The pH of the soil samples was analyzed with an AB151 pH meter (Fisher Scientific, Suwanee, GA) according to the manufacturer’s protocols for soils. The moisture content was determined using a Hydra ProbeÂ® Soil Sensor (StevensÂ® Water Monitoring System, Inc.) according to the manufacturer’s specification in the lab. The temperature of the sites was monitored using temperature probes (I-buttonÂ® Devices).
DNA Extraction and Quantification
Soil DNA was extracted with the Fast DNA Spin Kit for SoilÂ® (MP Bio, Solon, OH) (Mills et al., 2003) using the FastPrepÂ®-24 System homogenizer .The extracted DNA was quantified using the BioRad Fluorescent DNA Quantitation Kit and ModulusTM Microplate Multimode Reader. The DNA samples were diluted to a stock concentration of 20ng/Âµl and the remaining sample volumes were stored at -20CÂ°. Extracted DNA was checked for its quality on a 1% agarose yield gel.
The soil DNA was amplified using multiplex LH-PCR and two duplexes, one for bacteria and fungi and one for plant and archaea. Universal primers for the four taxa were used. 16S rRNA gene was used for bacteria and archaea (Suzuki et al., 1998, Delong, 1992 and Cocolin et al., 2001, the ribosomal internal transcribed spacer region (ITS) for fungi (White et al., 1990), and the chloroplast trnL intergenic region for plants (Taberlet et al., 1991). The forward primers were labeled with the blue 6-FAM dye. The optimized concentrations of PCR reagents for both duplexes are 1X reaction buffer, 2.5mM MgCl2, 250 Âµm dNTPs (Promega, Madison, WI), 1% BSA (fraction V, Fisher Scientific, Pittsburgh, PA), 1% DMSO (Promega, Madison, WI), various Âµm of primers, 2ng DNA, and 0.5 U DNA polymerase AmpliTaq Gold DNA PolymeraseTM (Applied Biosystems, Foster City, CA), and diethylpyrocarbonate-treated (DEPC) water to a final volume of 20ÂµL. Same program was used to amplify each duplex using a 9700TM thermocycler (Applied Biosystems, Foster City, CA). The following parameters were used; an initial 10 minute denaturing step at 95Â°C, 25 cycles of denaturation at 95Â°C annealing at 52Â°C and extension at 72Â°C each for 30 seconds with a final extension at 72Â°C for 10 minutes. The four taxa were each run individually and in duplex, with control DNAs and soil samples.
Capillary Electrophoresis (CE):
The amplified PCR products were used for amplicon length heterogeneity (LH-PCR). The sample for CE separation was prepared by mixing 0.5 Âµl of the PCR product with 9 Âµl of Hi-DiTM formamide containing a 96:1 ratio of Hi-DiTM and GeneScanTM LIZ 600TM (Applied Biosystems, Foster City, CA). GeneScanTM LIZ 600TM was fluorescently labeled size standard. DS-33 matrix and filter set G (6FAMâ„¢, VICâ„¢, NEDâ„¢ PETâ„¢, and LIZTM) were used (Applied Biosystems, Foster City, CA). The PCR products were denatured by heating at 95Â°C for two minutes and then immediately snap cooled on ice for five minutes. The samples were then electrokinetically injected at 15 kV for five seconds and run at 60Â°C on an ABI PrismTM 310 (Applied Biosystems, Foster City, CA) using Performance Optimized Polymer 4 (POP4) (Applied Biosystems, Foster City, CA) with laser power at 9.9mW and capillary length of 45.72 cm, and a run time of 28 minutes per sample.