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Abstract. The isolation of DNA is one of the most commonly used procedures in genetics, molecular biology and biochemistry. Nuclear and mitochondrial DNA can be isolated from all tissues of the body. In this particular laboratory for isolation of DNA we used the fish liver. Many protocols can be used for DNA isolation but in our lab we used phenol/chloroform extraction of DNA. For analysis of DNA we used the electrophoresis and spectroscopy of DNA. I received the following results for mtDNA: the concentration (μg/ml) was 0.0141 and 0.0048 for 260 nm and 280 nm respectively; the absorbance was 0.0001 and 0.0000 at 260 nm and 280 nm respectively, and the A260/A280 ratio was 0.0. The results for nuclear DNA: the concentration (μg/ml) was 7.9570 and 6.6339 for 260 nm and 280 nm respectively; the absorbance was 0.0796 and 0.0663 at 260 nm and 280 nm respectively, and the A260/A280 ratio was 1.2006. Those results indicated low concentration of mtDNA to contrast to the concentration of nuclear DNA. The DNA/protein ratio for nuclear DNA was not optimal but indicated that this sample did not have contamination of phenol. The low values of this ratio may indicate the protein contamination. Introduction. The isolation of DNA is one of the more commonly used procedures in genetics, molecular biology and biochemistry. Purified DNA is required for many application such as analysis of DNA structure and chemistry, examining DNA-protein binding, DNA hybridization, sequencing, PCR and gene cloning. The exact procedures for obtaining purified DNA vary depending upon the tissue source being used. However, several general principles apply to all techniques: 1. The disruption of the cells to release DNA. 2. Separation of the DNA from all other macromolecules as proteins in the lysate. 3. The inactivation of the enzymes that hydrolyze DNA. 4. Quantification of DNA. Most procedures start with cell disruption followed by deproteinization. For disruption of the cell membrane SDS is commonly used. In our laboratory exercise we also used non-idet TEKS to lyse the mitochondria membranes. The main differences in procedures lie in the deproteinization step. In our lab to purify the DNA from proteins we used phenol and chloroform. Phenol efficiently denatures proteins. To decrease the acidity of the phenol we use the phenol equilibrated with Tris buffer. Chloroform also acts as protein denaturant and, in addition it removes excess phenol. Both of those reagents also remove the lipids from the lysate.
Approximate Word count = 1605
Approximate Pages = 6.4
(250 words per page double spaced)
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