Dna extraction

Lee, Ha-Eun, Kim, Taehee & Ki, Jang-Seu, 2025, Description of Mychonastes koreanus sp. nov. (Sphaeropleales, Chlorophyceae) isolated from the Yun River, Republic of Korea, Phytotaxa 693 (4), pp. 255-269 : 256-257

DNA extraction , amplification, and assembly

The strain was harvested with 0.2 μm pore sized membrane filter (Millipore, Billerica, MA) and treated with 800 μL cetyltrimethylammonium bromide (CTAB) buffer (100 mM tris-HCl pH 8.0, 100mM Na2EDTA, 100 mM sodium phosphate pH 8.0, 1.5M NaCl, 1% CTAB). The sample was stored at -20°C until further experiment. Total genomic DNA was extracted following the CTAB method ( Richards et al. 1994).

A nuclear region of partial 18S to 28S rRNA including full ITS1 + 5.8S + ITS2 region (3,760 bp) was amplified with polymerase chain reaction ( PCR) with a primer set (forward: 18F01, 5′-CACCTGGTTGATCCTGCCAGTAG-3′; reverse: 28 R1318 , 5 ’-TCGGCAGGTGAGTTGTTACACAC-3′). The 20 μL PCR reaction mixture was composed of 12.8 μL sterile distilled water, 2 μL 10× Ex PCR buffer ( Takara Shuzo , Kyoto, Japan), 2 μL dNTP mix (4mM each), 1 μL of each primer (10 pmol), 0.2 μL Ex Taq polymerase (2.5U), and 1 μL of template. The PCR performing conditions were as follows: 94℃ for 3 min ; 40 cycles of 94℃ for 30 sec, 61℃ for 40 sec, and 72℃ for 2 min; and 72℃ for 10 min. The PCR product was confirmed with 1.5% agarose gel (Condalab, Madrid, Spain) stained with Midori Green under ultraviolet light. PCR products were purified with the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany) .

DNA was sequenced by the sanger sequencing method with the ABI3730 DNA sequencer using the same primer for PCR (Applied Biosystems, Foster City, CA). The complete sequence of the amplified DNA was obtained through primer walking. The obtained DNA fragments were assembled and edited with Sequencher 5.1 (Gene Codes Corporation, Ann Arbor, MI).