Dna extraction, , PCR

Mattapha, S., Suddee, S., Duangjai, S. & Kiewbang, W., 2022, Phanera mekongensis (Fabaceae: Cercidoideae), a new species from Thailand as supported by morphological and molecular evidence, Blumea 67 (2), pp. 113-122 : 114

DNA extraction, PCR amplification and sequencing

Leaf samples of the putatively new species and P. kockiana (Korth.) Benth. were collected from cultivated plants at Kasetsart University, Bangkok, and ten other species of Phanera and two species of Cheniella were collected in the field and preserved in silica gel. Total DNA was extracted following the 2 × cetyltrimethylammonium bromide (CTAB) procedure of Doyle & Doyle (1987) with minor modifications. We washed ground plant material with sorbitol buffer (Tel-zur et al. 1999) followed by centrifugation before incubating with CTAB buffer. The trnLF region of our samples was amplified using the universal primers c and f ( Taberlet et al. 1991). PCR was performed in a total reaction mixture of 50 μL containing 25 μL of 2 × DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 21 μL of nuclease-free water, 1 μL of bovine serum albumin (BSA (20 mg /mL), 1 μL of each primer (20 mmol/L) and 1 μL of template DNA . The PCR programme consisted of an initial 3 min pre-melt at 94 °C and 35 cycles of 45 sec denaturation at 94 °C, 45 sec annealing at 50 °C, and a 1 min extension at 65 °C, followed by final extension of 6 min at 65 °C. The inter- nal transcribed spacers (ITS) of nuclear ribosomal DNA were amplified using the 5F ( Möller & Cronk 2001) and 4R ( White et al. 1990) primers. As above, the PCR was performed in a total reaction mixture of 50 μL, but the volume of nuclease-free water was reduced to 20 μL and 1 μL DMSO was added. The PCR programme was the same as above, except that the extension step was conducted at 72 °C. All of the successfully amplified products were cleaned using FastAP Thermosensitive Alkaline Phosphatase and Exonuclease I (Thermo Fisher Scientific, Waltham, MA, USA). Ultimately, cleaned PCR products were sent to Macrogen (Seoul, Korea) for Sanger sequencing using the same primers as in the initial amplification. Raw sequences were edited and assembled using AutoAssembler v. 1.4.0 ( Applied Biosystems 1995). All newly generated sequences have been deposited in GenBank (Appendix).