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50 protocols using universal genomic dna kit

1

Genome Editing Validation by T7 Endonuclease I

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The GMECs were digested, half were used for T7 Endonuclease I (M0302L, NEB, Ipswich, MA, USA) cleavage assay [42 (link)], and the other half was cultured continuously. The cells genome was extracted using a Universal Genomic DNA Kit (CW2298S, CW Biotech, Beijing, China). Extraction of genomes from cells with a Universal Genomic DNA Kit (CW2298S, CW Biotech, Beijing, China). Utilizing the genome as the template, the genome fragment of about 500 bp near the sgRNA locus was amplified by PCR using PrimeSTAR Max DNA Polymerase (R045A, Takara Bio Inc., Otsu, Japan), then detected it in a 1% agarose gel. If the band was single, the solution be purified by a PCR Clean-Up Kit (AP-PCR-50, Axygen, Union City, CA, USA) according to the manufacturers instructions. The purified DNA fragments were annealed in NEB buffer 2 (B7202S, NEB, Ipswich, MA, USA), followed by the addition of 0.3 μL T7 Endonuclease I (M0302L, NEB, Ipswich, MA, USA), reaction at 37 °C for 30 min, and results of enzyme digestion were identified by 2% agarose electrophoresis. The selected DNA fragments were ligated into Pmd19-T vector (CB35526014, takara Bio Inc., Otsu, Japan), transferred to E. coli (CB171211, TIANGEN, Beijing, China), and monoclonal colonies were selected and sequenced, which were compared with the gene sequence of the control group.
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2

CRISPR Gene Editing Validation Protocol

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Plasmids encoding SpCas9 variants and guide RNAs were prepared based on the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene plasmid #42230).35 (link) 293 T cells were incubated at 37 °C for 72 h post-transfection before genomic DNA extraction. Genomic DNA was extracted using the Universal Genomic DNA Kit (CWBiotech) following the manufacturer’s protocol. The genomic region flanking the CRISPR target site was amplified by PCR (target sites and primers listed in Supplementary information, Fig. 2), and products were purified using GeneJET Gel Extraction Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. A total of 250 ng purified PCR products were mixed with 2 µL 10 × NEB 2 buffer (NEB) and ultrapure water to a final volume of 20 µL, and subjected to a re-annealing process to enable heteroduplex formation: 95 °C for 3 min, 95 °C to 85 °C ramping at −2 °C/s, 85 °C to 25 °C ramping at −0.1 °C/s, and 25 °C hold for 10 min. After re-annealing, products were treated with 1 µL T7 endonuclease at 37 °C for 15 min following the manufacturer’s recommended protocol, and analyzed on TBE-urea 7% PAGE. Gels were stained with EB for 3 min and imaged with Omega Lum C. Quantification was based on relative intensities of individual bands. Indel percentage was determined by the formula, % Indel = 100 × (1- (1- fraction cleaved) 1/2).
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3

Mitogenome Sequencing of Dolycoris Specimens

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A total of 15 Dolycoris specimens were sampled for mitogenome sequencing, representing three species from 15 locations in China (Table S1). Samples were collected from 2016 to 2022, preserved in 100% ethanol, and stored at −20 °C at the Institute of Entomology at Nankai University (Tianjin, China). We extracted the genomic DNA from the thoracic muscle using a Universal Genomic DNA Kit (CWBIO, Beijing, China). Whole mitochondrial genomes were sequenced individually using the 150 bp paired-end reads strategy via the Illumina NovaSeq 6000 platform. We used fastp [21 (link)] to remove low-quality reads. Finally, approximately two Gb of raw data was obtained for each individual.
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4

Quantification of Mitochondrial DNA

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Total DNA was extracted by the Universal Genomic DNA Kit (CWBiotech). Mitochondrial gene Cox1 and nuclear gene Pecam were selected as the representative genes of mitochondrial and nuclear DNA, respectively. Quantitative PCR was used to detect the copy number changes of Cox1.
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5

Genetic basis of H. pylori antibiotic resistance

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Genetic determinants of H. pylori antibiotic resistance to clarithromycin and levofloxacin were analyzed by high-throughput nucleotide sequencing technology. The total genomic DNA of H. pylori isolates was extracted using the Universal Genomic DNA Kit (Cwbiotech, Jiangsu, China). The extracted DNA was processed by DNA fragment enzyme, the DNA library was prepared by the rapid preparation kit, and sequenced on the Beijing Genomics Institute platform (MGISEQ-2000, MGI Tech Co. Ltd, Beijing, China). Trim Galore was used for quality control of the DNA sequence that was then compared to the reference genome by BWA; Picard was used for preprocessing, and finally, the mutation site was identified by bcftools. It was peptidyl transferase in the V domain of 23S rRNA related to clarithromycin resistance, and gyrA was related to levofloxacin resistance; all the genetic variations of 23S rRNA and gyrA were analyzed and compared to H. pylori 26,695. These variants were summarized and analyzed statistically with respect to the resistance phenotype using Fisher’s exact test.
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6

