Abi prism 3.1 bigdye terminator kit

The entire internal transcribed spacer (ITS) region was amplified with the fungalspecific primers ITS1F and ITS4 (Gardes and Bruns, 1993; (link)Chang et al., 2013; (link)Pei et al., 2014) (link). The PCR mixture contained 2 mL undiluted DNA template, 2 mL each primer (10 mM concentrations) and 25 mL Master Mixture (containing Mg and 1U Expand TM High Fidelity Polymerase from Sangon Biotech, China). The reaction mixture was topped-up with sterile distilled H 2 O to a total volume of 50 mL. The PCR was run using a DNA-Engine thermocycler (MJ Research, USA) and the regimen was as follows: a pre-denaturation step at 94°C for 2 min; 35 cycles of denaturation for 40 s at 94°C, annealing for 40 s at 56°C, and extension for 45 s at 72°C; and a final extension at 72°C for 10 min.
Successfully amplified products were purified with a PCR purification kit (TIANGEN, Bio-Chem Technology Group Company Ltd.) and re-amplified following the PCR protocol described above. The direct cycle sequencing was carried out with an ABI PRISM 3.1 BigDye terminator kit (Applied Biosystems, Foster City, CA, USA), using the same primers as in the initial PCR. Electrophoresis was carried out on an ABI PRISM 3100 genetic analyzer. The obtained sequences were arranged using Sequencher ® version 5.0 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI, USA) and deposited in the GenBank database.