Axyprep multisource genomic dna miniprep kit 50 prep

We took a ca. 1 mm² fragment of the thallus apex from each fresh or dry specimen to extract genomic DNA, following the instructions of the AxyPrep Multisource Genomic DNA Miniprep Kit 50-prep (Qiagen). Polymerase chain reactions (PCR) were performed in an automatic thermocycler (C 1000TM). Five markers, nrITS, nrLSU, RPB1, RPB2 and mtSSU, were chosen for our phylogenetic studies using the primers of ITS1f (Gardes and Bruns 1993 (link)) and ITS4a (Larena et al. 1999 (link)), LR0R (Rehner and Samuels 1994 (link)) and LR5 (Vilgalys and Hester 1990 (link)), gRPB1a (Stiller and Hall 1997 (link)) and fRPB1c (Matheny et al. 2002 (link)), RPB2-6f and RPB2-7cr (Liu et al. 1999 (link)), mrSSU1 mrSSU3R (Zoller et al. 1999 (link)), respectively. Amplifications were performed with a total volume of 25 μl, containing 12.5 μl 2× MasterMix [TaqDNA Polymerase (0.1 units/μl), 0.4 mM MgCl₂, 0.4 nM dNTPs] (Aidlab Biotechnologies Co. Ltd.), 0.5 μl of each primer, 10 μl ddH₂O and 1 μl of DNA. The PCR settings per locus are provided in Table 1. PCR products were sequenced by TsingKe Biological Technology using the same primers which had been used for amplification (Kunming, China).

Total genomic DNA was extracted from dried herbarium specimens using AxyPrep Multisource Genomic DNA Miniprep Kit 50-prep (Qiagen) according to the manufacturer’s instructions. ITS (nrDNA ITS1-5.8S-ITS2) and mtSSU (mitochondrial small subunit rDNA) were amplified by polymerase chain reactions (PCR) using the primer pairs ITS1F (Gardes and Bruns 1993 (link)), ITS4 (White et al. 1990 ) and mtSSU1/mtSSU2R (Zoller et al. 1999 (link)).
Amplifications were performed in a 25 μl volume comprising 12.5 μl of 2× MasterMix (TapDNA Polymerase, 0.1 units/μl; technologies Co. Ltd), 1.0 μl of each primer, 8.5 μl ddH₂O and 2 μl DNA. Conditions for the PCR were: initial denaturation at 94 °C for 4 min, 34 cycles at 94 °C for 1 min, 54 °C for 1 min and 72 °C for 1.5 min, with a final extension at 72 °C for 10 min. PCR products were sequenced in an ABI3730X using amplification primers manufactured by Tsingke (Kunming, China).
ITS and mtSSU sequences were assembled with Seqman 7.0 (DNAStar) and manually edited using Mega6. DNA sequences were aligned with MAFFT version 7 with default parameters (Katoh et al. 2005 (link)) via the online resource (http://mafft.cbrc.jp/alignment/server/index.html).