Abi prism 3730xl dna sequencer

To obtain the CHS and UFGT sequences, a polymerase chain reaction (PCR) analysis was performed in a MultiGene thermal cycler (Labnet International, Inc., Woodbridge, NJ, US) using 20 ng template DNA, 0.5–1 U Taq (Bernardo Scientific Corp., Taipei, Taiwan), 100 μM deoxyribonucleotide triphosphate, and 0.2 μM of each primer. Primers for CHS amplification were adapted from those used by Chiang et al. [19 (link)]. The primers for UFGT amplification were designed from the transcriptomic assembly of inflorescences buds used by Huang et al. [15 (link)]. The primer sequences for UFGT were ScUGT-F1: 5′-GTTCCAATGATAGCTCATGG-3′ and ScUGT-R1: 5′-GGAACATAGGCACTCAATTC-3′. The PCR program was set to 94 °C for 3 min for enzyme activation, followed by 35 cycles at 94 °C for 40 s, melting temperature for 40 s, and 72 °C for 80 s, with a 5-min final extension at 72 °C. The PCR products were sequenced directly in both directions using an ABI BigDye 3.1 Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). All sequences were visually checked via chromatograms using an ABI PRISM®3730XL DNA Sequencer (Perkin-Elmer, Foster City, CA, USA).

Leaf samples were collected from stone oak populations covering the areas of Nanling (E113°01′15′′, N24°58′51′′), Mangdang Shan (E118°07′33′′, N26°41′40′′), Emei (E103°16′27′′, N29°34′52′′), Chinfo Shan (E107°09′43′′, N29°01′04′′), Kunming Golden Temple (E102°46′10′′, N25°05′15′′), and botanical gardens (Additional file 1: Table S1). Total genomic DNA was extracted from leaf samples using a modified cetyl trimethylammonium bromide (CTAB) method (Doyle and Doyle 1987 ). The DNA samples were amplified by polymerase chain reaction (PCR) using primers specific for the regions of the atpB-rbcL spacer of the chloroplast (cp) genome and the nuclear ribosomal internal transcribed spacer (nrITS) of the nuclear genome. PCR products of the atpB-rbcL spacer were directly sequenced. The nrITS amplicons were cloned using the yT&A cloning kit (Yeastern Biotech, Taipei, Taiwan) and sequenced with M13F and M13R primers. Sequencing reactions were performed using the ABI BigDye 3.1 Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and the fragments were sequenced from both directions with the ABI PRISM^®3730XL DNA Sequencer (Perkin-Elmer, Foster City, CA, USA). All sequence polymorphisms were visually rechecked from the chromatograms.