Genomic DNA (2.5 ng/μl, 10 μl reactions), was amplified (35 cycles) in a GeneAmp 9700 thermal cycler using Top Taq mastermix kit (Qiagen) and 20 pmol of gene-specific primers (S4 Table ) [11 (link)]. Resulting PCR amplicons were enzyme-purified with ExoSAP-IT (USB Corporation, Cleveland, OH). Purified amplicons were direct cycle-sequenced in both directions with BigDye Terminator Ready Reaction Mix (v3.1) (Applied Biosystems/Life Technologies, Grand Island, NY) containing M13 forward or reverse sequencing primers, then ethanol precipitated and detected by capillary electrophoresis on a 3130xl Genetic Analyzer running Sequence Analysis (v.6.0) software (Applied Biosystems), and Chromas (v2.23) software (Technelysium, Tewantin, Queensland, Australia).
Chromas v2.23 software
Manufactured by Technelysium
Sourced in Australia
Chromas (v2.23) is a DNA sequence analysis software developed by Technelysium. It provides a user-interface for viewing and analyzing DNA sequence data.
Automatically generated - may contain errors
Lab products found in correlation
3130xl genetic analyzer, by Thermo Fisher Scientific (4 mentions)
Exosap it, by Thermo Fisher Scientific (4 mentions)
Geneamp 9700, by Qiagen (3 mentions)
Sequence analysis v 6.0 software, by Thermo Fisher Scientific (3 mentions)
Toptaq master mix kit, by Qiagen (3 mentions)
Bigdye terminator v3.1 ready reaction mix, by Thermo Fisher Scientific (2 mentions)
Big dye terminator ready reaction mix, by Thermo Fisher Scientific (1 mentions)
Amplitaq polymerase, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
4 protocols using chromas v2.23 software
1
PCR Amplification and DNA Sequencing Protocol
2
Genomic DNA PCR Amplification and Sequencing
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Genomic DNA (2.5 ng/μl, 10 μl reactions) was amplified (35 cycles) in a GeneAmp 9700 thermal cycler using Top Taq mastermix kit (Qiagen) and 20 pmol of gene-specific primers (Additional file 6 ). Resulting PCR amplicons were enzyme-purified with ExoSAP-IT (USB Corporation, Cleveland, OH). The purified amplicons were direct cycle-sequenced in both directions with BigDye Terminator Ready Reaction Mix (v3.1)(Applied Biosystems, Grand Island, NY) containing M13 forward or reverse sequencing primers, then ethanol precipitated and detected by capillary electrophoresis on a 3130xl Genetic Analyzer running Sequence Analysis (v.6.0) software (Applied Biosystems) and Chromas (v2.23) software (Technelysium, Tewantin, Queensland, Australia).
3
Sequencing of Human TRPM3 Gene
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Genomic sequence for TRPM3 (Gene ID: 80036) was obtained from the Ensembl human genome browser (http://www.ensembl.org/index.html ), and gene-specific M13-tailed PCR primers (Table S4 ) were selected from the NCBI re-sequencing amplicon (RSA) probe database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe ) or custom designed with Primer Quest (IDT.com) or Exon Primer (UCSC Genome Bioinformatics - http://genome.ucsc.edu ). Genomic DNA (2.5 ng/ul, 20 ul reactions), was amplified (35–40 cycles) in a GeneAmp 9700 thermal cycler using AmpliTaq polymerase (Applied Biosystems, Foster City, CA) and gene-specific primers (10 pmol). Resulting PCR amplicons were either enzyme-purified with ExoSAP-IT (USB Corporation, Cleveland, OH) or gel-purified with the QIAquick gel-extraction kit (Qiagen). Purified amplicons were direct cycle-sequenced in both directions with BigDye Terminator Ready Reaction Mix (v3.1) containing M13 forward or reverse sequencing primers then ethanol precipitated and detected by capillary electrophoresis on a 3130xl Genetic Analyzer running Sequence Analysis (v5.2) software (Applied Biosystems), and Chromas (v2.23) software (Technelysium, Tewantin, Queensland, Australia).
4
Sanger Sequencing of ARMC5 Mutations
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Genomic DNA from whole blood samples and removed adrenal gland tissues of some patients under surgery were extracted, and PCR primers surrounding each focal variant were designed by Primer3. PCR products were then sequenced on ABI 3730xl for genotype validation. For haploid phasing of ARMC5 mutations, long PCR primers encompassing candidate variants were designed, and PCR products were ligated into pGEM-T Easy vector for E. coli transformation. Five colonies were then picked up for Sanger sequencing from T7/SP6 primers. For Sanger sequencing, Genomic DNA (2.5 ng/ μL, 10 μL reactions) was amplified (35 cycles) in a GeneAmp 9700 thermal cycler using Top Taqmastermix kit (Qiagen) and 20 pmol of gene-specific primers. Resulting PCR amplicons were enzyme-purified with ExoSAP-IT (USB Corporation, Cleveland, OH). The purified amplicons were direct cycle sequenced in both directions with BigDye Terminator Ready Reaction Mix (v3.1) (Applied Biosystems, Grand Island, NY) containing M13 forward or reverse sequencing primers, then ethanol precipitated and detected by capillary electrophoresis on a 3130XL Genetic Analyzer running Sequence Analysis (v.6.0) software (Applied Biosystems) and Chromas (v2.23) software (Technelysium, Tewantin, Queensland, Australia).
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