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 96:662671. 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).

Primer

Sequence (5'-3')

Primer

Sequence (5'-3')

UBC807

AGA GAG AGA GAG AGA GT

UBC840

GAG AGA GAG AGA GAG AYT

UBC808

AGA GAG AGA GAG AGA GC

UBC842

GAG AGA GAG AGA GAG AYG

UBC810

GAG AGA GAG AGA GAG AT

UBC844

CTC TCT CTC TCT CTC TRC

UBC811

GAG AGA GAG AGA GAG AC

UBC854

TCT CTC TCT CTC TCT CRG

UBC812

GAG AGA GAG AGA GAG AA

UBC856

ACA CAC ACA CAC ACA CYA

UBC814

CTC TCT CTC TCT CTC TA

UBC873

GAC AGA CAG ACA GAC A

UBC815

CTC TCT CTC TCT CTC TG

UBC880

GGA GAG GAG AGG AGA

UBC834

AGA GAG AGA GAG AGA GYT

UBC881

GGG TGG GGT GGG GTG

UBC835

AGA GAG AGA GAG AGA GYC

 

 


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