site: Site B level of heavy metal contamination: Moderate
Treatment protocol
All samples were maintained on ice after collected, then stored at -80°C for further analysis.
Growth protocol
Sediment
Extracted molecule
genomic DNA
Extraction protocol
Given that high concentration of divalent metal ion may result in premature DNA precipitation during extraction, the sediment samples were pre-washed with 40mM EDTA (pH 7.2). The community DNA was extracted using the Soil DNA Kit (D5625-01; Omega Bioservices, Norcross, GA, USA) according to the manufacturer's instructions. Then DNA quality was checked by the absorbance ratios at A260/A280 and A260/A230 using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). Only when the A260/A280 ratio is larger than 1.7 and the A260/A230 ratio is more than 1.8, can the DNA be used for further analysis.
Label
Cy3
Label protocol
The DNA was labeled with fluorescent dye Cy3 (GE Healthcare) by random primer, then purified with a QIAquick purification kit (Qiagen), and dried in a SpeedVac (45°C, 45 min: ThermoSavant, Milford, MA, USA).
Hybridization protocol
DNA was resuspended into 27.5μl of DNase/RNase-free distilled water, and mixed with 42μl hybridization buffer which contains 1× Acgh blocking, 1× HI-RPM hybridization buffer, 10 pM universal standard DNA, 0.05μg/μl Cot-1 DNA, and 10% formamide, then incubated at 95 °C for 3 min, and kept in 37°C for 30min. Hybridizations process was implemented in GeoChip 5.0 arrays (60 K) at 67 °C in a Agilent hybridization oven for 24 h.
Scan protocol
Slides were washed by Agilent Wash Buffers at room temperature. Then the arrays using NimbleGen MS200 Microarray Scanner (Roche NimbleGen, Inc., Madison, WI, USA) at 633 nm by a laser power of 100% and 75% a photomultiplier tube (PMT).
Description
Microbial functional gene structure in sediment with heavy metal contamination
Data processing
Scanned images were quantified with the help of ImaGene® version 6.0 (BioDiscovery, Inc., Los Angeles, CA, USA). The mean signal intensity was determinted for each spot, and local background signals were automatically ducted.