Assessing Off-Target Effects of Nanocapsules

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To assess the off-target effects of nanocapsules, we predicted potential off-target sites based on the online database (https://cm.jefferson.edu/Off-Spotter/) according to the following acknowledged criteria (42 (link), 43 ): (i) Cas9 tolerates single-base mismatches in the protospacer adjacent motif (PAM)-distal region to a greater extent than in the PAM-proximal region. (ii) Three or more mismatched base pairs eliminated detectable Cas9 cleavage in the vast majority of loci.
Briefly, after being treated with ANCSS(Cas9/sgPLK1), the genomic DNAs of tumor, normal brain tissue, liver, and kidney were harvested from mice bearing U87MG or CSC2 GSCs, using the Universal Genomic DNA Kit (CWBIO, China) according to the manufacturer’s instructions. One hundred nanograms of genomic DNA was used as a template to perform PCR using primers designed against on-target and off-target sites. Purified DNA was amplified again by PCR with primers containing sequencing adapters and then sequenced and analyzed by Sangon Biotechnology Company (Shanghai, China) to detect indels around target sites.
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7

16S rRNA Gene Amplification and Sequencing

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According to the manufacturer's protocols, under aseptic conditions, genomic DNA was extracted from plasmodia using the Universal Genomic DNA Kit (CWBIO, China) according to the manufacturer's protocols. About 200 mg of plasmodia (collected from several individual plasmodia) were used for DNA extraction. DNA purity was examined on 1% agarose gels. NanoPhotometer spectrophotometer was used to detect sample purity; Qubit2.0 Flurometer was used to measure DNA sample concentration. The V3-V4 hypervariable region of the bacterial 16S ribosomal RNA gene was amplified by PCR (94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min) using primers 341F 5'–CCTACGGGNGGCWGCAG–3' and 805R 5'–GACTACHVGGGTATCTAATCC–3' [27 (link)], where the barcode was an eight-base sequence unique to each sample. PCR was performed in triplicate with 50 μL mixture containing 5 μL of 10 × Ex Taq Buffer (Mg2+ plus), 4 μL of 2.5 mM dNTPs, 1 μL of each primer (20 μM), 0.25 μL of TaKaRa Ex Taq (5 U/μL), and 2.5 ng of the template DNA. Amplicon sequencing targeting 16S V3–V4 region was performed by Annoroad company based on an Illumina HiSeq platform. Roughly 50,000, 250 bp paired-end reads were generated for each sample. Original data have been deposited into the NCBI SRA database with the accession number PRJNA600342.
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8

Sampling and DNA Extraction of Eurasian Apaludum

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We sampled 52 populations, comprising a total of 409 individuals between 1999 and 2018 across the natural distributional range of A. paludum in Eurasia. The latitude and longitude of each collection site were recorded using a handheld global positioning system (GPS) unit. All samples were preserved in 70–95% ethanol and stored in a freezer at −20 °C in the College of Life Sciences at Nankai University (NKU, Tianjin, China). Genomic DNA of each specimen was extracted from the entire body, excluding the abdomen and genitalia, of each specimen using a Universal Genomic DNA Kit (CWBIO). Detailed information for molecular voucher specimens is provided in supplementary tables S1–S4, Supplementary Material online, which includes sample codes, sample sizes, collection locations and data, longitude, and latitude.
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9

CRISPR-Cas9 Modification Detection

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After 72 h transfection, cells expressing green fluorescent protein were sorted out by flow cytometry (BD FACSAria Fusion). Genomic DNA was extracted using the Universal Genomic DNA Kit (CWBiotech) following the manufacturer’s protocol. The genomic region flanking the target site was amplified by PCR with GoTaq Green Master Mix (Promega). Equal amounts of PCR product were sent for NGS at the Hi-TOM platform.71 (link) Indels were analyzed by the Hi-TOM platform (http://121.40.237.174/Hi-TOM/). The primers used in this study are provided in Supplementary information, Table S5.
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10

Comprehensive Genomic DNA and RNA Isolation Protocols

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Total DNA was isolated from cells using the Universal Genomic DNA Kit (Cwbiotech). Isolated DNA was used for PCR using a Multiplex PCR MasterMix (Cwbiotech). The primers are listed in Supplementary Table 3.
Total RNA was isolated from cells using the RNApure Tissue & Cell Kit (Cwbiotech). Isolated RNA was used as a template for reverse transcription reactions using a HiFiScript cDNA Synthesis Kit (Cwbiotech).
Quantitative real-time PCR analyses were performed with a PCR mixture containing 1 μmol/L of each primer and SYBR Green Real-Time PCR Master Mixes (Thermo Fisher) using a CFX96 Real-Time PCR Detection System (Bio-Rad, USA). Each sample was run in triplicate, and the mRNA level of GAPHD was used as an internal control. The cycle threshold (Ct) value was normalized to the GAPDH level using the 2−△△Ct method. The primers are listed in Supplementary Table 4.
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