Thermo Scientific Gene JET PCR Purification Kit was used to purify the positive PCR products for Blastocystis, and sequencing was done using the primer pair RD5 and BhRDr, and Big-Dye® Terminator v3.1. Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was also used following the manufacturer’s instructions for the ABI Prism 310 genetic analysis. Sequences from Blastocystis isolates were corresponded with reference sequences in the GenBank database using online BLAST software at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST ). All sequences were aligned using the BioEdit software’s ClustalW tool50 (link). The phylogenetic tree for the sequences was made using the neighbor-joining approach51 (link) with the Molecular and Evolution Genetic Analysis X (MEGA10) program52 (link). Bootstrapping was used to assess the trustworthiness of the phylogenetic tree (1000 replicates). The Maximum-Likelihood approach with the Tamura-3 parameter substitution model was used with MEGA10 to calculate the evolutionary distances.
Ready reaction cycle sequencing kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ready Reaction Cycle Sequencing Kit is a laboratory equipment product that enables DNA sequencing. It provides the necessary reagents and components to perform cycle sequencing reactions, a fundamental technique in molecular biology and genetics.
Automatically generated - may contain errors
2 protocols using ready reaction cycle sequencing kit
1
Molecular Characterization of Blastocystis spp.
2
Molecular Phylogenetic Analysis of Blastocystis
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Genome DNA purification kit was used for purification of only 20 PCR products and the primer pair (RD5 and BhRDr) with Big-Dye® Terminator v3.1 was used for sequencing. Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) also was used following the manufacturer’s instructions of the ABI Prism 310 genetic analyzer.
Blastocystis isolates sequences were coincided with reference sequences in the GenBank database. The online BLAST program available at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST ) was employed. The ClustalW program of the BioEdit software was used to align all sequences [23 (link)]. The method of neighbor joining [24 (link)] utilizing the Molecular and Evolution Genetic Analysis v7 (MEGA7) software [25 ] was utilized to create the phylogenetic tree for the sequences.
Evaluation of the phylogenetic tree reliability was done using bootstrapping (1000 replicates). Computing the evolutionary distances was made by the Maximum–Likelihood algorithm with Tamura-3 parameter substitution model using MEGA7.
Blastocystis isolates sequences were coincided with reference sequences in the GenBank database. The online BLAST program available at the National Center for Biotechnology Information (NCBI) (
Evaluation of the phylogenetic tree reliability was done using bootstrapping (1000 replicates). Computing the evolutionary distances was made by the Maximum–Likelihood algorithm with Tamura-3 parameter substitution model using MEGA7.
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