Genomic DNA from all subjects was extracted from whole blood samples using 0.5 M ethylene diamine tetraacetic acid (EDTA) as an anticoagulant, using the QiaAmp DNA Mini Kit (Qiagen, Hilden, Germany). The DNA samples were stored at −20°C. Genotyping of rs12134493 and rs2078371 in TSPAN2 was performed using the Multiplex SNaPshot technique (Applied Biosystems by Life Technologies, Foster City, CA, USA). PCR amplifications were performed in a final volume of 25 μl, containing 50 mM MgCl2, 10 mM dNTP, 1 μM primers, and 5 units of Platinum Taq DNA polymerase. The PCR conditions used were 95°C denaturation for 2 min; followed by 33 cycles at 95°C for 20 s, 55°C for 20 s, and 72°C for 40 s; and a final extension step at 72°C for 5 min. The SNaPshot reaction was performed in a final volume of 5 μl (reaction mix 2.5 μl, PCR products 1.5 μl, and probe mix 1.0 μl). DNA sequencing was performed in a final volume of 10 μl containing 1 μl of SNaPshot purified product, 8.5 μl of deionized formamide, and 0.5 μl GeneScan-120 LIZ Size Standard, using an ABI PRISM 3730 DNA Sequencer (Applied Biosystems by Life Technologies). All sequence analyses were performed using GeneMapper4.0 DNA Sequencing Analysis software.
Abi prism 3730 dna sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI PRISM 3730 DNA Sequencer is a laboratory instrument designed for high-throughput DNA sequencing. It utilizes the capillary electrophoresis method to separate and detect fluorescently labeled DNA fragments. The instrument is capable of sequencing DNA samples with high accuracy and speed.
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Lab products found in correlation
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (5 mentions)
Qiaex 2 gel extraction kit, by Qiagen (4 mentions)
Pgem t easy vector, by Promega (3 mentions)
Wizard genomic dna purification kit, by Promega (3 mentions)
Abi prism 7900 ht fast real time instrument, by Thermo Fisher Scientific (3 mentions)
Genemapper 3, by Thermo Fisher Scientific (2 mentions)
Dna isolation kit, by Tiangen Biotech (2 mentions)
Abi prism snapshot method, by Thermo Fisher Scientific (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Multiplex snapshot technique, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Pgem t easy vector systems kit, by Promega (1 mentions)
Qiaamp viral rna kit, by Qiagen (1 mentions)
High pure pcr product purification kit, by Roche (1 mentions)
Bigdye v3.1 kit, by Thermo Fisher Scientific (1 mentions)
2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Recombinant taq dna polymerase, by Takara Bio (1 mentions)
Redextract n amp tissue pcr kit, by Merck Group (1 mentions)
Bigdye terminator mix v3, by Thermo Fisher Scientific (1 mentions)
41 protocols using abi prism 3730 dna sequencer
1
Genotyping TSPAN2 Variants in Blood Samples
2
Genomic DNA Extraction and STR Profiling
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Genomic DNA was extracted using the Chelex-100 protocol as described by Walsh et al. and quantified spectrophotometrically[24] (link). Multiplex PCR amplification was performed on approximately 1–3 ng of genomic DNA in a total reaction volume of 25 µl, consisting of 9.5 µl of the AmpFlSTR Identifiler PCR reaction mix, 0.5 µl of AmpliTaq Gold DNA polymerase, and 5.0 µl of the AmpFlSTRI dentifiler primer set. Amplification was carried out in a 9700 Perkin-Elmer DNA Thermal Cycler (Applied Biosystems) using 28 cycles under the following conditions (after an initial denaturation step of 11 min at 95°C): 94°C for 1 min, 59°C for 1 min, 72°C for 1 min (following the recommendations from the AmpFlSTR Identifiler PCR kit manufacturer's manual). The amplified DNA products were separated and detected using an ABI Prism 3730 DNA sequencer (Applied Biosystems). One microliter of PCR product was combined with 12 µl of formamide and 0.5 µl of size standard (GeneScan 500 LIZ). The resultant data analysis and allele designation were carried out using the GeneScan and Genotype software programs.
