Ecological Archives E094-178-A1

Laura T. Carney, Andrew J. Bohonak, Matthew S. Edwards, Filipe Alberto. 2013. Genetic and experimental evidence for a mixed-age, mixed-origin bank of kelp microscopic stages in southern California. Ecology 94:1955–1965. http://dx.doi.org/10.1890/13-0250.1

Appendix A. PCR reaction conditions and analysis.

Multiplex 1: Mpy-7, Mpy-8, Mpy-9, Mpy-17, Mpy-19, Multiplex 2: Mp-BC-9, Mp-BC-10, Multiplex 3: Mp-BC-4, Mp-BC-13, Mp-BC-16; Alberto et al. 2009). For each multiplex, PCR reactions in 15 µL contained ≈ 25 ng DNA, 2.5-10 µM of each primer, 60 µM of DNTPs, 2.0 mM of MgCl2, 1.5 µL 10x PCR buffer (200 mM Tris-HCL (pH 8.4), 500 mM KCL) and 0.3 µL U Taq DNA polymerase (Invitrogen, Life Technologies). Cycling conditions consisted of an initial denaturing step of 5 min at 94°C, followed by 24 cycles of “touchdown” PCR consisting of 30 s at 94°C, and then 30 s at either 60°C (multiplex 1), 55°C (multiplex 2) or 62°C (multiplex 3), each reduced 0.5°C each subsequent cycle, 30 s at 72°C, 10 additional cycles consisting of 30 s at 94°C, 30 s at 50°C and 45 s at 72°C and a final elongation step at 72°C for 20 min. All PCR reactions were performed on a GeneAmp 9700 thermocycler (PE Applied Biosystems). Fragment length was analyzed on an ABI PRISM 3130 DNA analyzer (Applied Biosystems) using GeneScan-500 LIZ standard. Allele sizes were scored with STRand (http://www.vgl.ucdavis.edu/informatics/strand.php) and reviewed for ambiguities and binned into allele classes using the R package msatAllele.1.03 (Alberto 2009).

Literature cited

Alberto, F. 2009. MsatAllele_1.0: An R package to visualize the binning of microsatellite alleles. Journal of Heredity 100:394–397.

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