Ecological Archives E096-064-A3

Xubing Liu, Rampal S. Etienne, Minxia Liang, Yongfan Wang, and Shixiao Yu. 2015. Experimental evidence for an intraspecific Janzen-Connell effect mediated by soil biota. Ecology 96:662–671. http://dx.doi.org/10.1890/14-0014.1

Appendix C. Detailed DNA isolating and PCR procedures, and the list of ISSR primers.

After genomic DNA isolating, we determined DNA concentration and purity by measuring ultraviolet (UV) absorption using a Pharmacia 2000 UV/Visible Spectrophotometer, and also assessed the quantity and integrity of DNA samples by 0.8% agarose gel electrophoresis.

Polymerase chain reactions (PCR) were performed in 20 µL reaction mixtures that contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.0 mM MgCl₂, 0.25 mM each dNTP, 0.5 µM primer, 1.5 U Taq DNA polymerase, 30 ng genomic DNA and DNA-free water. Amplifications were conducted in an MJ-Research PTC-100TM Peltier thermal cycler under the following cycle profile: 5 min of initial denaturation at 94°C, followed by 40 cycles of 94 °C for 30 s, 53 to 54 °C for 45 s, 72 °C for 90 s, and a final extension at 72 °C for 7 min. We included negative controls in each PCR set to check for the absence of contamination.

Table C1. Primers used in ISSR analyses of Castanopsis fissa and Canarium album (R = A, G; Y = C, T).

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