3
Influenza A Virus HA and M Gene Sequencing
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Viral cDNA sequencing of the Influenza A viruses was performed using freshly cultured isolates from the clinical specimens or nucleic acids from clinical samples. To analyze the phylogenetic relationships between the HA of the isolated pdm H1N1 and H3N2 serotypes, the HA and M genes were first examined via conventional RT-PCR using primers and a protocol as previously described [16 (link), 17 (link)]. Viral RNA was extracted from the freshly cultured isolates using an automated TANBead system (Taiwan Advanced Nanotech, Inc., Taiwan) or the QIAamp® Viral RNA Kit (Qiagen, Hilden, Germany). Further, the RT-PCR products were purified using a High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany) and cloned into the TA plasmid vector using a pGEM®-T Easy Vector Systems kit (Promega, Madison, WI, USA).
Plasmid DNA sequencing for the entire length of the cloned gene was performed using an ABI Prism 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Two or three different plasmid DNAs of the same Influenza A virus isolate were sequenced to determine the accuracy of the gene sequencing. The gene sequences were then assembled using the Basic Local Alignment Search Tool from the National Center for Biotechnology Information [18 (link)].
Plasmid DNA sequencing for the entire length of the cloned gene was performed using an ABI Prism 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Two or three different plasmid DNAs of the same Influenza A virus isolate were sequenced to determine the accuracy of the gene sequencing. The gene sequences were then assembled using the Basic Local Alignment Search Tool from the National Center for Biotechnology Information [18 (link)].
4
SNP Selection and Genotyping of NLRP3 Inflammasome
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In the process of SNP selection of NLRP3 inflammasome, we included in our study those SNPs with a MAF (Minor Allele Frequency) >0.05 in the Chinese Han population. MAFs were analyzed using online tools (http://www.ncbi.nlm.nih.gov/projects/SNP/ ). Finally, two SNPs (the NLRP3 rs10754558 and CARD8 rs2043211) were selected for further study. Genomic DNA was extracted from white blood cells using the commercially available DNA isolation kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. Genotypes for individual DNA samples were genotyped using the ABI PRISM-Snapshot method (Applied Biosystem, CA, USA). More detail information was shown in Supplementary Materials. Genotypes of 2 SNPs were identified by capillary electrophoresis (ABI PRISM3730 DNA Sequencer; Applied Biosystems). The results were analyzed with GeneMapper 3.0 software (Applied Biosystems). All SNapShot and PCR primers were listed in Supplementary Materials available online at http://dx.doi.org/10.1155/2016/3185397 .
5
PCR Identification of L. monocytogenes
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To validate the results of NGS, sequence-specific PCR identification of L. monocytogenes with a target fragment was carried out using the primers 5'-TATGTCGGGCAAGCG TTC-3' and 5'-GCGCTTGCGTGGTAATTC-3'. Sanger sequencing was performed with ABI PRISM 3730 DNA Sequencer (Applied Biosystems, Foster City, CA, USA) to validate the sequencing results. Finally, the sequences were aligned to the NT database with the online software NCBI Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome ).
6
Molecular Diagnosis of Usher Syndrome
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In Usher syndrome samples, where a major causative gene is involved, the Arrayed Primer Extension (APEX) approach plus direct gene sequencing was the molecular diagnosis procedure of selection. In all other cases, cosegregation analysis with the RD-chip allowed to highlight the best candidates for mutational screening. All the exons and exon–intron boundaries of selected genes were directly screened for mutations in each patient. Genomic DNA was amplified, purified on High Pure 96 UF Cleaning Plates (Roche) and sequenced using the BigDye v3.1 kit (Applied Biosystems, Inc.) in the ABI PRISM 3730 DNA sequencer (Applied Biosystems, Inc.).
All missense changes identified were verified in control population using the dbSNP database (Build 137,www.ncbi.nlm.nih.gov/projects/SNP/ ), the 1000 Genomes Project data (http://browser.1000genomes.org/index.html ), and ESP6500 data of the National Heart, Lung, and Blood Institute GO Exome Sequencing Project (http://evs.gs.washington.edu/EVS ). To validate unreported missense genetic variants, over one hundred matched controls were analyzed to discard rare non-pathogenic polymorphisms restricted to the Spanish population.
All missense changes identified were verified in control population using the dbSNP database (Build 137,
7
Amplification of AsSWEET CDSs from Allium Cultivars
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To amplify the AsSWEET CDSs, gene-specific primers were designed based on A. sativum cv. Ershuizao transcriptomic data (NCBI project accession number: PRJNA607255) (Supplementary Table S2 ). PCR amplification was performed with 30 ng cDNA from the roots of each cultivar at the following conditions: initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min, and final extension at 72 °C for 5 min. PCR products of the expected size were purified by using the QIAEX® II Gel Extraction kit (QIAGEN, Hilden, Germany), cloned in the pGEM®-T Easy vector (Promega, Madison, WI, USA), and sequenced (3–5 clones for each accession) on ABI Prism 3730 DNA Sequencer (Applied Biosystems, Waltham, MA, USA) using the same primers.
