Pcr purification kit

For DNA extraction, preserved spores in ethanol were centrifuged and washed two times with distilled water to remove the ethanol remnants. Genomic DNA was isolated from tissue homogenates using the Qiagen DNeasy Blood &Tissue Kit (Qiagen, Germany), following the manufacturer's recommended protocol for animal tissue. The gDNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Shanghai, China) at 260 nm.
The partial sequence of the SSU rDNA gene was amplified using a primer pair, MyxospecF (Fiala 2006 ) and 18R (Whipps et al. 2003 ) in a 25 µl reaction mixture, which comprised 30 ng template DNA, 1×PCR mixture (CWbiotech, Beijing, China), 10 pmol of each primer. Amplification conditions were initial denaturation at 94 °C for 4 min, followed by 35 cycles of 94 °C for 1 min, 46 °C for 50 s, 65 °C for 90 s, with a terminal extension at 65 °C for 10 min. The PCR products were electrophoresed in 1.2% agarose gels in Tris-borate-EDTA buffer gel stained with 1% ethidium bromide and then purified with a PCR purification kit (CWbiotech, China). The purified product was cloned into PMD-18T vector system (Takara, Dalian, China) and then sequenced with the ABI BigDye Terminator v 3.1 Cycle Sequencing Kit with an ABI 3100 Genetic Analyser.