Appendix A. Methods used for DNA isolation, PCR amplifications, and DGGE analysis of arbuscular mycorrhizal fungi.
Root material (80 mg) was added to a 2-mL-screw-capped polypropylene tube containing 600 µL of lysis solution, 300 mg of 1.0-mm-diameter zirconium beads (for the destruction of plant material) and 200 mg of 0.5-mm-diameter glass beads (BioSpec Products) (for the destruction of fungal material). Tubes were shaken three times for 30 s at 5,000 rpm in a minibeadbeater (BioSpec Products) and subsequently incubated at 65 ºC for 1 h. Samples were cooled to 37 ºC and treated with 3 µL of 10 mM RNAse solution for 15 min. Tubes were then cooled on ice for 5 min, 200 µL Puregene Protein Precipitation solution (Gentra Systems) added, mixed and centrifuged at 14,000 × g for 3 min. The supernatant was transferred to a new 1.5-mL microcentrifuge tube, and 600 µL ice-cold isopropanol was added. Tubes were inverted several times prior to centrifugation at 14,000 × g for 4 min at 4 ºC. The pellet was subsequently respuspended in 400 µL of TE (10 mM Tris, 1 mM EDTA, pH 8.0) for phenol-chloroform extraction, ethanol precipitation and washing (Sambrook et al. 1989). Pellets were allowed to air-dry and dissolved in 30 µL of 10mM Tris (pH 8.0) and stored at –20 ºC.
Approximately 30 ng of template DNA template was used for each PCR using the Expand High Fidelity DNA polymerase system (Boehringer, Mannheim, Germany), with the manufacturer’s recommended buffer, enzyme and nucleotide conditions. Reactions contained 10 pmol of each primer, and thermocycling used the following program: 94 °C, 2 min; 35 × (92 °C, 30 sec; 60 °C, 60 sec; 68 °C 45 sec + 1 sec per cycle); 68 °C, 5 min. PCR products (5 µL per reaction) were examined by standard 1.5 % (w/v) agarose gel electrophoresis with ethidium bromide staining, to confirm product integrity and estimate yield relative to known concentrations of Biozym Medium Ladder (Biozym Netherlands bv; Landgraaf, The Netherlands).
Gels contained 6% ( w/v) polyacrylamide (37:1 acrylamide:bis-acrylamide) 0.5 × TAE, and were 1.5 mm thick. (20 × 20 cm) The linear gradient used was from 25% - 35% denaturant, where 100% denaturing acrylamide is defined as containing 7 M urea and 40% formamide (Muyzer et al. 1998). To ensure well-polymerized slots, a 10 mL top gel containing no denaturants was added before polymerization was complete. All DGGE analyses were run using a D-Gene system (Bio-Rad Laboratories, Hercules, California, USA) at a constant temperature of 60 °C. Electrophoresis was for 10 min at 200 V, after which the voltage was lowered to 80 V for an additional 16 h. Gels were stained in MilliQ (Millipore B. V., Etten-Leur, the Netherlands) water containing 0.5 mg L-1 ethidium bromide and de-stained twice in MilliQ water prior to UV transillumination. Gel images were digitally captured using the ImaGo system (B & L; Maarssen, the Netherlands).
Muyzer, G., T. Brinkhoff, U. Nübel, C. Santegoeds, H. Shäfer, and C. Wawer. 1998. Denaturing gradient gel electrophoresis (DGGE) in microbial ecology. Pages 1-27 in A. D. L. Akkermans, J. D. van Elsas, and F. J. de Bruijn, editors. Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, Second edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, New York, USA