The Intestinal Microecology in Chronic Constipation

Project: Research project

Project Details

Description

The Intestinal Microecology in Chronic Constipation The Intestinal Microecology in Chronic Constipation ASU will extract DNA using the Qiagen Stool Kit (Qiagen) following the manufacturers instructions and confirm successful extraction by gel electrophoresis and Nano-Drop (NanoDrop ND-1000 spectrophotometer (NanoDrop Technology, Rockland, DE)) quantification. DNA will then be stored at -20C until use for pyrosequencing, and Bacteria and Archaea-targeted PCR. Generating 16S rDNA PCR amplicons for high-throughput pyrosequencing: At ASU, we will PCR-amplify samples for pyrosequencing. We will use bacterial primers 967F and 1046R to amplify the gene sequences for V6 region of the 16S rRNA (20). In order to combine and sort multiple samples in one 454 GS FLX run, we will design unique tags of five nucleotides to identify each sample. The resulting forward primer will be a fusion of the 454 life science adaptor A, the tag, and 967F (5-gcctccctcgcgccatcag-tag-CAACGCGAAGAACCTTACC-3). The reverse primer will be unchanged. The PCR conditions used will be 94C for 2 min, 30 cycles of denaturation at 94C for 30 s, 57C annealing for 45 s, and 72C for 1 min extension followed by a final extension at 72C for 2 min. The resulting PCR products will be cleaned by using QiaQuick spin columns to remove excess primer dimers and dNTPs. We will measure the concentrations of PCR amplicons by using a NanoDrop spectrophotometer.<5=
StatusFinished
Effective start/end date9/9/097/31/12

Funding

  • INDUSTRY: Domestic Company: $161,022.00

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