phase: Water land-use type: Residential land-use region: Res_Region2
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted from the about 5gm (wet weight) of sediment and 1.2L of water samples immediately after the samples were brought to the lab. Sediments were dewatered and DNA was extracted by a combination of mechanical, chemical and thermal lysis and chloroform-isoamyl alcohol purification using protocol from Zhou et al., 1996 (1). Water samples were filtered through sterile Millipore 0.22µm membrane filter (Thermo Fisher Scientific Inc.) to collect the biomass, followed by the DNA extraction protocol similar to the DNA extraction protocol for sediment samples. Humic substances were removed using OneStep™ PCR Inhibitor Removal Kit from Zymo Research Corporation (USA). The DNA quality and quantity was measured with PicoGreen using a FLUOstar Optima (BMG Labtech, Jena, Germany) before loading onto microarrays. amplification protocol: 0.5 - 1 ng template was used in each of 8 PCR tubes (8 different annealing temperatures were used). 300 nM primers (27F and 1492R for 16S rRNA amplicons), 200 uM each dNTP, 1 ug/uL BSA, and 1.25 U ExTaq (Takara) enzyme were used in the amplifications (95C for 3 min., 30 cycles of 95 (30 sec) ,48 - 58 C annealing temp. (30 sec), 72 C (2 min), followed by 72 C for 10 min., hold at 4 C).
Label
biotin
Label protocol
TDT enzyme (Promega; Madison, WI) and GeneChip Labeling Reagent (Affymetrix; Santa Clara, CA) were used to label fragmented amplicons.
Hybridization protocol
Arrays were hybridized overnight at 48degC and 60 rpm and then washed and stained using standard Affymetrix buffers/reagents.
Scan protocol
Images were captured on an Affymetrix GeneChip Scanner 3000 7G.
Data processing
CEL files were converted to CELa files and then processed using proprietary software (PhyCA).
