The DNA bands were excised from the gel and purified (Wizard SV Gel and PCR clean up System Promega, USA) using ABI PRISM BigDye Terminator version 3.1 (Applied Biosystem Foster City, USA CA.) purified product and same pair of primers (0.1 mM). The Sequence reactions were run on 3100 DNA analyzer (Applied BioSystem). Each DNA fragment was sequenced at least twice. Sequence assembly, editing, and homology analyses of open reading frame (ORF) of the B5R gene were carried out in the program DNASTAR Lasergene 8 (version 8.0.2 13; Madison, WI). All sequences were cut to equal length and aligned in BioEdit version 7.0.8 [28] using ClustalW method. A phylogenetic tree was constructed using Bayesian inference with the program MrBayes version 3.1.2 [29] .
Abi prism bigdye terminator
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism BigDye Terminator is a reagent kit used in DNA sequencing applications. It contains the necessary components for performing Sanger sequencing, including fluorescently-labeled dideoxynucleotides (BigDye Terminators) that enable the detection and analysis of DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 310, by Thermo Fisher Scientific (2 mentions)
Wizard sv gel and pcr clean up system, by Promega (1 mentions)
Abi prism 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Sephadex g50 resin, by Merck Group (1 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (1 mentions)
Abi 3730xl, by Thermo Fisher Scientific (1 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Amplitaq gold 360 master mix, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Abi prism 377, by Thermo Fisher Scientific (1 mentions)
Abi 377 sequencer, by Thermo Fisher Scientific (1 mentions)
3500xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Abi prism 3100 dna analyzer, by Thermo Fisher Scientific (1 mentions)
Sequencer 3, by Thermo Fisher Scientific (1 mentions)
Drhodamine terminator cycle sequencing ready reaction, by Thermo Fisher Scientific (1 mentions)
Abi prism 3130, by Thermo Fisher Scientific (1 mentions)
3730 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Sequencher v5, by Gene Codes (1 mentions)
Abi sequencing analysis software v 5, by Gene Codes (1 mentions)
22 protocols using abi prism bigdye terminator
1
DNA Sequence Analysis Workflow
2
PCR Product Sequencing and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PCR products were purified with the PCR Cleanup KIT (Abbott, Wiesbaden, Germany) and then sequenced on both strands using the BigDye® Terminator Cycle Sequencing Kit v.3.1 (Applied Biosystems). The reaction mixture for the sequencing reaction contained 4 μl ABI PRISM Big Dye Terminator (Applied Biosystem), 3.8 μl water, 4 μl BigDye® TerminatorBuffer 5X1 (Applied Biosystems), 3.2 μl primer (1 pmol) and 5 μl of purified cDNA (40 ng), for a total volume of 20 μl. The sequencing conditions were: one cycle at 96°C for 3 min and 25 cycles (96°C for 30 s, 50°C for 10 s, 60°C for 4 min). Sequencing primers were the same of PCR reactions (see Table 2 ). The sequence products were purified by gel filtration chromatography using Sephadex G-50 resin (Sigma-Aldrich, Missouri, United States), in order to eliminate excess primers and/or unincorporated dideoxynucleotides (dNTPs), and then separated on an automated sequencer (ABI PRISM-3130 Genetic Analyzer, Applied Biosystems).
3
Identification of Plasmid-Mediated Quinolone Resistance Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR amplicons of the qnr genes were sequenced using the specific primer as described above. Sequencing was carried out at Macrogen Inc. (Seoul, Korea) using an ABI PRISM® BigDye™ Terminator and ABI 3730XL sequencer (Applied Biosystem, USA). The qnr gene sequences of representative isolates were compared with known sequences in the NCBI Database by using BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/ ). The sequences were submitted to GenBank under accession numbers MK 414320, MK414318, MK414319, MK414320, MK414321, and MK414322.
4
TNFRSF14 and TP53 Mutation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was isolated from FFPE tissue samples by using the QIAamp FFPE Kit as described by the manufacturer (Qiagen, Hilden, Germany). The coding sequences covering the TNFRSF14 gene (exons 1–8) and the DNA-binding domain of TP53 (exons 4–8) were analyzed for the occurrence of mutations by direct sequencing of polymerase chain reaction (PCR) products amplified from genomic DNA using standard PCR conditions and cycling sequencing with the ABI PRISM BigDye Terminator chemistry (ThermoFisher Scientific, Schwerte, Germany), as previously published.14 (link),26 (link)
5
Influenza Genome Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted using QIAamp Viral RNA mini kit (Qiagen, Inc., Valencia, CA, USA) according to manufacturer’s instruction. Reverse-transcription of vRNA was carried out using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen Corporation, CA, USA) and Uni 12 primer (5’AGCRAAAGCAGG3’) [15 (link)]. Full-length genes were obtained by performing PCR using primers described previously [15 (link)–17 (link)], and with primers obtained from the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne (Dr. TBoon-Huan Tan, personal communication). Sanger sequencing was performed with ABI Prism Big DyeTerminator (ThermoFisher Scientific Inc.). The assembly and editing of raw sequence data were done using SeqMan (DNASTAR, Lasergene Version 7, Madison, USA). The sequences were deposited in GenBank and can be retrieved from the National Center for Biotechnology Information’s (NCBI) Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html ).
