method used in class shows how the nitrogenous bases of DNA absorb UV range. This findings also follow the Beer-Lambert Law. In further experiments, the group recommends that contamination during the TE buffer dilution, improper washing of cuvette sample, improper aspiration and disturbance of DNA suspension should be avoided so that these acts might not have an effect on the findings.

Canlas, Maria Katrina C. Cell& Molecular Biology Laboratory.Biology Department.Ateneo de Manila University. "Mini-prep Isolation of Plasmid DNA." Santa Monica College Homepage. EDVOTEK The Biotechnology Education Company. Web. 27 Feb. 2012. <http://homepage.smc.edu/bober_mary/Bio%2022/Mini-Prep%20plasmid%20DNA %20202.pdf>. Meyers RA, et al., editors. Encyclopedia of molecular biology and molecular medicine. 1st ed. Weinheim : VCH, c1996-1997. 1p. Rosemeyer, H. Chemistry & Biodiversity 2004, 1, 361. Organic Syntheses, Coll. Vol. 4, p.182 (1963); Vol. 35, p.34 (1955)

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