Genomic DNA was isolated using Easy DNA extraction kit (Invitrogen). Presence of human sequences was checked by PCR using Phusion Flash (Thermo Scientific) high-fidelity PCR Master Mix (30 cycles of 98°C for 1 s; 60°C for 5 s and 72°C for 15 s) with primers for the chromosome 17 alpha satellite (Key Resource Table ).
Easy dna extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Easy DNA Extraction Kit is a laboratory tool designed for the quick and efficient extraction of DNA from a variety of sample types. The kit provides a streamlined process for isolating genomic DNA, enabling researchers to prepare samples for further analysis and downstream applications.
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4 protocols using easy dna extraction kit
1
Genomic DNA Extraction and Verification
2
Genomic DNA Extraction and qPCR Standardization
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Strains of Salmonella enterica serovar Enteritidis PT4, E. coli (INCQS 00033), and S. aureus (INCQS 00186) obtained from the microbial culture collection of the National Institute of Health Quality Control (INCQS, Instituto Nacional de Controle de Qualidade em Saúde) of Oswaldo Cruz Foundation/RJ were used in this study. An isolated colony of each microorganism was inoculated in 1.0 mL of tryptic soy broth (TSB; HiMedia, Mumbai, India) and incubated at 37°C for 18–24 h. This bacterial suspension was then used for genomic DNA extraction using the Easy DNA Extraction Kit (Invitrogen, Carlsbad, CA, USA). The DNA from each strain was quantified at 260 and 280 nm using the Nano Drop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and was then used in conventional PCR to amplify the target gene of each strain and produce the qPCR standard curve.
3
Genomic DNA Extraction and Verification
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Genomic DNA was isolated using Easy DNA extraction kit (Invitrogen). Presence of human sequences was checked by PCR using Phusion Flash (Thermo Scientific) high-fidelity PCR Master Mix (30 cycles of 98°C for 1 s; 60°C for 5 s and 72°C for 15 s) with primers for the chromosome 17 alpha satellite (Key Resource Table ).
4
Bacterial Whole Genome Sequencing and ESBL Detection
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Bacterial genomic DNA (gDNA) was purified and its concentration determined using the Easy-DNA extraction kit Invitrogen® and the Qubit dsDNA Assay Kit Invitrogen® respectively. The gDNA library preparation was performed following Nextera® XT DNA Sample Preparation Guide [36 ]. In brief, each gDNA was tagmented (tagged and fragmented) by the Nextera® XT transposome. The transposome simultaneously fragments the input DNA and adds adapter sequences to the ends. Thereafter followed a limited-cycle PCR amplification whereby indexes required for cluster formation were added to each DNA piece. Then each gDNA library was normalized to ensure equal representation during sequencing. Equal volumes of the normalized library were combined, diluted in hybridization buffer, and heat-denatured prior to sequencing on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA). Whole genome sequence reads were analyzed using bioinformatics tools available on https://cge.cbs.dtu.dk/services/ . For this report’s purpose, the analyses included de novo reads assembly, species identification, and determination of blaCTX-M encoding ESBL.
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