6
Molecular Diagnostic Validation via Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm the results of the two molecular diagnostic methods, the PCR amplicons of all the clinical samples were sequenced using an ABI 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) and the ABI Prism BigDye Terminator (Applied Biosystems) system (CosmoGenetech, Republic of Korea). The primer set used to amplify the target ORF2 gene was 5′-TCTGAATTGTACATACATRGTTAYACGG-3′ (1070F) and 5′- TACCGYTGGAGAAGGAAAAATGG-3′ (1630R), which resulted in a 560-bp PCR product. The obtained sequences were compared with sequences in the National Center for Biotechnology Information (NCBI) GenBank database for species identification.
7
PCR and Sanger Sequencing of Genomic Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR reactions for the target were performed in a final volume of 20 μL, containing 10 μL of AmpliTaq Gold 360 Master Mix (Applied Biosystems), 500 nM of each primer (forward primer: 5’-GTTTGGGGGTGTGTGGTCT-3’ and reverse primer: 5’-CCTAGCCCCTTGTGGACATA-3’ ), and 20 ng of isolated genomic DNA from the frozen tissue specimen. Thermal cycling conditions included preincubation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, 72°C for 20 s, and an additional incubation at 72°C for 7 min. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and processed for cycle sequencing with ABI PRISM BigDye Terminator (version 3.1, Applied Biosystems, CA, USA) and the same primers used in the first PCR. Sequence data were generated using the 3130xl Genetic Analyzer. All tumor samples were confirmed by direct sequencing of the PCR product for both DNA strands. We used “4Peaks” to view and edit the sequence trace files (http://nucleobytes.com/4peaks/ ).
8
Plasmid DNA Sequencing with ABI BigDye
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA sequencing reactions were performed on a plasmid template using the ABI Prism BigDye terminator cycle sequencing ready reaction kits (Applied Biosystems, California, USA) and an ABI PRISM 377 DNA sequencer (Applied Biosystems).
9
Sequencing and Analysis of G12 RV Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First round amplicons of three G12 RV strains were chosen for sequencing using ABI Prism Big Dye Terminator cycle sequencing ready reaction kit (Applied Bio-systems, USA). The sequences were resolved using an automated DNA sequencer (ABI Prism 310 genetic analyser, Applied Bio systems, USA). Access to sequencher software (Gene Codes Corporation, version 5.4.6, accessible at http://www.genecodes.com ) was provided by CDC, USA and was used to read and analyse the sequences.
10
Identifying Mutations in fliF Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
It has been determined that SJW3060 carried mutation only in fliF gene (Yamaguchi, unpublished data). The original mutation of SJW3060 was determined by DNA sequencing analysis. The DNA fragment containing fliF was amplified by polymerase chain reaction (PCR) using two primers surrounding the fliF gene and the genome DNA prepared from SJW3060 as a template. DNA sequencing samples were prepared using the purified PCR product as a template, the appropriate sequence primers, and ABIPRISM big dye terminator (Applied Biosystems).
The second mutation sites of the revertants were determined by exhaustive survey of the DNA sequence of the regions indicated by the mapping results. The entire length of Region I, II, or III was amplified by PCR using HindIII site-conjugated primers and the genome DNA prepared from each revertant, and then the PCR products were cloned into pBR322. The inserted DNAs were digested by combinations of appropriate restriction enzymes, and the resultant fragments were cloned into pHSG395. DNA sequencing samples were prepared using the plasmids as a template, M13 and the appropriate sequence primers, and ABIPRISM big dye terminator. DNA sequencing was performed using an ABI377 sequencer (Applied Biosystems).
The second mutation sites of the revertants were determined by exhaustive survey of the DNA sequence of the regions indicated by the mapping results. The entire length of Region I, II, or III was amplified by PCR using HindIII site-conjugated primers and the genome DNA prepared from each revertant, and then the PCR products were cloned into pBR322. The inserted DNAs were digested by combinations of appropriate restriction enzymes, and the resultant fragments were cloned into pHSG395. DNA sequencing samples were prepared using the plasmids as a template, M13 and the appropriate sequence primers, and ABIPRISM big dye terminator. DNA sequencing was performed using an ABI377 sequencer (Applied Biosystems).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!