Dna extraction

Atria, M., Eurlings, M., Baker, W. J., Dransfield, J. & Welzen, P. C. van, 2020, Phylogenetic analysis of the Calamus javensis complex (Arecaceae: Calamoideae) in Malesia, Blumea 65 (3), pp. 205-211 : 206

DNA extraction , amplification and sequencing

DNA extraction from the silica gel-dried leaves and herbarium vouchers was done using the magnetic bead-based isolation procedure (NucleoMag® 96 Plant kit, Macherey-Nagel; https:// www.mn-net.com), carried out on an automated KingFisher extractor (https://www.thermofisher.com/nl/en/home.html).

Two DNA regions were amplified, the nuclear 5S spacer and the chloroplast marker mat K. Primers ( Table 1) for this study were designed specifically for the group based on the work of Baker et al. (2000b) using Geneious v. 10.1.3 (http://www.geneious. com). Primers for the 5S spacer ( Table 1) were M13-R435R (R2) and M13F-ITS103F (F2). The mat K sequences were amplified from total genomic DNA using the designed primers M13R-831R mat K3 (M3) and M13F-578F mat K3 (F3). M13 tails (M13F TGTAAAACGACGGCCAGT, M13R CAGGAAACAGC- TATGAC) used are from Messing (1983).

The Polymerase Chain Reactions (PCRs) were carried out with an end volume of 25 μl containing 5 μl of 5 × Phire green reaction Buffer (F-527, Thermo Fischer Scientific), ultrapure water 10.5 μl, 1 μl of each 10 μM forward and reverse primer, 1 μl of 100 mg /ml Polyvinylpyrrolidone (PVP), 0.5 μl of 2 U/μl Phire™ Hot Start II DNA Polymerase (F-122S, Thermo Fischer Scientific), 1 μl of 10 mM dNTP, and 1 μl of DNA template.

The amplifications were conducted in a 96+ Grad 1000S thermocycle, programmed as follows: initial denaturation step at 98 °C for 30 sec, followed by 35 cycles of denaturation steps at 98 °C for 5 sec, an annealing step at 55 °C for 5 sec, extension step at 72 °C for 15 sec; final extension at 72 °C for 1 min.

PCR results were checked in standard 1 % agarose gel electrophoresis. Gels were stained and immersed in 0.5 μg/ml ethidium bromide solution for 30 min, visualized and recorded on a Gel Doc Systems (Bio-Rad, Barcelona, Spain; https://www. bio-rad.com/). All selected PCR amplification products were sent to BaseClear (https://www.baseclear.com). The resulting chromatograms were then assembled and edited using Sequencher™ 4.1.4 (Gene Codes Corp., Ann Arbor, Michigan, USA; https://genecodes.com/). To ensure that the DNA isolated was not contaminated, all sequences were BLAST-searched in GenBank. The sequence results of all markers were submitted to the NCBI GenBank sequence database (see Appendix 1).