8
Genetic Analysis of AR Gene in PCOS Patients
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Genomic DNA was isolated from the peripheral blood samples of patients with PCOS using the DNeasy Blood kit (cat. no. 69504; Qiagen, Inc.) according to the manufacturer's protocol. The entire coding regions and the adjacent exon/intron boundaries of the AR gene were amplified by PCR with 10 sets of primer pairs (Table II ). In brief, for each PCR amplification reaction, ~50 ng total DNA was used in a final volume of 30 µl, with the following amplification protocol: Initial pre-denaturation step at 94˚C for 3 min, followed by 35 cycles of denaturation at 94˚C for 30 sec, annealing at different temperatures (52-60˚C; Table II ) for 30 sec and extension at 72˚C for 30 sec; final extension at 72˚C for 7 min. PCR was performed using a Thermal Cycler 2720 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR amplification products were then purified and sequenced on an ABI Prism 3730 DNA sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.). An additional independent PCR amplification and DNA sequencing experiment was performed with samples of patients with PCOS harboring potential AR mutations. PCR amplification and DNA sequencing were performed as described above. The identified mutations were confirmed by bidirectional sequencing on ABI 3730 Prism DNA sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.).
9
Genetic Variant Analysis of LOXL1 in XFG
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Genomic DNA was extracted from all the subjects by using a Wizard Genomic DNA Purification Kit (Promega, Southampton, U.K.) according to the manufacturer’s instructions. In XFG patients and controls, the region of the LOXL1 gene harboring the SNPs, rs1048661 (R141L) and rs3825942 (G153D), was amplified using the primers, 5-ATTCGGCTTTGGCCAGGT-3' and 5-GAACTGCTGCGGGTAGGA-3. Bidirectional cycle sequencing was performed using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Warrington, UK) and electrophoresed on an ABI PRISM 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA) (conditions available on request). Sequencing results were analyzed manually using the sequence analysis software SeqScape version 2.1.1 (Applied Biosystems, Foster City, CA, USA).
Statistical analysis was performed using Pearson’s Χ2 test (adjusted by Yates correction where necessary) to compare patient and control groups for possible associations between SNP alleles, genotype, and haplotype frequencies with the disease state. Odds ratios (ORs) were calculated using binary logistic regression, taking a 95% confidence interval (95% CI) into account. P < 0.05 was considered as statistically significant. ORs, and 95% CIs were calculated for each haplotype compared to all the other haplotypes. Hardy–Weinberg equilibrium was assessed using c2 test.
Statistical analysis was performed using Pearson’s Χ2 test (adjusted by Yates correction where necessary) to compare patient and control groups for possible associations between SNP alleles, genotype, and haplotype frequencies with the disease state. Odds ratios (ORs) were calculated using binary logistic regression, taking a 95% confidence interval (95% CI) into account. P < 0.05 was considered as statistically significant. ORs, and 95% CIs were calculated for each haplotype compared to all the other haplotypes. Hardy–Weinberg equilibrium was assessed using c2 test.
10
DNA Extraction and COX1 Amplification of Corbicula uncinata
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About 500 C. uncinata were collected from the culture medium, suspended in lysis buffer (10 mM Tris–HCl, pH 8.0; 1 M EDTA, pH 8.0; 0.5% sodium dodecyl sulfate; 60 μg/ml proteinase K), and incubated at 55 °C for 12–20 h. DNA was extracted using the REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, USA). The COX1 gene was amplified with two primers listed in Additional file 1 : Table S1. Polymerase chain reaction (PCR) was conducted in a 20-µl reaction mixture containing 7.4 µl ddH2O, 10 µl 2 × PCR buffer (Mg2+, dNTP plus, Takara, Dalian, China), 0.6 µl of each primer, 0.4 µl recombinant Taq DNA polymerase (250 U/µl, Takara, Dalian, China), and 1 µl DNA template. PCR conditions were as follows: initial denaturation at 98 °C for 2 min, followed by 40 cycles at 98 °C for 10 s, 50 °C for 15 s, 68 °C for 1 min/kb, and a final extension at 68 °C for 10 min. PCR amplified products were sequenced on an ABI PRISM® 3730 DNA Sequencer (Applied Biosystems, USA).
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