Genomic DNA was extracted from peripheral blood samples collected from participants using the QIAamp Blood DNA Mini Kit (QIAGEN, USA) according to the manufacturer’s protocol. After amplification using 2X polymerase chain reaction (PCR) MasterMix polymerase (Tiangen, China) by ABI9700 PCR (Life technology, USA), the products were captured and purified with Panel probe (Illumine Inc., USA), then directly sequenced on the ABI 3500 automated DNA sequencer (Life technology, USA). Mutations were detected using next generation sequencing (NGS) and subsequently confirmed using Sanger sequencing. We investigated 40 genes reported to be associated with Gitelman syndrome or Bartter syndrome, including CLCNKA, CLCNKB, BSND, LAMC, LAMB, ALMS, EDAR, CASR, DSP, BFSP2, TMEM67, PLEC, KIF7, SLC12A3, KRT9, KRT10, KRT14, PNPLA6 and so on. To assess the pathogenicity of the variants, those variants were analyzed with PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/ ), Mutation Taster (http://mutationtaster.org ), PROVEAN and SIFT (http://provean.jcvi.org/ ) as alignment reference databases. Gene mutation databases such as 1000 Genomes, dbSNP, and Uniprot were used for reference genome alignment.
Abi 3500 automated dna sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 3500 automated DNA sequencer is a laboratory instrument designed for DNA sequencing. It utilizes capillary electrophoresis technology to separate and detect DNA fragments labeled with fluorescent dyes. The ABI 3500 is capable of performing highly accurate and reliable DNA sequence analysis.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp dna blood mini kit, by Qiagen (4 mentions)
Abi9700 pcr, by Thermo Fisher Scientific (3 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
Primestar max dna polymerase system, by Takara Bio (3 mentions)
Mastermix polymerase, by Tiangen Biotech (2 mentions)
Pcr master mix, by Tiangen Biotech (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using abi 3500 automated dna sequencer
1
Genetic Variant Analysis in Kidney Disorders
2
Genetic Profiling of Neurofibromatosis Type 1
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Informed consent was obtained from all family members before peripheral blood samples were collected. Genomic DNA was extracted from 0.2 mL blood samples with a QIAamp DNA Blood Mini Kit (QIAGEN, Germantown, MD, USA) following the manufacturer protocol. We designed primers flanking all 60 coding exons of the NF1 gene using the web-based version of the Primer 5.0 program (http://www.premierbiosoft. com/). All exons were amplified from the genomic DNA of each participant by PCR using 2X PCR Master Mix (Tian Gen, BeiJing, China). The amplified products were sequenced on an ABI 3500 automated DNA sequencer (Life Technologies, Carlsbad, CA, USA). Nucleotide sequences were then compared pairwise with published NF1 sequences (RefSeq accession Nos. NM_000267.3 and NG_009018.1). Mutations were named according to the nomenclature recommended by the Human Genomic Variation Society (http://www.hgvs.org/). Experiments were performed at the molecular genetics facility of the Guangzhou Golden Field Medical Institute. The genetic variant was checked against the Human Gene Mutation Database (HGMD; http://www.hgmd.cf.ac.uk) and published literature available on to determine its novelty.
3
Genetic Profiling of Blood Samples
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Genomic DNA was extracted from peripheral blood sample using the QIAamp Blood DNA Mini Kit (QIAGEN, USA) according to the manufacturer's protocol.
After ampli cation using 2X polymerase chain reactions (PCR) MasterMix polymerase (Tiangen, China) by ABI9700 PCR (Life technology, USA), the products were captured and puri ed with Panel probe (illumine Inc. USA), then directly sequenced on the ABI 3500 automated DNA sequencer (Life technology, USA). Mutations of SLC12A3 were detected using next generation sequencing (NGS) and subsequently con rmed using Sanger sequencing.
After ampli cation using 2X polymerase chain reactions (PCR) MasterMix polymerase (Tiangen, China) by ABI9700 PCR (Life technology, USA), the products were captured and puri ed with Panel probe (illumine Inc. USA), then directly sequenced on the ABI 3500 automated DNA sequencer (Life technology, USA). Mutations of SLC12A3 were detected using next generation sequencing (NGS) and subsequently con rmed using Sanger sequencing.
4
Genomic DNA Extraction and ABCD1 Mutation Analysis
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Genomic DNA was extracted from peripheral blood sample using the QIAamp Blood DNA Mini Kit (QIAGEN, USA) according to the manufacturer's protocol. After ampli cation using 2X polymerase chain reactions (PCR) MasterMix polymerase (Tiangen, China) by ABI9700 PCR (Life technology, USA), the products were captured and puri ed with Panel probe (illumine Inc. USA), then directly sequenced on the ABI 3500 automated DNA sequencer (Life technology, USA). Mutations of ABCD1 were detected using next generation sequencing (NGS) and subsequently con rmed using Sanger sequencing.
5
Sanger Sequencing of SNV Loci
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Sanger sequencing analysis was conducted to confirm the location of specific SNVs in the detected genes. PCR was performed using the PrimeSTAR Max DNA polymerase system (Takara Bio, Kusatsu, Japan). Thereafter, PCR-amplified products were extracted using the QIAquick Gel Extraction Kit (QIAGEN). Sequencing in the reverse direction was undertaken according to the manufacturer’s instructions (BigDye; Applied Biosystems, Warrington, UK). Sequencing of the products was performed using the ABI 3500 automated DNA sequencer (Applied Biosystems).
6
Amplification and Sequencing of pncA Gene in Mycobacterium tuberculosis
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The deoxyribonucleic acid (DNA) was extracted using the Quick-DNA™ Miniprep Kit. The pncA gene was amplified using published primers; forward (5 = GGCGTCATGGACCCTATA 3 =) and reverse (5 = GTGAACAACCCGACCCAG 3 =) [21 (link)]. The amplified product yielded a full length pncA gene consisting of 561 base pairs (bp), plus 90bp downstream and 30bp upstream. Both positive and negative control were used during the PCR reaction. Sequencing reaction was performed in the ABI 3500 automated DNA sequencer (Applied Biosystems) using the BigDye Terminator v3.1 cycle sequencing kit and forward primers. The sequences were analyzed using the BioEdit sequence alignment editor with M. tuberculosis H37Rv as a wild type reference strain.
7
Sanger Sequencing of SNV Loci
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Sanger sequencing analysis was conducted to confirm the location of specific SNVs in the detected genes. PCR was performed using the PrimeSTAR Max DNA polymerase system (Takara Bio, Kusatsu, Japan). Thereafter, PCR-amplified products were extracted using the QIAquick Gel Extraction Kit (QIAGEN). Sequencing in the reverse direction was undertaken according to the manufacturer’s instructions (BigDye; Applied Biosystems, Warrington, UK). Sequencing of the products was performed using the ABI 3500 automated DNA sequencer (Applied Biosystems).
8
Sanger Sequencing of SNV Loci
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Sanger sequencing analysis was conducted to confirm the location of specific SNVs in the detected genes. PCR was performed using the PrimeSTAR Max DNA polymerase system (Takara Bio, Kusatsu, Japan). Thereafter, PCR-amplified products were extracted using the QIAquick Gel Extraction Kit (QIAGEN). Sequencing in the reverse direction was undertaken according to the manufacturer’s instructions (BigDye; Applied Biosystems, Warrington, UK). Sequencing of the products was performed using the ABI 3500 automated DNA sequencer (Applied Biosystems).
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