HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Figures Only Full Text Free Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Flagel, L. Articles by Matthews, P. D. Search for Related Content PubMed Articles by Flagel, L. Articles by Matthews, P. D. Agricola Articles by Flagel, L. Articles by Matthews, P. D. Related Collections Soybean Crop Genetics

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY NOTE

Inexpensive, High Throughput Microplate Format for Plant Nucleic Acid Extraction

Suitable for Multiplex Southern Analyses of Transgenes

^a Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, St. Paul, MN 55108
^b BASF Plant Science, 26 Davis Drive, Research Triangle Park, NC 27709
^c Crop Improvement, S.S. Steiner, Inc., 655 Madison Ave, New York, NY 10021

^* Corresponding author (pmatthews{at}hopsteiner.com)

To achieve high throughput analysis of genomic DNA from small amounts of plant tissue, a standard nucleic acid extraction was formatted to microplates by linking a novel series of interlocking plates. The protocol integrates steel pellet tissue homogenization and unique liquid handling and phase separation techniques with minimal special equipment. The method was validated for difficult plant DNAs, such as hop (Humulus lupulus L.), soybean [Glycine max (L.) Merr.], and tobacco (Nicotiana tabacum L.), with agarose gel separations of restriction fragments and Southern blot analysis of transgene integrants in soybean. The range of success rates for Southern blots was 73 to 92% per sample per plate (n = 2016 samples). Variation in absolute yield was quantified by PicoGreen microplate flourimetry. The variation in yield among samples per plate was small enough to obviate individual sample concentration adjustment before Southern analysis. Average absolute yield for the barley (Hordeum vulgare L.) mapping population based on flourimetry was 6.8 µg ± 0.85 SE, n = 96; providing enough pure, stable DNA for many individual polymerase chain reaction (PCR) reactions. Intersample cross-contamination and suitability for PCR analysis were validated by simple sequence repeat (SSR) display of a previously characterized barley mapping population. Cost calculation from a materials detail was U.S. $0.17 per sample extraction. Although the implementation requires some skill and practice, we find it to be a robust, adaptable, and versatile alternative to expensive commercial kits.

Abbreviations: CTAB, cetyltrimethylammonium bromide • PCR, polymerase chain reaction • SSR, simple sequence repeat