PCR products were purified using ExoSap-IT (Affymetrix/USB, Santa Clara, CA, USA). Thereafter, cycle sequencing was carried out using BigDye® Terminator v1.1 (for IDH1/2) or v3.1 (for BRAF) Cycle Sequencing Kits (Applied Biosystems). Following purification, the electrophoresis and analyses were conducted using a PRISM® 3100 Genetic Analyzer (Applied Biosystems). Raw data were analyzed using the phred/phrap/consed package (http://www.phrap.org/consed/consed.html#howToGet ) for sequence determination.
Cycle sequencing kits
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cycle Sequencing Kits are a set of reagents and consumables used for DNA sequencing. The kits contain all the necessary components, including primers, polymerase, and labeled nucleotides, to perform automated DNA sequencing using the chain-termination method.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator v1, by Thermo Fisher Scientific (1 mentions)
Prism 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye xterminator purification kit, by Thermo Fisher Scientific (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Abi prism bigdye terminator v 3, by Thermo Fisher Scientific (1 mentions)
Abi prism 310 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Bigdye terminator version 3, by Thermo Fisher Scientific (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Abi 3730xl sequencer, by Thermo Fisher Scientific (1 mentions)
Ex taq, by Takara Bio (1 mentions)
Jm109 competent cells, by Takara Bio (1 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using cycle sequencing kits
1
Sequencing of IDH1/2 and BRAF
2
Multilocus PCR for Toxoplasma Clonal Types
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To investigate the clonal type of B1 gene-positive samples, multilocus PCR was carried out using nine markers: SAG1, 3′SAG2, 5′SAG2, SAG3, BTUB, GRA6, altSAG2, C29-2, and L358, based on the method described by Su et al. (2010 (link)).
Nested and multilocus PCR were carried out in a C1000 Thermal Cycler (Bio-Rad). Real-time PCR amplification was performed in a thermal cycler CFX-96 (Bio-Rad). As positive controls, RH (type I), ME49 (type II), and C56 (type III) DNA isolates of T. gondii strains, and as a negative control nuclease-free water, were used.
DNA sequencing of amplicons was performed using ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA), with the use of Abi Prism Big Dye Terminator v. 3.1. Cycle Sequencing Kits and Big Dye XTerminator Purification Kit (Applied Biosystems). Sequences were analyzed using Geneious v. 11.1.4. software (Geneious Co., Wellington, New Zealand) and compared with the sequences deposited in NCBI database using Blast.
Nested and multilocus PCR were carried out in a C1000 Thermal Cycler (Bio-Rad). Real-time PCR amplification was performed in a thermal cycler CFX-96 (Bio-Rad). As positive controls, RH (type I), ME49 (type II), and C56 (type III) DNA isolates of T. gondii strains, and as a negative control nuclease-free water, were used.
DNA sequencing of amplicons was performed using ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA), with the use of Abi Prism Big Dye Terminator v. 3.1. Cycle Sequencing Kits and Big Dye XTerminator Purification Kit (Applied Biosystems). Sequences were analyzed using Geneious v. 11.1.4. software (Geneious Co., Wellington, New Zealand) and compared with the sequences deposited in NCBI database using Blast.
3
TA Cloning and Sequencing of Genes
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TA cloning was performed to isolate several genes from A. papillare and A. donei, whose sequences were not indetified. First, target sequences were amplified with ExTaq (Takara, Ohtsu, Japan) using several primers (Supplementary Table S1 ). The PCR products were ligated into the pGEM-T Easy Vector (Promega, Madison, WI, United States), which was then transformed into JM109 competent cells (Takara, Ohtsu, Japan). Plasmids were extracted, and cycle sequencing reactions were conducted using ABI BigDye Terminator version 3.1 and Cycle Sequencing Kits with T7 or SP6 primers, followed by capillary electrophoresis in an ABI 3730xl sequencer (Applied Biosystems, Foster City, CA, United States).
4
Trypanosome Identification via Molecular Sequencing
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The PCR products were puri ed and 20µl of the PCR products was sequenced using Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). As described by Applied Biosystems, Product of PCR were sequenced using Applied Biosystems Cycle Sequencing Kits (BigDye Terminator v1.1 and v3.1 kits). The sequence data obtained were viewed on Finch Trace Viewer v.1.4.0 (Applied Biosystems, Foster City, CA, USA), while anking regions of high Noise-to-Signal ratio were trimmed off the sequence to improve the accuracy and precision of sequence data obtained. Ambiguous nucleotides were edited and replaced with conventional ones based on the highest peak recorded on the electropherogram. Each edited sequence were BLAST searched against the DNA sequence database (NCBI) and/or the published databases for various trypanosome species (Tri-Tryp) explicitly for trypanosomes. Sequences of the ITS-1 region were aligned using ClutalW against known sequences in order to con rm species identity. Molecular Evolution Genetic Analysis Version 7.0.2.6 (MEGA7) was used to construct phylogenetic tree to observe their evolutionary trend and variation over time.
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