For promoter analysis, 1 kb of the TbREF upstream sequence was amplified using primers P-TbREF-DP HindIII fw and P-TbREF-DP XhoI rev (Table S4). The GFPER gene was isolated from pGJ1029 (kindly provided by Guido Jach, Phytowelt GreenTechnologies GmbH, Cologne, Germany) using the XhoI and XbaI restriction sites, and transferred to binary plasmid pLab12.10 (Post et al., 2014) . The TbREF PCR products were digested using HindIII and XhoI, and transferred into the corresponding restriction sites of pLab12.10. The integrity of each construct was verified by Sanger sequencing using an ABI PRISM â 3730 genetic analyzer (Life Technologies).
Abi prism 3730 genetic analyzer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 3730 Genetic Analyzer is a high-throughput DNA sequencing instrument designed for automated, high-quality DNA sequencing. It utilizes capillary electrophoresis technology to separate and detect fluorescently labeled DNA fragments, enabling efficient and accurate DNA sequencing.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (7 mentions)
Abi prism bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (5 mentions)
Bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (5 mentions)
Ex taq polymerase, by Takara Bio (3 mentions)
Lasergene v7, by DNASTAR (3 mentions)
Fungus genomic dna extraction kit, by Sangon (3 mentions)
Universal pcr master mix, by Thermo Fisher Scientific (3 mentions)
Expin pcr purification kit, by GeneAll (2 mentions)
C1000 thermal cycler, by Bio-Rad (2 mentions)
Seqscape v2, by Thermo Fisher Scientific (2 mentions)
Hot firepol dna polymerase, by Solis BioDyne (2 mentions)
Big dye terminator cycle v1.1 sequencing kit, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator v3.1 kit, by Thermo Fisher Scientific (2 mentions)
Favorprep gel pcr purification kit, by Favorgen Biotech (2 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Genemapper software version 4, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna blood mini kit, by Qiagen (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Tprofessional standard thermocycler, by Analytik Jena (2 mentions)
87 protocols using abi prism 3730 genetic analyzer
1
Cloning and Sequencing of TbREF Promoter
2
Generating TbREF cDNA Construct
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A 350 bp fragment of the TbREF cDNA sequence (nucleotides 761-1110) was amplified by PCR using primers REF RNAi 2 KpnI fw and REF RNAi 2 XhoI rev (Table S1). The cDNA fragment was inserted into the KpnI and XhoI sites of Gateway vector pENTR4 (Life Technologies, www. lifetechnologies.com), and transferred to pFGC5941 (Wahler et al., 2009) using Gateway â LR Clonase â II Enzyme mix (Life Technologies). The integrity of the construct was verified by Sanger sequencing using an ABI PRISM â 3730 genetic analyzer (Life Technologies).
3
Phylogenetic Analysis of Leishmania HSP70 Gene
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PCR products of the HAP70 gene related to 36 Leishmania isolates were purified and sequenced using an ABI PrismTM 3730 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) by the Macrogen Company (Seoul, South Korea). Sequencing was carried out by the same primer pair. Consensus sequences from forward and reverse reads were trimmed and edited using the BioEdit 5.0.9 [25 ]. Furthermore, HSP70 sequences of 37 reference strains available in GenBank database were taken from NCBI (http://www.ncbi.nlm.nih.gov ) and included in the analyses. All the original and retrieved sequences were aligned together, first applying the ClustalW algorithm implemented in the MEGA 6.0 [26 (link)], and then manually. Applying the corrected Akaike Information Criterion (AICc) [27 ] in jModelTest 2.1.10 [28 (link)], the best-fit models of nucleotide substitution was estimated. The maximum likelihood (ML) and neighbor joining (NJ) phylogenetic trees were constructed using the MEGA software. The HSP70 gene sequence of Trypanosoma rangeli (GenBank accession No. EF108422) was used as an outgroup. Using Arlequin 3.11 [29 (link)], basic parameters of genetic diversity were estimated. Using the same software, demographic histories were examined based on 2 neutrality tests, Tajima’s D [30 (link)] and Fu’s Fs [31 (link)].
4
Phylogenetic Analysis of Parasite Sequences
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PCR products were purified and sequenced using an ABI PrismTM 3730 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) by the Macrogen Company (Seoul, South Korea). The sequences of each samples were edited and aligned with reference sequences by Bio-Edit software. In the next step, the sequences of each samples were blasted for finding regions of similarity between sequences. The phylogenetic tree was created with MEGA (ver; 10) software using the Maximum Likelihood method. The percentage of trees in which the associated taxa clustered together is shown next to the branches. In addition, branch confidence in clades in each tree was assessed using a bootstrap analysis with 1000 replicates (Kumar et al., 2018 (link)). Sequences were deposited in GenBank under accession numbers MW464190– MW464194 for Cox1 and MW460010– MW460014 for Nad1 genes. To draw the phylogenetic network, G1 (n = 72) and G3 (n = 20) sequences from Kinkar et al. (2018) (link) and current isolates (n = 5) were analyzed. Phylogenetic networks were generated using Network v4.6.1.5 (Bandelt et al. 1999 (link); http://www.fluxusengineering.com , Fluxus Technology Ltd., 2004).
