Inexpensive, high throughput microplate format for plant nucleic acid extraction: Suitable for multiplex southern analyses of transgenes

L. Flagel, J. R. Christensen, C. D. Gustus, K. P. Smith, P. M. Olhoft, D. A. Somers, P. D. Matthews

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

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 homegenization 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 mioroplate 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 popalation 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.

Original languageEnglish (US)
Pages (from-to)1985-1989
Number of pages5
JournalCrop Science
Volume45
Issue number5
DOIs
StatePublished - Sep 2005

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