COI DNA barcoding (Baetidae)

Knowledge graph centered on COI DNA barcoding (Baetidae) with 35 nodes and 127 connections. Top connected: Rushes, aquatic insects, Ambystoma, phenotypic plasticity, Ambystoma tigrinum.

Method synopsis

DNA extraction using modified Chelex protocols followed by PCR amplification and sequencing of the mitochondrial cytochrome oxidase I gene to assess genetic variation within and among mayfly populations.

Synthesized from method descriptions across 2 papers 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 — Are populations of mayflies living in adjacent fish and fishless streams genetically differentiated? (2005), Freshwater Biology. Implementations in other papers (listed below) may differ.

1. Mitochondrial DNA extraction
   Modified Chelex protocols to extract total genomic DNA from each B. bicaudatus individual. Used residual tissue from allozyme analysis, transferred tissue to clean 1.5 mL Eppendorf tubes with 500 μL of 5% chelex solution, ground with plastic micro-pestle, added 7 μL proteinase K (20 mg mL⁻¹), vortexed and incubated overnight at 54°C.
   Quantities: Individual extractions for each B. bicaudatus specimen, 500 μL chelex solution per sample, 7 μL proteinase K per sampleDuration: Overnight incubation at 54°CConditions: 54°C incubation temperature
   Equipment: 1.5 mL Eppendorf tubes, 5% chelex solution, plastic micro-pestle, proteinase K
2. PCR amplification
   Heated samples at 96°C for 20 min, centrifuged at 16,000 g for 2 min at room temperature, then subjected to polymerase chain reaction. Amplified approximately 710 bp fragment of cytochrome oxidase subunit I gene (COI) using primers LCO1490 and HCO2198. Cleaned DNA according to manufacturer's instructions and sequenced using an Applied Biosystems 377 automated sequencer.
   Quantities: ~710 bp fragment amplified per sampleDuration: 20 min heating, 2 min centrifugationConditions: 96°C heating, room temperature centrifugation, 16,000 g centrifugal force
   Equipment: PCR equipment, LCO1490 primer, HCO2198 primer, Applied Biosystems 377 automated sequencer

Typical Equipment

• Eppendorf tubes
• chelex solution
• proteinase K
• PCR equipment
• LCO1490 primer
• HCO2198 primer
• Applied Biosystems 377 automated sequencer

Output Measurements

• DNA sequences
• haplotype frequencies
• sequence divergence

Papers Using This Protocol (2)

Species Studied With This Protocol