Soil samples were taken from Ansio (S4) and Artxanda (S10 and S14) in the Basque Country of Spain during October and November 2002. DNA was extracted using the FastDNA SPIN kit for soil (Q-Biogene, Carlsbad, CA). All PCR amplifications used the same Bacteria
-specific forward primer, 63f (5′-CAG GCC TAA CAC ATG CAA GTC [9
]), with a GC clamp attached at the 5′ end (13
), which was purified by polyacrylamide gel electrophoresis (PAGE) by Integrated DNA Technologies (Coralville, IA). The universal reverse primer was 517r, 5′-ATT ACC GCG GCT GCT GG (13
). The reverse primer was synthesized alone or with the addition of a 5′-terminal Cy5, Cy3, or 6-carboxyfluorescein (FAM).
Fluorophore-labeled primers were purified by high-pressure liquid chromatography (HPLC). Primers were dissolved in sterile water to a concentration of 100 μM between December 2003 and April 2004 and were stored frozen (−20°C). The PCR amplifications described here were conducted in August 2004. There was no noticeable effect of long-term storage on fluorophore stability. PCR (25 cycles) amplified a ~490-bp 16S rRNA gene fragment from 1 μl of diluted soil DNA (0.625 ng/μl) in a PTC-200 thermal cycler (MJ Research, Waltham, MA), following a previously described protocol (8
). PCR products were quantified by UV transillumination on a 1.5% agarose gel stained with ethidium bromide.
DGGE was performed using the Bio-Rad D-Code system (Bio-Rad, Hercules, CA) according to the manufacturer's directions using 6% (37.5:1) polyacrylamide gels with a denaturing gradient of 40 to 70% and a nondenaturing polyacrylamide top-up. Standard markers were generated with equal-volume mixtures of PCR products from 10 16S rRNA gene fragments cloned from cultured isolates or previously run soil DGGE fingerprints (8
). For gels in which standards were run with samples, 4 μl (160 ng) of the standard mixture and 100 ng of each soil PCR product were loaded in each lane. Electrophoresis was carried out for 14 h at 60°C and 85 V. After electrophoresis, gels were either imaged directly or first stained with SYBR Green I (Molecular Probes, Eugene, OR) at a 1:10,000 dilution for 1 h and then destained for 15 min in 1× Tris-acetate-EDTA buffer. Gel images were obtained at 100-μm resolution with a Typhoon 9400 variable mode imager (Amersham Biosciences, Piscataway, NJ) or by charge-coupled device image capture from UV transillumination with an AlphaImager 1200 (Alpha Innotech, CA) using a SYBR Green I filter. Typhoon scans were done using the excitation laser and emission filter recommended by the manufacturer for each fluorophore. All gels were scanned with photomultiplier tube voltages set to maximize signal without saturating fingerprint bands.