Abstract
Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.
Highlights
DNA extraction is a routine procedure in most plant laboratories
Transgenic plants are usually generated using an Agrobacterium tumefaciens-mediated transformation procedure, where the bacteria carry an engineered binary plasmid harboring the gene of interest for integration into the plant genome
Lysozyme is often added to the cell lysis solution, while the isolated DNA is usually re-transformed into E. coli to propagate before subsequent restriction digestion verification [2]
Summary
DNA extraction is a routine procedure in most plant laboratories. Molecular cloning involves DNA purification from E. coli, from PCR and restriction digestion mixtures, and from agarose gel slices containing DNA fragments. Genomic DNA often needs to be extracted from plant tissues to facilitate subsequent PCR, sequencing or DNA blot analysis. The extraction of the binary plasmid from A. tumefaciens is notoriously tricky due to the low plasmid copy number in Agrobacterium and the recalcitrance of the bacteria strain to cell lysis [1] To solve these problems, lysozyme is often added to the cell lysis solution, while the isolated DNA is usually re-transformed into E. coli to propagate before subsequent restriction digestion verification [2]. By using the cheap chemical compound silicon dioxide as a DNA binding matrix, we have been able to develop individual DNA purification sub-protocols for plasmid miniprep from E. coli or A. tumefaciens, extraction of DNA fragments from PCR mixtures, restriction digests or agarose gels, and extraction of genomic DNA from plant tissues. We have extensively simplified and streamlined these sub-protocols to optimize time and labor efficiency, as well as minimize the effective chemicals to achieve maximal long-term saving without sacrificing the quality of DNA products
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