(BioTechniques 1993 article Vol. 14(5):748-749)
C. Neal Stewart, Jr., and Laura E. Via
Biology Department
Virginia Polytechnic Institute and State University
Blacksburg, VA 24061
Internet:nstewart@uga.cc.uga.edu
INTRODUCTION
Many DNA isolation techniques widely employed by plant molecular biologists use a CTAB (hexadecyltrimethylammonium bromide) extraction buffer coupled with reusable tissue homogenization systems such as a mortar and pestle (3,4,10). These procedures, though simple, typically use large amounts of buffer (10 ml), utilize nondisposable homogenizers and require ethanol washes. The risk of cross contamination associated with reusing homogenizers and vessels is unacceptable if the DNA isolated will be amplified in PCR or RAPD (random amplified polymorphic DNA, 11) experiments. Recent DNA extractions methods developed to avoid potential contamination disrupt cells by biochemical means (1), leaf squashes (7) or sodium dodecyl sulfate mini preps (5). However, the biochemical lysis method and the leaf squash method are complicated and/or do not yield sufficient DNA for many replicate reactions. The SDS procedure is similar to the protocol described here, but the CTAB buffer should be more amenable to plant material containing polysaccharides (4, 6).
The procedure presented is a modification of the Doyle and Doyle CTAB method (4) scaled to fit in microcentrifuge tubes with reagents (6) added to increase separation of polysaccharides from the DNA. Milligram amounts of leaf tissue is ground using a cordless drill driven pipette tip (as devised by authors) in a microcentrifuge tube with hot CTAB buffer. A single chloroform isoamyl alcohol (24:1) extraction is followed by a single isopropanol precipitation. This simplified, quick, and inexpensive CTAB procedure yielded sufficient template for >100 reactions using only disposable homogenizers and vessels, and requiring only one transfer of the DNA solution thereby reducing potential DNA cross contamination. The RAPD fingerprinting technique is thought to be sensitive to the quality of the DNA template (11). To show the success of the presented CTAB mini prep method, DNA samples were isolated from 5 plant and 1 fungus species by both the Doyle and Doyle method (4) followed by purification through a CsCl ethidium bromide gradient and the described miniprep procedure. The DNA samples isolated by the two methods were used in RAPD reactions and the fingerprints compared. The DNA isolated by the described CTAB miniprep method compared favorablely to the control CsCl cleaned DNA for use in RAPD reactions.
MATERIALS AND METHODS
DNA was extracted from 5 plant species (American chestnut, Castanea dentata; American cranberry, Vaccinium macrocarpon; geranium, Pelargonium x hortorum; Peters Mountain mallow, Iliamna corei; and peanut, Arachis hypogaea) and 1 fungus (Russula sp.) by two methods. A standard CTAB genomic DNA isolation method (4) followed by ultracentrifugation through a cesium chloride/ethidium bromide gradient (8) was used as a control isolation method against which the following method was judged. For the control method 0.33 grams of plant or fungal tissue was processed in 10 ml CTAB buffer and the resulting DNA was resuspended in 100 l of TE (10 mM Tris HCl, 1 mM EDTA, pH 7.4).
