Pcr quick spin pcr product purification kit

Five representative specimens were selected and used for further molecular analyses. Apothecial discs were mainly used for DNA extraction. Samples were ground with a Mini-Beadbeater-16 (3450 RPM, 115V, 10 A; Biospec products) and then extracted with a NucleoSpin Plant II Kit according to the manufacturer's instructions (Macherey-Nagel, Duren, Germany). PCR amplifications were conducted using Amplitaq DNA polymerase with buffer conditions. The following primers were used for PCR amplifications: mtSSU1 and mtSSU3R (sequences as designed by Zoller et al. [18 ]) for amplification of mtSSU; nuLSU_artho_2F (5′-CCTTCGACGAGTCGAGTTG-3′), nuLSU_artho_2R (5′-GTGAGTTGTTACACACTCCT-3′) for nuLSU; RPB2-7cF and RPB2-11aR (as designed by Liu et al. [19 (link)]) for RPB2. PCR conditions for nuLSU and RPB2 are as described in a previous study [13 ]. The following program was used for amplification of mtSSU: initial denaturation for 5min at 94℃ followed by 30 cycles of 94℃ for 30 sec, 54℃ for 39 sec, 72℃ for 7 sec, and then final extension at 72℃ for 7 min. The amplified DNA was concentrated and purified using a PCR quick-spin PCR Product Purification Kit (INTRON Biotechnology, Inc., Seongnam, Korea). Sequencing analysis was performed.

Four representative S. saccata specimens were selected and
used for further molecular analyses. Lichen thalli with apothecial discs were mainly used
for DNA extraction. Samples were ground and extracted with DNeasy plant mini kit (Qiagen,
Valencia, CA, USA) according to the manufacturer’s instructions. PCR amplifications were
conducted using Amplitaq DNA polymerase (ThermoFisher, Watham, MA, USA). The following
primers were used for PCR amplifications: mtSSU1 and mtSSU3R for mtSSU [29 ]; NS17UCB, NS20UCB for nuSSU [30 ]; LIC24R [31 ] and LR7 [32 ] for nuLSU; fRPB2-7cf
and fRPB2-11aR for RPB2 [33 (link)]. PCR conditions
for nuSSU are as described in a previous study [34 (link)]. The following program was used for amplification of nuLSU: initial
denaturation for 4 min at 94 °C, followed by 30 cycles of 94 °C for 40 s, 52 °C for 40 s,
72 °C for 50 s, and then a final extension step at 72 °C for 8 min. Amplified DNA was
concentrated and purified using a PCR quick-spin PCR Product Purification Kit (INTRON
Biotechnology, Inc. Sungnam City, Korea) for sequencing analysis.

Total DNA was extracted directly from the lichen thalli using the DNeasy Plant Mini Kit (QIAGEN, Dusseldorf, Germany). The partial 18 s rRNA–ITS1–5.8s rRNA–ITS2–partial 28 s rRNA region was amplified with universal primers ITS1F [15 (link)], ITS4 [16 ], or LR5 [17 (link)]. PCR amplification was carried out using a Takara JP/TP600 PCR machine (Takara Bio Inc., Kusatsu, Japan) under the following conditions: initial cycle of 5 min at 94 °C, followed by 30 cycles of 30 seconds at 94 °C, 30 seconds at 55 °C, 10 min at 72 °C, and then finally 10 min at 72 °C. The partial β-tubulin sequence was amplified with Bt3-LM and Bt10-LM [18 ] or Bt-PJS-F1(5′-GCCTCTGGCAAATATGT-3′) and Bt-PJS-R1(5′-AATTCTGGCACGGTGACC-3′).
β-tubulin samples were prepared in the same way as ITS samples. The PCR reaction of β-tubulin was carried out in a 2720 thermal cycler PCR machine (Applied Biosystems, Singapore, Singapore) under the following conditions: initial cycle of 5 min at 94 °C, followed by 30 cycles of 30 seconds at 94 °C, 30 seconds at 55 °C initially, and then 52 °C for the remaining cycles, 1 min at 72 °C, and finally 72 °C for 5 min [4 (link)].
PCR products were purified using a PCR quick-spin PCR Product Purification Kit (INTRON Biotechnology, Inc., seoul, South Korea) and then sent to Genotech Cooperation (Daejeon, Korea) for sequencing.