Titlebook: New Nucleic Acid Techniques; John M. Walker Book 1988 Springer Science+Business Media New York 1988

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Oligonucleotide Synthesis Using the Manual Phosphotriester Method,is because of the availability of highly reactive mononucleotides and specially developed coupling catalysts. In optimized systems, this allows for synthesis of oligomers greater than 50 bases in length.

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Electrophoresis of RNA Denatured with Glyoxal or Formaldehyde,uction allowed for major advances, most particularly in the molecular biology of eukaryotic organisms. The method had, nevertheless, two significant disadvantages in that the gels (composed of acrylamide at very low concentrations) were mechanically fragile, and the migration of RNA molecules did no

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Northern Blotting,A is first separated by electrophoresis under denaturing conditions (..), followed by blotting onto a suitable filter. Specific sequences are then detected by hybridization. The RNA species are both immobilized on the filter and denatured so that when the filter is immersed in a solution containing

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Hybrid-Arrested Translation,, a specific messenger RNA in a cell-free system. In this respect the approach is similar to “hybrid selection,” also known as “hybrid release” (..), in which messenger RNAs homologous to cDNA clones are specifically trapped as RNA/DNA hybrids on nitrocelloulose filters. By washing the filters at sp

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In Vitro Translation and Analysis of Early Events in Protein Synthesis Initiation in Nonreticulocyties). Both these systems have a number of advantages, including the ease and cheapness of preparation, the relative absence of RNAse, the high level of reinitiation of ribosomes onto mRNA, the high fidelity of translation, and the maintenance of activity upon long-term storage. Both of them have als

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Use of Elutips to Purify DNA,l techniques for manipulation of DNA. The Elutip-d columns commercially available are manufactured by Schleicher and Schuell. Each column consists of a pipet tip, such as may be used on a 0–200 μL automatic pipet, that is filled with an insoluble matrix.

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Plasmid Preparation on Sephacryl S1000, for plasmid purification, all of which have common features; namely, (.) growth of plasmid-containing bacteria, (.) lysis of bacteria, (.) low-speed centrifugation to remove the majority of bacterial DNA and protein, (4) an ultracentrifugation step in cesium chloride and ethidium bromide to separat

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Electrophoresis of DNA in Nondenaturing Polyacrylamide Gels,ly adequate. As the size of the fragments of interest decreases to below 1000 base pairs (bp), however, the resolution deteriorates significantly, and an alternative is required. Polyacrylamide gels from 5% acrylamide upward provide a convenient system for analysis of fragments down to 10 bp. This t

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Use of 35S Nucleotides for DNA Sequencing,d are known as the Maxam-Gilbert method of chemical cleavage (.) and the dideoxy chain termination method developed originally by Sanger (.). One of the most critical steps in either of these two methods is the radiolabeling of the DNA such that the sequencing gel “l(fā)adder” of reaction products may b