Titlebook: E. coli Plasmid Vectors; Methods and Applicat Nicola Casali,Andrew Preston Book 20031st edition Springer Science+Business Media New York 20

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Chemical Transformation of ,,ence is restricted to a subset of bacteria, methods for the chemical induction of a competent state in otherwise nontransformable bacteria are an important tool in bacterial genetics. For these species, competence refers to the ability to take up and propagate plasmid DNA, usually with no sequence specificity for uptake.

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Cosmid Packaging and Infection of ,,, usually ColE1, which regulates cosmid replication and copy number within the . host (.,.), and restriction sites that facilitate cloning of the desired DNA fragments and insert verification by restriction digestion (..).

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Isolation of Plasmids from , by Alkaline Lysis,lasmid DNA is concentrated from the supernatant by ethanol precipitation. Using this procedure, 2–5 μg of DNA can be obtained from a 1.5-mL culture of . containing a pBR322-derived plasmid, and three- to five-fold higher yields can be expected from pUC-derived plasmids (.).

ODIUM

modest

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Anthology

Isolation of Cosmid and BAC DNA from ,,nd sequencing. Numerous standard protocols have been developed to isolate supercoiled plasmids, but of these, the alkaline lysis protocol remains the most suitable approach to isolate large-insert clones such as cosmids and BACs.

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Preparation of Single-Stranded DNA from Phagemid Vectors,e phagemids as ssDNA into viral particles, which are subsequently released into the culture medium (.). ssDNA isolated from these virions is suitable for use as a template in a variety of procedures with no further manipulation, as denaturation is not required prior to its use as a template.

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