Genomic DNA was prepared from patients' peripheral blood. The 47 FLNA coding exons (exon 2 to exon 48) and their intron–exon boundaries (Genbank, NC_000023.11 ) were amplified, with informed consent. The entire FLNA coding sequence and flanking splice sites (reference sequence XM_011531131.1) were amplified by PCR. PCR products were sequenced on an ABI 3730 XL automatic sequencer and were analyzed with Seqscanner Software 2 (Applied Biosystems, Foster City, Calif., USA) for potential sequence variations by direct sequencing of PCR products using an ABI Prism Big-Dye Terminator Cycle Sequencing Kit version 3.1 and ABI 3730 XL Avant sequencer (Applied Biosystems, Foster City, Calif., USA).
Abi prism bigdye terminator cycle sequencing kit version 3
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The ABI Prism BigDye Terminator Cycle Sequencing Kit version 3.1 is a reagent kit designed for automated DNA sequencing. It contains the necessary components, including fluorescently labeled dideoxynucleotides, to perform cycle sequencing reactions.
Automatically generated - may contain errors
Lab products found in correlation
Multiscreen pcr 96 filter plate, by Merck Group (2 mentions)
Exosap it, by Thermo Fisher Scientific (2 mentions)
Abi prism 3100 sequencer, by Thermo Fisher Scientific (2 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Abi 3730xl automatic sequencer, by Thermo Fisher Scientific (1 mentions)
Dreamtaq master mix, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Bioworld Technology (1 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions)
Sequencher, by Gene Codes (1 mentions)
Shrimp alkaline phosphatase, by Roche (1 mentions)
Hotstartaq master mix, by Qiagen (1 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (1 mentions)
Mutation surveyor software, by SoftGenetics (1 mentions)
7 protocols using abi prism bigdye terminator cycle sequencing kit version 3
1
Genetic analysis of FLNA gene
2
Sanger Sequencing of Genetic Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The candidate variants were validated by direct Sanger sequencing in patients as well as their parents. Primers for PCR and sequencing were provided by PHUSA Biochem Company (Can Tho, Vietnam). For PCR amplification, 10 ng of total genomic DNA was used as a template in 20 μl of reaction mixture containing 1X Neb Master mix (New England Biolabs, Ipswich), 0.8 μl of each primer (10 pmole), and 8.4 μl of deionized water. The thermocycling was 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 58°C for 30 s, 68°C for 20 s, and a final extension at 68°C for 5 min. The PCR products were purified using Multiscreen PCR 96 Filter Plate (Merck‐Millipore) and sequenced by ABI Prism BigDye Terminator Cycle Sequencing Kit Version 3.1 (Applied BioSystems) on ABI 3500 Genetic Analyzer (Applied BioSystems).
3
Comprehensive Genetic Analysis of CYP2D6 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sanger sequencing method was used to identify nucleotide variants in the promoter, entire 9 coding exons and their flanking regions of CYP2D6 gene. Primers provided by PHUSA Biochem Company (Can Tho, Vietnam) were listed in Supplementary Table 1. Polymerase chain reaction (PCR) reaction was performed with a total volume of 20 μL containing: 10 ng total genomic DNA, 1× DreamTaq Master mix (Thermo Fisher Scientific, Waltham, Massachusetts), 10 pmole each primer, 1 μL DMSO (Bioworld, Dublin, Ohio), and 16.5 μL deionized water. The thermo cycle was as following: denaturation at 95 °C for 5 minutes, following by 40 cycles of 95 °C for 15 seconds, 58 °C for 30 seconds, 72 °C for 40 seconds to 2 minutes, and a final extension at 72 °C for 5 minutes. For exon 5 to exon 8, the thermo cycle was as following: denaturation at 95 °C for 2 minutes, following by 35 cycles of 95 °C for 30 seconds, 59 °C for 30 seconds, 72 °C for 2 minutes, and a final extension at 72 °C for 5 minutes. PCR products were purified using Multiscreen PCR 96 Filter Plate (Merck-Millipore, Burlington, Massachusetts) and were then sequenced using ABI Prism BigDye Terminator Cycle Sequencing Kit Version 3.1 (Applied Biosystems), on an ABI genetic analyzer 3500 (Applied Biosystems).
4
POLE Exonuclease Domain Mutation Screening
5
Genotyping CYP2D6 Gene Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from 300 μl peripheral blood using a GoldMagMini Whole Blood Genomic DNA Purification Kit (GoldMag Ltd). The purity of the extracted DNA reached above 99 %. Our PCR primers, which were described in a previous study, were designed to amplify 2000 bp of the 5’ flanking regions, all exons, and all introns of the CYP2D6 gene [10 (link)]. The 10 μl PCR system contained 5 μl Hotstar Taq Master Mix, 1 μl genomic DNA (20 ng/μl), 0.5 μl each primer pair (5 μM), and 3 μl deionized water. The PCR reaction conditions were as follows: the thermal profile consisted of denaturation at 95 °C for 15 min, followed by 35 cycles of denaturation at 95 °C for 30 s, 60 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 3 min and subsequent storage at 4 °C. We detected the PCR products by agarose gel electrophoresis and directly sequenced them using an ABI Prism BigDye Terminator Cycle Sequencing Kit version 3.1 on an ABI Prism 3100 sequencer (Applied Biosystems).
6
Genetic Profiling of CYP2J2 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood samples were collected from each individual. Genomic DNA was extracted using the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Ltd, Xi’an, China) according to the manufacturer's protocol. Polymerase chain reaction (PCR) primers were designed to amplify 2000 bp of the promoter and all exons of the CYP2J2 (Table 1 ). PCR was performed with 10 μL reactions, 1 μL template DNA and 5 μL HotStar Taq Master Mix (Qiagen, Germantown, MD), 0.5 μL primer (5 μM), and 3 μL deionized water. The thermal cycling conditions were initial denaturation for 15 minutes at 95°C followed by 35 cycles of denaturation at 95°C for 30 seconds, 55 to 64°C for 30 seconds, and 72°C for 1 minute, and a final extension step at 72°C for 3 minutes. PCR products were incubated with 0.5 μL shrimp alkaline phosphatase (Roche Diagnostics, Basel, Switzerland), 8 μL HotStar PCR product, and 1.5 μL deionized water (to a total volume of 10 μL), at 37°C for 30 minutes, followed by heat inactivation at 80°C for 15 minutes. Purified PCR products were directly sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Kit version 3.1 (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA) using an ABI Prism 3100 sequencer (Applied Biosystems, Thermo Fisher Scientific, Inc.).
7
Sanger Sequencing of Mutant DNA
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!