Microsatellite genotyping (Polemoniaceae)

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Method synopsis

PCR amplification of microsatellite loci followed by capillary electrophoresis for allele sizing and polymorphism assessment.

Synthesized from method descriptions across 3 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 — 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. PCR amplification
   PCR was performed using Eppendorf Mastercycler thermocyclers with initial denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, then final extension at 72°C for 4 min
   Quantities: 12.5 μL total reaction volume containing 1.5 mM MgCl2, 0.02 mM of each dNTP, 0.4 μM unlabeled primer, 0.1 μM dye-labeled primer, 0.4 μM dye-labeled primer, and 50 ng DNADuration: 4 min initial + 35 cycles (1.5 min each) + 4 min final = approximately 56.5 minutesConditions: Temperature cycling as specified
   Equipment: Eppendorf Mastercycler thermocyclers
2. Fragment analysis
   PCR products were analyzed using capillary electrophoresis with fluorescent detection and sized using ROX 500 size standard
   Quantities: Not specifiedDuration: Not specifiedConditions: Not specified
   Equipment: ABI 3730xl sequencer, ROX 500 size standard, GeneMarker software

Typical Equipment

• Eppendorf Mastercycler thermocyclers
• ABI 3730xl sequencer
• ROX 500 size standard
• GeneMarker software

Output Measurements

• allele sizes
• heterozygosity
• number of alleles per locus

Papers Using This Protocol (3)

Species Studied With This Protocol

Places Where Used