ABSTRACT
In unit 1, a total of 27 experiments have been included in which isolation of DNA from different tissues, either plant or plasmid, is at the heart of molecular biology because all the genetic information is carried in the linear sequence of nucleotides in DNA. Several protocols have been developed for the isolation of genomic DNA, but selective protocols are described in Isolation of Total Genomic DNA for plant, plasmid, yeast, and bacteria. Purification of Genomic DNA describes purification of nucleic acids, as this is the first step in most molecular biology studies and in all recombinant DNA techniques. DNA is extracted from samples such as bacteria or plant and animal cells by dissolving the sample in a buffer and then added to an absorbent material. In Isolation of RNA from Plant Tissues, we describe 2 procedures for the isolation of RNA. In both procedures, a high pH of the extraction buffer and the presence of a chelating agent (EDTA and EGTA, respectively) are used to prevent RNA degradation. Quantitative Determination of DNA and RNA by a Spectrophotometric Method can also be employed for judging purity of DNA and RNA preparations. Agarose Gel Electrophoresis is a routinely used technique in the laboratory in which DNA fragments are separated based on their molecular weight. Several techniques, such as as Electrophoresis of DNA: Linear, Circular, and Super Coiled, have been developed for the analysis of nucleic acids (both RNA and DNA), but agarose gel electrophoresis is an ideal technique for analysis of nucleic acids. The mobility of nucleic acids in agarose gels is influenced by the agarose concentration and the molecular size and molecular conformation of the nucleic acid. Hybridization and Autoradiography of DNA describes autoradiographic detection of a plant DNA fragment, which bears sequence homology with the probe and the radioactive spot on the membrane. Prior to hybridization, the membrane is prehybridized with salmon-sperm DNA to block the nonspecific binding sites where the probe may otherwise get bound spuriously. PCR is a very different approach to isolation of a DNA segment. Rather than a lengthy series of manipulation involving cells, PCR is a test-tube reaction that is carried out simply by mixing together the appropriate reagents and incubating them in a thermal cycler, a piece of equipment that enable the incubation temperature to be varied over time in a preprogrammed manner. Reverse Transcriptase PCR (RT-PCR) is the PCR amplification of c-DNA (or complementary DNA, which is complementary to mRNA) as it involves a reverse transcription step prior to amplification. Isolation of pUC18 Plasmid from TOP 10—pUC 18 E. coli Cells is based on the principle that exposure of bacterial suspensions to the strongly anionic detergent at high pH opens the cell wall, denatures chromosomal DNA and proteins, and releases plasmid DNA into the supernatant. Transformation of the Desired Bacterial Strain with Plasmid DNA is done to introduce a foreign plasmid DNA and to use the transformed bacteria to amplify the foreign DNA. Purifying pUC18/Hind III/Eco R I Digest by Gel Elution works best for DNA fragments ranging in size from 0.5–5.0 kb. In the Restriction Digestion of pUC 18 and A DNA experiment, pUC 18 and A DNA are double digested with EcoRI and Hind III. Enzyme Linked Immunosorbent Assay (ELISA) is an immunological technique used to detect proteins and is based on the ability of low molecular weight antibodies to couple with enzymes/secondary antibodies and conjugate with an enzymes (e.g., alkaline phosphatase), which then can be visually detected by reaction with an appropriate substrate (e.g., PNPP). Isolation of lambda (λ) DNA with a quick method and also using single-stranded M13 DNA isolation using phenol completes the process of plasmid analysis.
