Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures.
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 require standardization 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-positive and Gram-negative 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.
Institute for Innovation Design and Sustainability, Robert Gordon University, Aberdeen, UK. k.minas@rgu.ac.uk
Konstantinos
K
Minas
Neil R
NR
McEwan
Charles Jamie
CJ
Newbold
Karen P
KP
Scott
D000704:Analysis of Variance; D000818:Animals; D001826:Body Fluids / Q000737:chemistry; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000737:chemistry; D057166:High-Throughput Screening Assays; D030821:Plants, Genetically Modified; D012417:Rumen / Q000737:chemistry; D012639:Seeds / Q000737:chemistry; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet
D002593:Cetrimonium Compounds; D004247:DNA; D000077286:Cetrimonium