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Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.

Plant methods (2014-07-24)
Adam Healey, Agnelo Furtado, Tal Cooper, Robert J Henry
ABSTRACT

Next-generation sequencing technologies rely on high quality DNA that is suitable for library preparation followed by sequencing. Some plant species store large amounts of phenolics and polysaccharides within their leaf tissue making genomic DNA extraction difficult. While many DNA extraction methods exist that contend with the presence of phenolics and polysaccharides, these methods rely on long incubations, multiple precipitations or commercially available kits to produce high molecular weight and contaminant-free DNA. In this protocol, we describe simple modifications to the established CTAB- based extraction method that allows for reliable isolation of high molecular weight genomic DNA from difficult to isolate plant species Corymbia (a eucalypt) and Coffea (coffee). The simplified protocol does not require multiple clean up steps or commercial based kits, and the isolated DNA passed stringent quality control standards for whole genome sequencing on Illumina HiSeq and TruSeq sequencing platforms.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Polyvinylpyrrolidone, average mol wt 10,000
Sigma-Aldrich
2-Mercaptoethanol, BioUltra, for molecular biology, ≥99.0% (GC)