Application of an inexpensive and high-throughput genomic DNA extraction method for the molecular ecology of zooplanktonic diapausing eggs

We describe the application of a simple, low-cost, and effective method of DNA extraction (hot sodium hydroxide and Tris, HotSHOT) to the diapausing propagules of continental aquatic invertebrates for its use in PCR amplification. We illustrate the use of the technique in cladocerans, rotifers, anostracans, notostracans, and copepod diapausing eggs. We compare the performance of the HotSHOT technique to the currently most widely used method for DNA extraction of zooplankton eggs and individuals, the chelating resin (or Chelex) technique. The HotSHOT technique overcomes several of the problems posed by Chelex and permits easy optimization for its use with 96-well plates for high-throughput DNA extraction and subsequent genetic characterization. We foresee a wide use of this technique in the future from DNA barcoding of diapausing stages to the genetic characterization of the diapausing egg banks of continental aquatic invertebrates. *Corresponding author: E-mail [email protected] Acknowledgments We thank D. Lunt for suggesting the use of HotSHOT, for technical help, and for providing old Daphnia samples. We are grateful to T. Zierold for providing Triops samples and F. Amat for providing Artemia cysts. Rob Hammond is kindly thanked for his constructive comments on an earlier version of this manuscript. We also thank the valuable comments and suggestions by two anonymous reviewers. This study was partly funded by a NERC Advanced Fellowship to AG. JM was supported in his stay in Hull by the Spanish Ministerio de Educación y Ciencia project (project CGL2006-05085/BOS) to A.J. Green, C. Rico, F. Hontoria and J. Figuerola. The Spanish Ministerio de Educación y Ciencia supported JMP during his stay in the University of Hull. Limnol. Oceanogr.: Methods 6, 2008, 218–222 © 2008, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS