16S rRNA gene clone library (Bacteria)

Knowledge graph centered on 16S rRNA gene clone library (Bacteria) with 17 nodes and 48 connections. Top connected: Microbes on mountainsides: contrasting elevational, Lepidoptera, Bacteria, elevational gradients, small mammals.

Method synopsis

PCR amplification of Acidobacteria 16S rRNA genes from soil DNA followed by cloning into vectors and Sanger sequencing to characterize bacterial community composition and phylogenetic diversity.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure from a recent peer-reviewed implementation

Steps below were extracted from the most recent peer-reviewed implementation of this protocol in the corpus — Microbes on mountainsides: contrasting elevational patterns of bacterial and plant diversity (2008), PNAS. Implementations in other papers (listed below) may differ.

1. Extract bacterial DNA and perform PCR amplification
   DNA extracted using MoBio Power Soil DNA Isolation kits. Triplicate PCRs carried out on each soil extract using Acidobacteria-specific PCR primers. PCR conditions: 3 min at 94°C, 25 cycles of 30 s at 94°C, 30 s at 50°C, 60 s at 72°C, and a final extension of 5 min at 72°C.
   Quantities: Triplicate PCRs per soil sample, 25 PCR cyclesDuration: PCR protocol: 3 min initial + 25 cycles + 5 min final extensionConditions: Laboratory conditions with specified temperature cycling
   Equipment: MoBio Power Soil DNA Isolation kits, Acidobacteria-specific PCR primers, PCR thermal cycler
2. Construct clone libraries and sequence analysis
   Purified PCR products gel-purified using MinElute PCR Purification Kit and cloned using TOPO-TA cloning kit. Ninety-six clones from each soil sample were selected for sequencing. Plasmid purification and sequencing of cloned PCR products performed at Qiagen Genomic Services. Total of 2,239 cloned 16S sequences were aligned using NAST alignment tool.
   Quantities: 96 clones per soil sample, 2,239 total cloned 16S sequencesDuration: Not specifiedConditions: Laboratory sequencing conditions
   Equipment: MinElute PCR Purification Kit, TOPO-TA cloning kit, Qiagen Genomic Services sequencing, NAST alignment tool
3. Phylogenetic analysis of bacterial sequences
   Sequences aligned and phylogenetic trees constructed using maximum-likelihood methods with phyloML program. Microbial phylogenetic tree grouped sequences into phylotypes with >99% sequence similarity cutoff using PHYLIP and DOTUR programs. Phylogenetic diversity measured using classical NRI and NTI metrics.

Typical Equipment

• MoBio Power Soil DNA Isolation kits
• Acidobacteria-specific PCR primers
• TOPO-TA cloning kit
• MinElute PCR Purification Kit
• phyloML program
• PHYLIP program
• DOTUR program

Output Measurements

• 16S rRNA gene sequences
• bacterial phylotypes
• phylogenetic diversity

Papers Using This Protocol (1)

Species Studied With This Protocol