RT-PCR analysis for CABYV isolates was carried out as described above and the resulting RT-PCR fragment (the full-length CP gene) for CABYV was cloned into pCR4TOPO vector (Invitrogen, USA). The candidate cDNA clones were selected using digestions of restriction endonucleases, according to standard protocols. The entire cDNA insert was sequenced using a BigDye termination cycle sequencing kit (Applied Biosystems, USA) with M13 forward/reverse primers. The comparisons of determined nucleotide sequence and deduced amino acid sequence were analyzed using the Jukes-Cantor index and the neighbor-joining method with 1000 bootstrap replications in the MEGA 6.0 and DNASTAR package software (Choi et al., 2011; Tamura et al., 2013 (link)). Phylogenetic tree of CABYV CP sequences was constructed using DNASTART package software.
Bigdye termination cycle sequencing kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BigDye Termination Cycle Sequencing Kit is a laboratory product used for DNA sequencing. It contains reagents necessary for the Sanger sequencing method, which is a widely used technique for determining the precise order of nucleotides in a DNA molecule.
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Lab products found in correlation
2 protocols using bigdye termination cycle sequencing kit
1
Cloning and Sequencing of CABYV CP Gene
2
Sequence Analysis of Chrysanthemum and Tomato Viroids
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Leaves (0.1 g) of CSVd-infected chrysanthemum and tomato plants were ground in buffer B (0.1 M Tris-HCl, pH 9.0, 5 M urea, 0.1 M NaCl, 0.1 M 2-mercaptoethanol, and 10 mM EDTA). The homogenate was clarified by centrifugation, and the supernatant was extracted with 0.5 vol of phenol/chloroform (1:1). Total RNA was recovered by ethanol precipitation and resuspended in sterile water. RT-PCR analysis for CSVd progeny was carried out as described above and the resulting RT-PCR fragment (full-length) was cloned into pCR4TOPO vector (Invitrogen, USA). The entire cDNA insert was sequenced using a BigDye termination cycle sequencing kit (Applied Biosystems, USA) with M13 forward/reverse primers. The determined nucleotide sequences comparisons were calculated using the Jukes and Cantor index in the MEGA 5.0 Software (Choi et al., 2011 (link)).
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