Molecular Ecology Notes (2007) 7, 191–193 doi: 10.1111/j.1471-8286.2006.01587.x © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd TECHNICAL ARTICLE A fast and inexpensive DNA extraction/purification protocol for brown macroalgae GALICE HOARAU, JAMES A. COYER, WYTZE T. STAM and JEANINE L. OLSEN Department of Marine Benthic Ecology and Evolution, Centre for Ecological and Evolutionary Studies, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands Abstract Here we describe a rapid method for extracting DNA from dried brown algae material using a microtitre plate system in conjunction with a milling instrument. The method allows the preparation of nuclear and organelle DNA of quality suitable for polymerase chain reaction amplification. It combines high throughput with low cost per sample: DNA from 192 samples can be extracted in c. 3 h for < $0.40 per sample, nearly tenfold cheaper than commercially available kits. Furthermore, by using microtitre plates, efficient storage and downstream processing is facilitated. Keywords: brown algae, DNA extraction, heterokonts, high throughput, Phaeophyceae Received 15 June 2006; revision accepted 18 September 2006 Extracting polymerase chain reaction (PCR)-amplifiable DNA from brown algae is notoriously difficult as poly- saccharide and polyphenolic compounds, which can be strong enzyme inhibitors, are often co-extracted (Mayes et al. 1992; Jin et al. 1997). Protocols involving extraction with cetyltrimethyl ammonium bromide (CTAB), precipit- ation with chloroform-isoamyl alcohol and purification with either gradient-centrifugation or glass-binding (e.g. Sephaglas, Amersham Biosciences) techniques, have been developed to overcome the problems (Coyer et al. 1994, 2002b; Phillips et al. 2001). While these methods yield PCR- quality DNA, they are time-consuming and cannot be automated, thereby precluding their usage in high throughput projects. Methods employing Chelex resin are quick and inexpensive and have been successfully used in some brown and red algal species (Billot et al. 1998; Cohen et al. 2004), but not in other genera of brown algae (e.g. Fucus, Turbinaria, Undaria; G. Hoarau, unpublished data). Further- more, as DNA extractions with Chelex resin have a limited shelf life, extractions must be repeated regularly (e.g. Laminaria, Billot 1999; fish, G. Hoarau, unpublished data). The limited shelf life and species-specific success therefore preclude routine use of Chelex resin for DNA extraction in brown algae. Alternatively, several commercial DNA extraction kits for plants are available (e.g. Nucleospin plant kit, Clontech; DNeasy 96 Plant Mini Kit, QIAGEN) and have been successfully used with brown algae (Engel et al. 2003; Billard et al. 2005). DNA extraction with kits is much faster and can be automated, but the cost is often prohibitive for large-scale projects. The increased costs associated with greater samples sizes is an important consideration when funding sources become limited. In view of these problems, we developed a DNA extraction/ precipitation/purification method for brown algae that, when combined with a mixer mill for pulverizing dried samples, is fast, automated, and inexpensive. Reagents included: 1 The CTAB extraction buffer was 2% CTAB, 1.4 m NaCl, 20 mm EDTA (pH 8), 100 mm Tris-HCl (pH 8), 0.1% polyvinylpolypyrrolidone (PVPP), 0.2% β-mercaptoethanol (added freshly) (Coyer et al. 1994). 2 The binding buffer consisted of 6 m NaI and 0.1 m Na 2 SO 3 , clarified by filtration through a 0.45-μm membrane filter (Whatman). 3 Silica fines were prepared by placing 20–30 g of silica (silica gel 60 0.015–0.040, Merck) into c. 500 mL of milliQ- filtered de-ionized water and stirring for c. 1 h. After stirring, the silica was allowed to settle for c. 15 min. The supernatant (= fines) was transferred to 50 mL plastic tubes and centrifuged for 5 min at 1250 g. Note that the silica that settled by gravity when preparing the fines can still be used for other DNA extraction protocols Correspondence: Dr Galice Hoarau, Fax: +31 503632261; E-mail: g.g.hoarau@rug.nl