Theory Agarose gel electrophoresis is a widely used procedure in various areas of biotechnology. This simple and precise analytical procedure is used in the research, biomedical and forensic laboratories. It is used for (i) determining the size of DNA molecules in the range of 500 to 30,000 base pairs, (ii) to analyze DNA fragments generated by restriction enzymes, and (iii) to separate other charged biomolecules such as dyes, RNA and proteins. Principle DNA molecules are negatively charged at neutral or alkaline pH and migrate towards anode when an electric field is applied. Charge/ mass ratio of nucleic acid is unity, thus migration occurs largely on the basis of molecular size of the DNA molecules.
21 Fresh semen samples were diluted with phosphate-buffered saline (PBS) to 2x106 sperm/mL. Fifty µL were incubated with 100 µL lysing reagent for 15 seconds and then 2 mL of PI was added and mixed with tube. Tube acquisition was done by flowcytometry immediately after staining. The intensity of its emission corresponds to the DNA content. Flowcytometric analysis displays a constant and characteristic bimodal nonartifactual DNA pattern indicating the presence of two different populations.
2.7 Polymerase Chain Reaction (PCR) In 1971, Dr. Har Gobind Khorana’s group described the idea to amplify a DNA (Templeton, 1992). Later on in 1985, Kary B. Mullis invented the Polymerase Chain Reaction (PCR) which is used today in different fields, such as scientific research, clinical diagnostics and criminal investigations. PCR is a molecular biology tool that is used to amplify a fragment of DNA to generate thousands to millions of copies. This technique is based on an enzymatic reaction which is controlled by thermal cycling, where every cycle consists of heating and cooling steps. The generated DNA fragments after every cycle are used as templates for the next cycle.
Abstract In this experiment, the isolation, characterization, and determination of concentration and purity of deoxyribonucleic acid or DNA from Allium Cepa or onion was performed. DNA was isolated through the use of a homogenizing solution. The absorbance ratio was 1.5, which indicates protein contamination. Moreover, the characterization of its components was conducted through the use of different chemical tests. The DNA gathered by the group bore positive results only on Test for Deoxyribose; compared to the standard solution, which bore positive results on all chemical tests, namely, Test for Deoxyribose, Test for Phosphate, Test for Purines, and test for Pyrimidines.
Approximately 2 gm, nearest to 0.1 mg, oven dried cornhusk fibres, were weighed out accurately in weighing bottle and transferred to a 100 ml beaker. 40 ml of cold (10-15˚C) 72% sulphuric acid was added gradually to the fibres in small increments while stirring the mixture and macerating the fibres with a small glass rod. The beaker was kept in a bath at 2 ± 1˚C for dispersion of material. After the specimen was dispersed, beaker was covered with a watch glass and kept in a bath at 20 ± 1˚C for 2 hours. Mixture was stirred frequently to ensure complete
However, all proteins are constructed from the same set of 20 amino acids linked in unbranched polymers. The covalent bond that exists between amino acids is called peptide bond, hence a polymer of amino acids is named polypeptide. A protein is a biological functional molecule made up of one or more polypeptides which is folded and coiled into unique three-dimensional structure. In laboratory, it is important to measure the concentration of proteins for research investigations. Biuret test is adopted to quantify proteins in fluid by using a spectrophotometer.
Protein synthesis Introduction Translation or protein synthesis is a central process of central dogma of molecular biology. It deals with production of proteins or chains of amino acids by making use of a mRNA as a template, ribosomes as protein synthesizing machinery and tRNA’s as carriers of amino acids during the translation process Living cells devote about 90 % of their chemical energy to synthesis of proteins and only about 10 % to other biosynthetic processes. More than 35% of the dry weight of the cell consists of ribosomes, proteins involved in translation process and tRNA molecules. This suggests that protein synthesis is an important process for the survival of microorganisms Protein synthesis process in
Measurement of lipid peroxidation TBARS, a measure of lipid per oxidation, was measured as described by Ohkawa . Briefly, 1 ml of suspension medium was taken from the 10% tissue homogenate. 0.5 ml of 30% Trichloroacetic acid (TCA) was added to it, followed by 0.5 ml of 0.8% thiobarbituric acid (TBA) reagent. The tubes were covered with aluminium foil and kept in shaking water bath for 30 minutes at 80°C. After 30 minutes, tubes were taken out and kept in ice-cold water for 30 minutes.
Twenty tablets were weighed accurately and powdered. An amount of the powder equivalent to 5 mg of amoxicillin trihydrate (content of one tablet) was dissolved in 60 ml of diluent. The solution was stirred for 10 min using a magnetic stirrer and filtered into a 100 ml volumetric flask through 0.45µ nylon membrane filter. The residue was washed 3 times with 10 ml of diluent and then the volume was completed to 100 ml with the same solvent. This solution was diluted with diluents to gae a concentration of 0.1 mg/ml solution each of Amoxicillin trihydrate.
The bacteria were heat-killed, and these respective components were extracted and the composition resulted in being similar to that of DNA. They also treated the bacteria with multiple enzymes, such as trypsin, chymotrypsin, ribonuclease and deoxyribonucleodepolymerase, where it was found that only the deoxyribonucleodepolymerase inhibited the formation of smooth Pneumococcus colonies. [Downie. A. W. (1972)] Thus, they confirmed that DNA was the transformation principle in Griffith's experiments. The Avery and MacLeod experiment was replicated in the laboratories at the University of the Witwatersrand.