Abi prism bigdye terminator cycle sequencing ready reaction

Samples of CSF in which virus was detected by D-RT-PCR were submitted to sequencing analysis according to the method of Sanger. The sequencing reaction was performed using the kit ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Life Technologies, Carlsbad, California, United States) according to the manufacturer's instructions. Sequencing was completed using the Genetic Analyzer ABI 3100 (Applied Biosystems, Life Technologies, Carlsbad, California, U.S).
The sequences were analyzed using the program Sequencher 3.0 Sequencing Software (Ann Arbor, Michigan, United States) and compared with the genomic database GenBank (National Center for Biotechnology Information—NCBI- Bethesda, Maryland, U.S).

Those gene fragments present only in the mRNA profile of roots infected with P. cinnamomi and those that showed an increase or decrease in their expression were eluted from the gel. Rectangular pieces of Whatman paper were placed into tubes with the piece of excised gel in 40 μl water and kept on ice for 10 min. After hydration, the tubes were placed at 95°C for 15 min and transferred to ice. The mixture was centrifuged for a few seconds, the supernatant was collected and 5 μl of the solution were used as target DNA, in reamplification reactions (Habu and Iida 1998 (link)). These were performed with the selective primers used to generate the corresponding cDNA-AFLP profile. The re-amplified PCR products were run on a 2% agarose gel, excised and purified with the Qiaquick PCR Purification Kit (Qiagen) and cloned into the pCRII Topo vector, with the TA Cloning Kit (Invitrogen). Manufacturer’s instructions for these kits were followed throughout. The fragments were sequenced using the kit ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction (Perkin Elmer/Applied Biosystems) in an automatic sequencer ABI PRISM 377 (Perkin-Elmer) at Macrogen Company (Seoul, Korea).

A BlastN search was performed (www.ncbi.nlm.nih.gov/BLAST/) on the complete, non-redundant GenBank nucleotide database for ortholog of phd2 in other fish species. A multiple sequence nucleotide alignment was carried out on coding sequences to design the primers. The specific sequences were obtained through the Sanger protocol using ABI 3130 (Applied Biosystems, CA, USA) following the ABI PRISM^® Big Dye™ Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, CA, USA) protocol. The obtained sequences were analyzed through ABI 3130 Sequence Analyzer software (Applied Biosystems, CA, USA) for their electropherograms quality parameters. The contigs were generated using the BlastN validated tool for the detection of nucleotide homology on NCBI (www.ncbi.nlm.nih). Primers were designed using Oligo Explorer 1.5 software. The annealing temperature was optimized by gradient PCR. phd2 primers sequence used in Quantitative real-time PCR (qPCR) assays was: Forward primer: 5´-AAGTTGTCGGTTAGTAGGGC-3´ and Reverse primer: 5´-TCGNTCTGCGGCTTCTCCA-3´.