Genomic DNA was extracted from buccal epithelial cells using a commercially available DNA isolation kit (PrepFiler® Express Forensic DNA Extraction Kit, Applied Biosystems, Life Technologies Polska, Warsaw, Poland) according to the manufacturer’s instructions. The amplification of the ADRB3 sequence of 440 base pairs (bp) in length, including the rs4994 polymorphism, was performed by PCR, using 5′-CGCCCAATACCGCCAACA CCAGT-3′ as the forward primer and 5′-CGCGGCCGACACGACCCACAC-3′ as the reverse primer. Subsequently, the PCR amplification products were purified using Exonuclease I and FastAP Thermosensitive Alkaline Phosphatase (ThermoFisher Scientific Inc., Waltham, MA, USA) according to manufacturer procedures. The sequencing of the products used BigDye® Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Life Technologies Polska, Warsaw, Poland). Electrophoresis and analyses were performed according to manufacturer procedures, using an ABI PRISM 3100-Avant machine (Data Collection Software v2.0 and Sequencing Analysis Software v5.4; Applied Biosystems).
Prepfiler express forensic dna extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PrepFiler Express™ Forensic DNA Extraction Kit is a lab equipment product designed for the extraction of DNA from forensic samples. It is a standardized and automated solution for efficient DNA recovery.
Automatically generated - may contain errors
Lab products found in correlation
Quantifiler trio dna quantification kit, by Thermo Fisher Scientific (2 mentions)
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
Exonuclease 1, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Automate express forensic dna extraction system, by Thermo Fisher Scientific (1 mentions)
5 protocols using prepfiler express forensic dna extraction kit
1
Genomic DNA Isolation and ADRB3 Sequencing
2
Forensic DNA Extraction and Quantification
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Sample swabs were collected from volunteer donors under informed consent, with a protocol approved by the Bioethical Committee of the Universidade de Santiago de Compostela (USC-30/2021 and USC-58/2022). When required, DNA extractions were performed using the PrepFiler Express Forensic DNA Extraction Kit (Applied Biosystems, AB) on the AutoMate Express Nucleic Acid Extraction System (AB), following the recommended protocol. DNA quantification was performed using the Quantifiler Trio DNA Quantification Kit (AB) on a 7500 Real-Time PCR System (AB). A total of 15 reference DNA samples including six DNAs extracted from donor buccal swabs, five Coriell cell-line DNA controls (HG00403, NA06994, NA07000, NA07029 and NA18498) and four DNAs as part of two kinship testing scenarios, half-siblings -C1.1 and C1.2 -and samples from an avuncular related pair -C2.1 and C2.2 -were analyzed at input amounts ranging from 5 to 10 ng. A single non-template control was included to assess levels of non-specific amplification. A dilution series of DNA control 007 was prepared for input quantities of 5 ng, 1 ng, 0.5 ng, 250 pg, 125 pg and 62.5 pg, to evaluate sensitivity to low level input DNA. Blood, semen, and buccal swabs were collected in duplicate from a single donor and analyzed to evaluate the direct PCR approach.
3
Forensic DNA Proficiency Validated
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Genomic DNA was extracted using PrepFiler Express™ Forensic DNA Extraction Kit (Thermo Scientific, US) following the manufacturer’s guidelines. The extracted DNA was quantified using Quantifiler® Trio DNA Quantification Kit (Thermo Scientific, US) and QuantStudio™ 3 Real-Time PCR System (Thermo Scientific, US) according to the manufacturer’s instructions. Further, the concentration of DNA samples wwas adjusted to 1.0 ng/µl using TE buffer and stored at − 20 °C until further use. The authors have passed the Academia Iberoamericana de Criminalística y EstudiosForenses (AICEF) DNA Proficiency test of the de BIOLOGIA y QUÍMICA FORENSE (GITAD), Spain (http://gitad.ugr.es/principal.htm ).
4
DNA Extraction from Blood Samples
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The study cohort consisted of 999 unrelated individuals (673 males and 326 females), collected together within the NEXT project, funded by the National Centre for Research and Development, grant number DOB-BIO7/17/01/2015. The study was approved by the Ethics Committee of the Jagiellonian University in Kraków (decision no. KBET/122/6120/11/2016), and all volunteers gave written informed consent prior to their inclusion in the study. Recruitment of the participants was carried out in the Police Academy in Szczytno.
Whole blood was collected from the volunteers and subjected to DNA extraction using the PrepFiler Express™ Forensic DNA Extraction Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Quantification of the extracted samples was performed using the Quantifiler™ Human DNA Quantification Kit or the Plexor® HY System.
Whole blood was collected from the volunteers and subjected to DNA extraction using the PrepFiler Express™ Forensic DNA Extraction Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Quantification of the extracted samples was performed using the Quantifiler™ Human DNA Quantification Kit or the Plexor® HY System.
5
Genomic DNA Extraction and Quantification
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A total of 95 samples were collected (54 males and 41 females) from the central Indian population. The blood samples were collected from the participants after obtaining written informed consent. The peripheral liquid blood samples collected in K 2 EDTA vials were stored at 4 °C until further use. Genomic DNA was extracted from the blood samples by following recommended protocol of the manufacturer using AutoMate Express™ Forensic DNA Extraction System (Thermo Scientific, USA) and PrepFiler Express™ Forensic DNA Extraction Kit (Thermo Scientific, USA). Extracted genomic DNA was subjected to quantification using QuantStudio™ 5 Real-Time PCR System (Thermo Scientific, USA) using Quantifiler® Trio DNA Quantification Kit (Thermo Scientific, USA). Further, the DNA samples were normalized to 1.0 ng/µl in TE buffer and stored at -20 °C until further use.
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