Appendix C. Methods for DNA extraction, PCR, sequencing, and sequence assembly followed Arnold et al. (2007).
To extract total genomic DNA, small quantities of mycelium (ca. 75µL in volume) were removed directly from active cultures on 2% MEA, ground with a sterile plastic pestle in 500µL SDS extraction buffer (0.15 M NaCl, 50mM Tris [pH 8.0], 10mM Na2EDTA, and 2% SDS), and incubated 12 h at room temperature. Samples were treated with 500µL of phenol:chloroform:isoamyl alcohol, mixed briefly, and centrifuged at 10000 G for 15 min. DNA was precipitated from the reserved supernatant using absolute isopropanol (-20°C; 0.6:1 isopropanol:aqueous volume), pelleted by centrifugation (5 min at 10000 G), washed in 70% ethanol (-20°C), dried for 40 min in a rotary vacufuge, and eluted in 50µL of sterile water. Extraction products were diluted 1:10 for use in PCR.
PCR amplification of the nuclear ribosomal internal transcribed spacer region (ITSrDNA) was performed in 25 µL reaction volumes containing 2.5 µL dNTPs (10 µM), 2.5 µL bovine serum albumin (BSA), 2.5 µL PCR buffer, 1.25 µL of each primer (10µM), 13.875 µL water, 0.125 µL Taq polymerase, and 1.0 µL of diluted template. Primers ITS5 or ITS1F, and ITS4 or LR3, were used to amplify ITSrDNA ( see http://www.lutzonilab.net). Cycling reactions were run on PTC-200 Thermal Cyclers (MJ Research) using the following parameters: 4 min at 95°C; 35 cycles of 30 s at 95°C, 30 s at 50°, 52°or 54°C, and 90 s at 72°C; 10 min at 72°C. Gel electrophoresis using SYBR Green demonstrated single bands for all products.
PCR products were cleaned using Qiagen or Microcon PCR purification columns and sequenced in 10 µL reaction volumes (1.5 µL of BigDye, 2.5 µL of Big Dye buffer, 1 µL of primer, 3.5 µL of sterile water, and 1.5 µL of PCR product). Amplified DNA was sequenced for both forward and reverse reads on an ABI 3700 automated sequencer using primers ITS5 or ITS1F and ITS4.
Contigs were assembled automatically using the applications phred and phrap (Ewing et al. 1998), facilitated by BioPython scripts written by Frank Kauff, Duke University (available through FL). All sequencing reads were checked manually before submission to BLAST searches of the NCBI GenBank database for provisional identification at higher taxonomic levels.
LITERATURE CITED
Ewing , B., L. Hillier, M. Wendl, and P. Green. 1998. Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Research 8:175–185.
Arnold , A. E., D. A. Henk, R. L. Eells, F. Lutzoni, and R. Vilgalys. 2007. Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR. Mycologia (in press).