Optimisation of DNA extraction for AFLP analysis of mycorrhizal fungi of terrestrial orchids caladeniinae and drakaeinae

Published DNA extraction methods present a number of problems when applied to mycorrhizal fungi of native Australian terrestrial orchids. Grinding with liquid nitrogen shears the DNA, and other pulverisation methods yield too little DNA. We found that freezing the fungal sample with liquid nitrogen, with no grinding, followed by the Qiagen DNeasy extraction procedure produced good yields of high-molecular-weight DNA. The DNA was then used for amplified fragment length polymorphism (AFLP) fingerprinting. Good fingerprints were produced by restriction with EcoRI/MseI enzymes, the use of preamplification primer mix II (for small genomes), and a 2-base extension MseI primer (m-cc) with 3-base extension EcoRI primers in the selective amplification. This protocol may be of general utility for other fungi with similarly fragile DNA. © 2004 International Society for Plant Molecular Biology.