Solid phase technique for selectively isolating nucleic acids
a solid phase technique and nucleic acid technology, applied in the direction of nucleic acid reduction, microorganisms, biochemical apparatus and processes, etc., can solve the problem of limited throughput, achieve high ionic strength, improve the purity of exogenous dna of microparticles, and facilitate automation
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example 1
Isolation of Plasmid DNA Using PEG-Induced Separation and Paramagnetic Microparticles
[0063] This example provides a procedure, using the method described herein, to simultaneously process 96 individual plasmid miniprep samples from bacterial host cells comprising a pOT plasmid. The teaching of the instant disclosure provides ample guidance to allow an investigator of ordinary skill to modify this example to perform routing experimentation to derive a modification of this method that is capable of isolating exogenous DNA produced by the expression of alternative vectors (e.g., cosmids, BACs, P1s etc), in either high-copy- or low-copy-number, in numerous alternative host cells. Plasmid (e.g., exogenous) DNA was purified from the host cells by producing a mixture host cell lysate; preclearing the lysate of high molecular weight endogenous (e.g., host cell genomic) DNA by selectively precipitating it under conditions which promote its adsorption, but not the adsorption of pOT plasmid ...
example 2
PEG-Induced Size Selection of Sheared DNA for Shotgun Library Construction
[0082] Shortgun sequencing strategies enable the de novo determination of an unknown nucleotide sequence. The method imposes no limitation on the size of the starting DNA molecule whose sequence is to be determined and requires no prior knowledge of the nucleotide sequence of the DNA fragments selected as inserts for cloning. According to protocols which are well-known to those of skill in the art, the starting DNA molecule, whose sequence is to elucidated, s fragmented, either by enzymatic digestion or by physical shearing (e.g., using a nebulizer, sonicator or hydroshearing) to produce a shattered DNA library typically comprising 0.5-5 kb fragments. The shotgun strategy then requires that a subfraction of these fragments characterized by a narrow size range (e.g., 0.5-1.0 kb, 0.8-1.5 kb, 1.0-1.5 kb) be selected for use as inserts into an appropriate DNA sequencing vector. The nucleotide sequences of the re...
example 3
Use of PEG-Induced Size Selection to Prepare Post Nucleotide Sequencing Reaction Extension Products for Capillary Electrophoresis
[0093] A conventional sequencing reaction comprises a mixture of a DNA template, numerous extension products, excess terminators or primers and nucleotides (e.g., both deoxy- and dideoxynucleotides) admixed with reagents (e.g., salts or alcohols). It is well known that the quality of the DNA sequence data, as assessed by average read length and unedited accuracy, is a direct correlate of the purity of the extension products used for electrophoretic analysis. Purity is an important factor for all sequencing methods, and is particularly crucial to the success of automated dye-labeled dideoxynucleotide sequencing methods. The following method has been used to prepare extension products (e.g., Sanger sequencing products) for capillary electrophoresis: [0094] Transfer a 20 ul aliquot of detemplated fluorescently labeled dye terminator or dye primer sequencing...
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