From dgalbraith@rbg.ca Sat Dec 2 11:39:57 2000 Received: from mxu2.u.washington.edu (mxu2.u.washington.edu [140.142.32.9]) by lists.u.washington.edu (8.9.3+UW00.05/8.9.3+UW99.09) with ESMTP id LAA271234 for ; Sat, 2 Dec 2000 11:39:56 -0800 Received: from exchserver.rbg.ca (rbg.ca [207.236.61.195]) by mxu2.u.washington.edu (8.9.3+UW00.02/8.9.3+UW99.09) with ESMTP id LAA25509 for ; Sat, 2 Dec 2000 11:39:55 -0800 Received: by EXCHSERVER with Internet Mail Service (5.5.2650.21) id ; Sat, 2 Dec 2000 14:40:25 -0500 Message-ID: <5619D6434A1AD411859D00E018C2BA8304A5D4@EXCHSERVER> From: David Galbraith To: "'CONSBIO Conservation Biology List'" Subject: Suggestions for Field Preservation of Plant Tissue for Subsequent DNA Studies Date: Sat, 2 Dec 2000 14:40:24 -0500 MIME-Version: 1.0 X-Mailer: Internet Mail Service (5.5.2650.21) Content-Type: text/plain; charset="iso-8859-1" Suggestions for Field Preservation of Plant Tissue for Subsequent DNA Studies (reprinted from CBCN Newsletter, Vol. 5, No. 3, Nov 2000) - David Galbraith The use of molecular genetic markers in studies of biodiversity and conservation biology are gaining importance as the lab methods become more affordable and more powerful all the time. Just as in computer science, however, the maxim "garbage in, garbage out" holds for genetics studies. Although there have been many advances in recovering degraded DNA from various sources, fresh specimens that have been stored properly are always the best choice. As plant tissues are biochemically much more complex than animal tissues, common methods used for animal tissues don't work well for plants. In order to gather suggestions on how to preserve tissues from different workers, I recently asked Internet list servers on conservation biology (CONSBIO) and plant collections in botanical gardens (AABGA-COL) for recommended non-refrigerated, non-toxic field methods for preserving plant tissues for subsequent DNA extraction. Two methods seem to be in use at this time. Dr Steve Rogstad (steven.rogstad@uc.edu, University of Cincinnati, Cincinnati, OH) uses a buffer composed of CTAB and NaCl. This buffer does not require refrigeration, but unfortunately CTAB is toxic. Dr. Rogstad noted: "Several methods have been proposed for preserving DNA, and the method you choose in part depends on what markers you want, or how you want to use the DNA. E.g., for cloning or minisatellites, you need large, undegraded DNA. In our hands, the Taxon method (Rogstad, 1992) works well for most, perhaps NOT ALL (test if you can), leafy spp. Drying in silica gel, and certainly preserving in alchohol can degrade the DNA, but it can still be used to amplify shorter (ca. <5000bp??) sequences." Most frequently, researchers have reported good results using of clean, fresh, young tissue by dehydration in new silica gel. Young tissue (young shoots, leaves) is generally recommended to avoid secondary metabolites that build up in plant tissues with time; new silica gel is recommended to avoid any possible cross-contamination with earlier samples. It should be noted that silica gel, while not toxic, is an irritant to mucus membranes. Inhalation of silica gel dust must be avoided by the use of appropriate respiratory filters. Also, silica gel that has been exposed to ornamental plants (such as used for drying flowers) may contain bound pesticides and should be handled appropriately. Dr. Catherine A. Paris at University of Vermont (cparis@zoo.uvm.edu), Dr. Scott Zona at the Fairchild Tropical Garden (szona@fairchildgarden.org), Shaun Belt at the U.S. National Arboretum (nasb@SUN.ARS-GRIN.GOV) and Mary Pfauth, Portland State University (mpfauth@TELEPORT.COM) all reported using or having colleagues that use silica gel dessication. Dr Zona provided a very helpful report: "Most field botanists are using desiccants for bringing back plant tissue for later DNA extraction. Drierite (anhydrous CaSO4--available from Fisher Scientific) is excellent and very effective. It dries thumb-nail sized pieces of leaves in just hours. Unfortunately, it is difficult to re-use--a very hot oven is required to drive off the moisture. The other alternative is silica gel. It's cheaper and more readily available and is easy to re-use. However, it doesn't pull water out of specimens as quickly as Drierite (so use smaller bits of tissue). Both products come with indicator dyes to show when they are too moist to be useful. Both are non-toxic." "No specific [DNA extraction] protocol [is needed] ... just rinse off the Drierite and extract in CTab as usual." References: Rogstad, S. H. 1992. Saturated NaCl-CTAB solution as a means of field preservation of leaves for DNA analysis. Taxon 41:701-708 Dr Rogstad's method using the NaCl-CTAB buffer is available on the Web at: http://fp.bio.utk.edu/mycology/mt-NaCTAB.htm .