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Fundamental molecular techniques for rhizobia

Reeve, W.G., Tiwari, R.P. and Poole, P.S. (2016) Fundamental molecular techniques for rhizobia. In: Howieson, J.G. and Dilworth, M.J., (eds.) Working with rhizobia. Australian Centre for International Agricultural Research, Canberra, pp. 221-243.

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Abstract

Working with DNA is now a fundamental skill in working with rhizobia. It is necessary for typing strains using PCR methods and for sequencing activities ap¬plied to understanding genomes; their structure, how they function, and their taxonomic position.

Nucleic acid purification is the separation of nucleic acids from proteins, cell wall debris and polysaccharide after lysis of cells. For rhizobia, we provide here a num¬ber of commonly used methods for the extraction of genomic and plasmid DNA. Methods for extraction of total RNA are presented in Chapter 13. The CTAB method (Protocol 11.1.1) has been used extensively for extraction of total genom¬ic DNA for DNA sequencing while Protocol 11.1.2 gives higher yields but gener¬ally with slightly lower purity. Plasmid DNA can be differentially displayed using Protocol 11.2.1 for determination of replicon number. This method allows locali¬sation of genes to replicons, confirmation of genome assemblies and identification of genetic changes. The plasmids can subsequently be purified from low melting point gels using GELase (Epicentre, http://www.epibio.com/item.asp?id=297). Protocol 11.2.2 presents a method to recover introduced plasmids from rhizobia (i.e. complementing plasmids) for transformation into Escherichia coli prior to restriction analysis. Protocol 11.2.3 provides an alternative method to the GELase procedure for purifying plasmids but has not been tested as extensively.

Publication Type: Book Chapter
Murdoch Affiliation: Centre for Rhizobium Studies
Publisher: Australian Centre for International Agricultural Research
Copyright: © Australian Centre for International Agricultural Research (ACIAR) 2016
URI: http://researchrepository.murdoch.edu.au/id/eprint/34586
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