Abi prism bigdye terminator v3.1 kit

The mutants were identified using molecular typing methods. Yeast genomic DNA was isolated using Qiagen DNeasy Kit following manufacturer’s instructions. Fungal LSU (large ribosomal subunit) and ITS (internal transcribed spacer) regions were amplified using standard PCR reaction [20 (link)]. The primers for the ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 regions (5′-TCCTCCGCTTATTGATATGC-3′) were used in the PCR amplification. For the LSU region, the primer LR7 (5′-TACTACCACCAAGATCT-3′) and 5.8 SR (5′-TCGATGAAGAACGCAGC-3′) were used to amplify the fragment. After amplification, the products were purified by using a geneO-spin PCR product Purification kit (GeneOmbio Technologies, Pune, India) and were directly sequenced using an ABI PRISM BigDye Terminator V3.1 kit (Applied Biosystems, USA). DNA sequencing was performed using primers used for PCR of LSU and ITS regions. The sequences were analyzed using Sequencing Analysis 5.2 software. BLAST analysis was performed at BlastN site at NCBI server (http://www.ncbi.nlm.nih.gov/BLAST).

Bacterial genomic DNA was isolated using geneO-spin Microbial DNA Isolation Kit (geneOmbio Technologies, Pune, India). This DNA was used as template for PCR analysis using the primers 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R: 5′-TACCTTGTTACGACTT-3′. The amplification conditions were 95°C for 10 min, 57°C for 1 min, 72°C for 90 sec, and final amplification at 72°C for 10 min. The PCR products were purified by using a geneO-spin PCR Product Purification Kit (geneOmbio Technologies, Pune, India) and were directly sequenced using an ABI PRISM BigDye Terminator V3.1 Kit (Applied Biosystems, USA). The sequences were analyzed using Sequencing Analysis 5.2 software. BLAST analysis was performed at BlastN site at NCBI server (http://www.ncbi.nlm.nih.gov/BLAST) and evolutionary relationship of 4R was deduced by constructing phylogenetic tree.