Dna extraction

Hsu, K. - M., Tsai, J. - L., Chen, M. - Y., Ku, H. - M. & Liu, S. - C., 2013, Molecular phylogeny of Dioscorea (Dioscoreaceae) in East and Southeast Asia, Blumea 58 (1), pp. 21-27 : 23

DNA extraction , amplification and sequencing

DNA was extracted from fresh leaves, dried leaves, or herbarium sheets using a Puregene DNA Purification kit (Gentra Systems, Minneapolis, MN, USA). Four gene products were amplified by primers trnL -5 (5’-CGAAATCGGTAGACGC- TACG-3’) and IGS-3 (5’-ATTTGAACTGGTGACACGAG-3’) for trnL-F, matK -F (5’- ACCCCATCCCATCCATCTGGAAAT-3’) and matK -R (5’-TATCCAAATACCAAATGCGTCCTG-3’) for matK, rbcL -F (5’-GTTGGATTCAAAGCTGGTGTTAAAGAT-3’) and rbcL -R (5’-CGTCCCTCATTACGAGCTTG-3’) for rbcL, and atpB -2 (5’-AGCGTTGTAAATATTAGGCATCTT-3’) and rbcL -2 (5’-ATCTTTAACACCAGCTTTGAATCCAAC-3’) for atpB-rbcL, respectively. A total volume of 50 μl PCR reaction contained 1 μl of template DNA (50–100 ng extracted genomic DNA ), 1 μl of 10 mM of each primer, 2.5 μl of PCR buffer, 1 μl of 10 mM dNTPs, 2.5 μl of 25 mM MgCl 2 and 1 U of Taq polymerase. PCR reactions were performed in a PCR thermocycler (GeneAmp 9700 PCR system;Applied Biosystems, Foster City, CA, USA) and carried out in the following conditions: an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 52 °C for 1 min and 72 °C for 2 mins, with a final extension of 72 °C for 7 min. The PCR amplified products were checked on a 1 % agarose gel electrophoresis stained with ethidium bromide. Using Micro-Elute DNA Clean /Extraction Kit (GeneMark, Taiwan), the PCR products were purified and dissolved in 10 μl ddH 2 O. The purified PCR products were sequenced with the

PCR primer pairs in both directions by an ABI Model 3100 DNA sequencer (Applied Biosystems, USA) with BigDye terminator cycle sequencing reagent (Applied Biosystems, USA).