Optimization of a high-throughput ctab-based protocol for the extraction of QPCR-grade dna from rumen fluid, plant and bacterial pure cultures

Konstantinos Minas, Neil Ross McEwan, Jamie Newbold, Karen P. Scott

Research output: Contribution to journalArticlepeer-review

150 Citations (SciVal)

Abstract

The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread, thus, validation and cross-laboratory comparison of data requires standardisation of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis, and further purification of DNA with phenol:chloroform:isoamyl- alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram +ve and Gram ?ve bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications.
Original languageEnglish
Pages (from-to)162-169
Number of pages8
JournalFEMS Microbiology Letters
Volume325
Issue number2
DOIs
Publication statusPublished - Dec 2011

Keywords

  • genetically modified
  • DNA extraction
  • rumen fluid
  • qPCR inhibitors
  • 96-well plate format

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