Microsatellite enrichment and primer development (Polemoniaceae)

Knowledge graph centered on Microsatellite enrichment and primer development (Polemoniaceae) with 16 nodes and 43 connections. Top connected: Ipomopsis, I. aggregata, Ipomopsis aggregata, Ipomopsis aggregata ssp. aggregata, I. tenuituba.

Method synopsis

Development of microsatellite primers through genomic library construction, enrichment, cloning, sequencing, and primer design for population genetic studies.

Synthesized from method descriptions across 1 paper using this protocol.

Procedure as described in the canonical source

Steps below were extracted from the paper that introduces this protocol — Microsatellite loci in <i>Ipomopsis aggregata</i> (Polemoniaceae) and cross-species applicability for ecological genetics studies (2012), American Journal of Botany. Implementations in other papers (listed below) may differ.

1. DNA extraction and digestion
   Single plant DNA from Poverty Gulch, Colorado was digested with RsaI endonuclease
   Quantities: One individual plantDuration: Not specifiedConditions: Not specified
   Equipment: RsaI endonuclease
2. Microsatellite enrichment
   DNA was enriched for microsatellite-containing fragments using biotinylated oligonucleotides
   Quantities: Not specifiedDuration: Not specifiedConditions: Not specified
   Equipment: biotinylated oligonucleotides
3. Fragment cloning
   Recovered fragments were cloned using TOPO TA Cloning vector and transformed into chemically competent Escherichia coli TOP10 competent cells
   Quantities: Several hundred individual colonies were PCR-amplified and sequencedDuration: Not specifiedConditions: Not specified
   Equipment: TOPO TA Cloning vector, Escherichia coli TOP10 competent cells

Typical Equipment

• RsaI endonuclease
• biotinylated oligonucleotides
• TOPO TA Cloning vector
• Escherichia coli TOP10 competent cells
• Primer3 software

Output Measurements

• microsatellite primer sequences
• allele sizes
• polymorphism metrics

Papers Using This Protocol (1)

Species Studied With This Protocol