Abstract
In double-stranded DNA sequencing the two DNA strands must first be separated to enable the primer to bind to the priming site. This may be done by treating the DNA with alkali. Conventionally, use of the alkali denaturation method involves neutralization of the sample by acid treatment followed by ethanol precipitation and recovery of the DNA by centrifugation. The time-consuming process of ethanol precipitation may be avoided by neutralizing the alkali-treated DNA and recovering the sample in its original volume by passage through a spin-dialysis column. The use of spin-dialysis confers the additional advantage of cleaning-up the template by removing traces of low molecular weight compounds that may interfere with the sequencing reactions. The method used here to prepare plasmid is a variation on the boiled-lysis method (1) using a rapid approach avoiding phenol extraction.
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Reference
Holmes, D S and Qulgley, M. (1981) A rapid bollmg method for the preparatlon of bacterlal plasmlds. Anal. Bmchem. 114, 193
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© 1993 Humana Press Inc. Totowa, New Jersey
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Murphy, G. (1993). Plasmid Sequencing. In: Griffin, H.G., Griffin, A.M. (eds) DNA Sequencing Protocols. Methods in Molecular Biology™, vol 23. Humana Press. https://doi.org/10.1385/0-89603-248-5:137
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DOI: https://doi.org/10.1385/0-89603-248-5:137
Publisher Name: Humana Press
Print ISBN: 978-0-89603-248-4
Online ISBN: 978-1-59259-510-5
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