Optimizing DNA Barcoding Techniques in Plant Research
This document outlines the essential processes involved in optimizing DNA barcoding techniques within plant research. Key procedures highlighted include DNA extraction from minimal tissue samples, effective archival methods, and best practices for PCR and sequencing. It emphasizes the importance of pH levels in DNA stability, dilution strategies to mitigate contamination, and tailored PCR cycles for specific DNA regions. Detailed references and resources are also provided for further exploration of these methodologies in a botanical context.
Optimizing DNA Barcoding Techniques in Plant Research
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Presentation Transcript
Making Barcoding work in Plants David Erickson Department of Botany Smithsonian Institution ericksond@si.edu
Processes • DNA extraction • Archival • PCR • Sequencing • Analysis
Processes • DNA extraction • Disruption • LESS tissue is better: 0.05g dry = 2 hole punches • We use either 96 well plate (=$5 each) with 5mm stainless ball bearing – grind with tissue-lyser • Costar brand plates with 8 strip caps from Qiagen; beads from Qiagen • Single tube with lysing matrix ($2/sample) – forensic samples • http://www.qbiogene.com/fastprep/lysing_tubes.shtml • Extraction • GUSCN+Silica membrane • Ivanova, Fazekas, Hebert (2008) Plant Mol Biol Reporter • (DOI 10.1007/s11105-008-0029-4) • Archival • PCR • Sequencing • Analysis
Processes • DNA extraction • DNA Archival • pH matters….8.0 or higher is better using Tris-HCl • DNA is an acid so it will change the pH of the solution as you mix the Tris with your DNA – • check it if you want it to last? • PCR • Sequencing • Analysis
Processes • DNA extraction • DNA Archival • PCR • Dilution is the solution to Pollution • Dilute DNA 10x-100x before PCR • When PCR fails, we re-dilute 10x and try again • Taq does not matter much • Exception is repeat regions, then use phusion taq • New England Biolabs • (Fazekas et al. 2010. Biotechniques 48:277-285) • Sequencing • Analysis
Processes • DNA extraction • DNA Archival • PCR • Sequencing • Lots of cycles for rbcL and matK help (40+ cycles are good) • Lots of cycles are less good for non-coding DNA (psbA-trnH) • Use ExoSap mixture for purification of PCR (0.3ul ExoSap + 10ul PCR) • Typical Seq reaction: 0.5ul BC, 1.75ul 5X Seq buffer, 5ul PCR, 0.02ul 100uM primer, 2.73 H2O • Analysis
Processes • DNA extraction • DNA Archival • PCR • Sequencing • Analysis • Its Complicated…