5
Sequencing and Phylogenetic Analysis of Isolates
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The PCR products were sequenced in one direction by the primer V9G using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on an automated DNA sequencer (ABI PrismTM3730 Genetic Analyzer, Applied Biosystems) according to the instructions of the manufacturer. The obtained sequence data were imported into MEGA software (version 6), ambiguous regions were edited manually to improve alignment accuracy, and final identification of isolates was performed by comparing the obtained sequences with the reference sequences of the National Center for Biotechnology Information database.
Sequences were subjected to BioEdit software (version 7.0.5) for pairwise comparisons and multiple alignments to determine intra- and inter-species similarities and differences in nucleotides. The Maximum Likelihood method was applied to the phylogenetic analysis using unambiguously aligned sequences with the Tamura-Nei parameter with substitution model as implemented in the MEGA software (version 7) [ 29
]. Bootstrap values equal to or greater than 70% were considered significant.
Sequences were subjected to BioEdit software (version 7.0.5) for pairwise comparisons and multiple alignments to determine intra- and inter-species similarities and differences in nucleotides. The Maximum Likelihood method was applied to the phylogenetic analysis using unambiguously aligned sequences with the Tamura-Nei parameter with substitution model as implemented in the MEGA software (version 7) [ 29
]. Bootstrap values equal to or greater than 70% were considered significant.
6
RT-PCR Amplification and Sequencing of RdRp Gene
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A 315-bp fragment from the RNA-dependent RNA polymerase (RdRp) gene was amplified using a semi-nested PCR assay (10 (link)). The resulting amplicons from the second amplification round of four specimens were sequenced in both directions using the same primers used for amplification in an ABI PrismTM 3730 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) by a commercial Company (Macrogen, Seoul, South Korea). The generated sequences were BLASTed against similar sequences available in the GenBank database, and the similarities were obtained.
7
DNA Sequencing and Homology Analysis
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PCR products were sequenced in both directions using an ABI PrismTM 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) by the Macrogen Company (Seoul, South Korea). In order to conduct homology analysis, DNA sequencing using the Basic Local Alignment Search Tool (BLAST) from the National Center for Biotechnology Information homepage (NCBI) was blasted with reference sequences available in the GenBank database. Nucleotide sequence data were deposited to the GenBank using BankIt under the accession number MZ458337-MZ458347.
8
Validating Monoallelic Gene Expression
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To validate the monoallelic expression of the tested genes identified in this study, polymerase chain reaction (PCR) and direct sequencing were performed using DNAs and synthesized cDNAs with specific primer sets (Table S1 ). PCR products were purified and Sanger sequencing was performed using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (ver. 3.0; Life Technologies Corp., Grand Island, NY, USA) and an ABI PRISM® 3730 Genetic Analyzer (Life Technologies Corp., Grand Island, NY, USA). The sequence analysis for SNP loci was performed using SeqMan software (DNASTAR).
9
GSTP1 and TS 3'UTR Genotyping
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GSTP1 (I105V) and TS 3'UTR (agttcat variant) genotyping was performed by Sanger sequencing. Purified pellets were dissolved in Hi-Di Formamide (Life Technologies) and analyzed using an ABI PRISM 3730 Genetic Analyzer (Life Technologies). Chromatograms were analyzed by SeqScape V2.5 (Life Technologies) and manual review.
10
Aspergillus Genomic DNA Extraction and Amplification
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Genomic DNA was extracted from isolated Aspergillus using a modified cetyltrimethylammonium bromide extraction protocol [33 ]. For the primer sets, Bt2a/Bt2b for BenA and CF1/CF4 or cmd5/cmd6 for CaM, were used [34–36 (link)]. PCR was performed in a C1000 thermal cycler (Bio-Rad, Richmond, CA, USA) with previously described methods [37 (link)]. The PCR products were purified using the ExpinTM PCR Purification Kit (GeneAll Biotechnology, Seoul, Korea), according to the guideline. DNA sequencing was performed using the PCR primers at Macrogen (Seoul, Korea), using an ABI Prism 3730 genetic analyzer (Life Technologies, Gaithersburg, MD, USA).
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