I starmax 2 pcr master mix system

Fungal genomic DNA was extracted from mycelia cultured in 5 mL PDB at 25 °C for 7 days. Mycelia from wild-type and transformants were harvested, lyophilized, and ground using Mini-Beadbeater-24 (Biospec Products, Bartlesville, OK, USA) after freezing in liquid nitrogen. Genomic DNA was purified using NucleoSpin Plant kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instruction.
PCR reactions were carried out using genomic DNAs of 14 randomly selected transformants in an ABI 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). PCR amplification of the hph and eGFP genes was carried out using 20 ng of genomic DNA and 10 pmol of each primer (Table 1) using i-StarMAX II PCR master mix system (iNtRON Biotechnology Inc., Seongnam, Korea). The amplification conditions were as follows: (a) initial denaturation at 94 °C for 3 min; (b) 30 cycles of 30 s denaturation at 95 °C, 30 s annealing at 56 °C, and 1 min elongation at 72 °C, and (c) 10 min elongation at 72 °C. PCR products were resolved via electrophoresis using a 1% agarose gel, stained with Ecodye™ DNA staining solution (Solgent Co. Daejeon, Korea), and visualized under UV light.

We tested the efficacy of various rRNA regions, which were amplified from the extracted DNAs and primer pairs listed in Supplementary Table 3 using the i-StarMAXII PCR master mix system (iNtRON Biotechnology, Seongnam, Korea), or the AccuPower PCR PreMixs (Bioneer, Seoul, Korea). A Takara PCR thermal cycler MP (Takara, Tokyo, Japan), or a 96-well GenAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA) was used in a regular 30 cycle PCR reaction. Amplified PCR products were purified using MEGAquick-spin Total Fragment DNA Purification Kit (iNtRON Biotechnology) prior to sequencing, and forward and reverse strands were sequenced using the same PCR amplification primers. The generated sequences of fungi and algae from lichen were submitted to GenBank (http://www.ncbi.nlm.nih.gov/).