All were obtained from the same reputable supplier at different dates, and paired with primer R907 (Teske et al., 1996). Soil community PCR was performed with

a 25-μL reaction mixture containing Protein Tyrosine Kinase inhibitor 1.25 U GoTaq polymerase (Promega), 1 × PCR buffer, 1.5 mM MgCl2, 0.4 mg mL−1 bovine serum albumin, 4 μM each primer, 200 μM dNTPs, and 10 ng of template DNA. Pure-culture PCR was performed using a 30-μL reaction mix using the above concentrations of reagents. Thermocycling conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 45 s, and 72 °C for 1 min, and a final elongation at 72 °C for 7 min. After PCR, 1 μL of PCR product was resolved in a 1.5% agarose gel to confirm product size and the negative control. DNA concentrations were determined throughout by fluorometry using the HS dsDNA kit and Qubit Fluorometer (Invitrogen). A total of 300 ng of PCR product was loaded into each lane for soil community DGGE, while 50 ng of DNA was loaded for pure-culture DGGE. A denaturing gradient of 35–65% denaturants [100% denaturants is a mixture of 7 M urea and 40% (v/v) formamide] (Muyzer et al., 1993) was used in 6% (w/v) polyacrylamide gels. Electrophoresis was performed in 1 × Tris-acetate EDTA buffer at 60 °C

and at a constant voltage of 70 V for 16 h using a DCode system (BioRad). The DGGE gels were stained in a 1 : 2000-diluted SybrGold (Molecular Probes) in water Ribociclib datasheet Florfenicol for 30 min. Gel images were captured

using a ChemiDoc XRS (BioRad), and analyzed using quantity one software (BioRad). The background was subtracted using a rolling disk set at 20, and band density at positions was converted to intensity per Rf value between 0 and 1. After normalizing for total intensity across lanes, data were input into the past software package and analyzed using multivariate principal component analysis (Hammer et al., 2001). PCR product from each primer set was ligated into pGEM-T Easy Vector (Promega) and transformed into E. coli JM109 competent cells. A total of 10 clones from each primer set reaction were chosen at random for sequencing. The DNA sequences were determined using the chain termination method by the Nevada Genomics Center, using the T7 primer. Vector sequence and 16S rRNA gene sequences downstream of the respective primer were removed manually. The melting temperature (Tm) was calculated with biomatch (Promega), using the base-stacking algorithm. Empirical observations of differences in DGGE profiles generated using separate GC-clamp primer batches lead us to suspect variation in performance of distinct batches. Therefore, we PCR-amplified two soil DNA extracts using three batches (N1–N3) of the same GC-clamp primer, paired with the same reverse primer R907. To compare the effect of a longer template-directed sequence, primer G1 had the same GC-clamp sequence but an 18- rather than a 15-base 16S rRNA gene sequence (Muyzer et al., 1993).