Restriction and DNA modification enzymes were purchased from New England Biolabs or Fermentas. Plasmid DNA was extracted from E. coli or Sulfolobus cells using an AxyPrep Plasmid Miniprep Kit (Wujiang, China). Polymerase chain reaction (PCR) products were purified by using an Axygen PCR Clean-up Kit, and DNA bands fractionated from the agarose gel were extracted by using an Axygen DNA extraction kit. Total DNA was prepared from Sulfolobus by using an Axygen Genomic DNA Miniprep Kit. The oligonucleotides were synthesized by Invitrogen (Shanghai, China) and were also used for DNA sequencing.
Genomic dna miniprep kit
Manufactured by Corning
Sourced in United States
The Genomic DNA Miniprep Kit is a laboratory product designed for the rapid and efficient extraction and purification of genomic DNA from a variety of sample types, including bacterial, plant, and animal cells. The kit utilizes a spin-column format and includes all necessary reagents and buffers for the DNA isolation process.
Automatically generated - may contain errors
Lab products found in correlation
Mitochondria isolation kit, by Beyotime (2 mentions)
Pcr cleanup kit, by Corning (1 mentions)
Dna extraction kit, by Corning (1 mentions)
Axyprep pcr cleanup kit, by Corning (1 mentions)
Vahts universal dna library prep kit for v2, by Illumina (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Ion torrent pgm, by Thermo Fisher Scientific (1 mentions)
Ampure xp reagent, by Beckman Coulter (1 mentions)
Mitochondrial isolation kit, by Abcam (1 mentions)
Sequencing primer adaptor, by Illumina (1 mentions)
Quant it dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 analyzer, by Agilent Technologies (1 mentions)
Paired end dna sample prep kit, by Illumina (1 mentions)
Biophotometer plus spectrophotometer, by Eppendorf (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Sequenom massarray platform, by CapitalBio (1 mentions)
Cfx96 system, by Bio-Rad (1 mentions)
10 protocols using genomic dna miniprep kit
1
Molecular Techniques for DNA Manipulation
2
Fine-Scale Mapping of Insecticide Resistance
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For fine-scale mapping of resistance, we generated a second F2 segregation generation. As described above, we started with a mass cross between a single female from DH-R and a single male from the DH-S strain. The F2 segregating population was obtained by crossing F1 population. The progeny of F2 population were put on diet treated with 0.5 μg Vip3Aa per cm2 diet. Ninety-six survivors were selected and named F2-R. Genomic DNA was extracted individually using a Multisource Genomic DNA Miniprep Kit (Axygen, New York, USA) according to the manufacturer’s recommendations.
Based on the SNPs calculated from the genomic resequencing data and the reference genome data, we designed primers specific for the exons of functional genes spanning 10.01 to 10.63 Mb on chromosome 27. PCR amplification and sequencing were performed for parent moths. SNPs that were homozygous in DH-S and different homozygous SNPs in DH-R were selected as informative markers. To evaluate genetic linkage with resistance for each marker, we used the chi-square test to check for significant deviation between the observed and expected genotype frequencies (rr:rs:ss = 1:2:1).
Based on the SNPs calculated from the genomic resequencing data and the reference genome data, we designed primers specific for the exons of functional genes spanning 10.01 to 10.63 Mb on chromosome 27. PCR amplification and sequencing were performed for parent moths. SNPs that were homozygous in DH-S and different homozygous SNPs in DH-R were selected as informative markers. To evaluate genetic linkage with resistance for each marker, we used the chi-square test to check for significant deviation between the observed and expected genotype frequencies (rr:rs:ss = 1:2:1).
3
Genomic DNA Extraction and Sequencing
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Genomic DNA (gDNA) was extracted from cells and 20 mg fresh liver tumor tissues using the Genomic DNA Miniprep Kit (AxyGEN, USA) according to the manufacturer’s instructions. As done previously [44 (link)], 35 targeted sites of gDNA and eccDNAs were amplified by PCR with primers listed (Additional file 1 : Table S1). PCR products were purified by AxyPrep DNA Gel Extraction Kit or AxyPrep PCR Cleanup Kit (AxyGEN). Purified PCR products of each target were combined into a tube with equal volume. One microgram of PCR product mixture was used to establish DNA fragment libraries by VAHTS Universal DNA Library Prep Kit for Illumina V2 (Vazyme Biotech, China) and sequenced on Illumina HiSeq 2500.
4
Genetic Analysis of Hypertrophic Cardiomyopathy
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Genomic DNA was isolated from the peripheral whole blood of 18 HCM patients and 100 healthy controls using a genomic DNA Miniprep kit (AxyPrep; Axygen, Union City, CA, USA) following the manufacturer's protocol. OD260/280 of DNA samples was ~1.8 after purification with AMPure XP reagents (Beckman Coulter, Fullerton, CA, USA). The DNA concentration was measured using the Qubit dsDNA HS assay kit (Life Technologies, Carlsbad, CA, USA). Whole coding exons and in exon-intron boundaries of 19 HCM-related genes were amplified and then sequenced on Ion Torrent PGM (Life Technologies). These genes were MYH7, myosin binding protein C (MYBPC3), α-actinin 2 (ACTN2), desmin (DES), α-galactosidase A (GLA), lysosome-associated membrane protein 2 (LAMP2), LIM domain-binding 3 (LDB3), α-myosin heavy chain (MYH6), regulatory myosine light chain 2 (MYL2), regulatory myosine light chain 3 (MYL3), TAZ, myopalladin (MYPN), AMP-activated protein kinase (PRKAG2), sodium channel, voltage-gated type V α-subunit (SCN5A), Titin-cap (TCAP), troponin I 3 (TNNI3), troponin T 2 (TNNT2), α-tropomyosin 1 (TPM1), and vinculin (VCL).
5
Quantifying Cytosolic Mitochondrial DNA
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Mitochondria were isolated from mDPC6T cells using a mitochondrial isolation kit (BioVision, USA). After the mitochondria were discarded, the cytoplasm was prepared to isolate cytosolic mtDNA using a genomic DNA miniprep kit (Axygen, USA). PCR quantification was then carried out to analyze mitochondrial DNA and nuclear DNA in the cytoplasm. Mitochondrial DNA was marked by tRNA-LeuUUR, while nuclear DNA was characterized by β-microglobulin genes. The ratio of mtDNA to nDNA in the cytoplasm represented the copy number of cytosolic mtDNA.
6
DNA Extraction, Fragmentation, and Sequencing
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Total genomic DNA was extracted using a Genomic DNA Miniprep Kit (Axygen) following the manufacturer’s instructions. The DNA quality was then evaluated by agarose gel electrophoresis and a BioPhotometer Plus spectrophotometer (Eppendorf, Germany).
Subsequently, the two samples were sonicated to produce DNA fragments ranging from 100 to 500 bp. After end repairing, phosphorylating and A-tailing with Paired-End DNA Sample Prep kit (Illumina, USA), the DNA was ligated to an Illumina sequencing primer adaptor. Then the fragments were used for methylated DNA immunoprecipitation (MeDIP) enrichment using a Magnetic Methylated DNA Immunoprecipitation kit (Diagenod, Belgium) following the manufacturer’s recommendations and the qualifying DNA was used for PCR amplification. Then bands between 220 and 320 bp were excised from the gel and purified with a QIAquick Gel Extraction Kit (Qiagen, Germany). The products were quantified with a Quant-iT™ dsDNA HS Assay Kit (Invitrogen, USA) using an Agilent 2100 Analyzer (Agilent Technologies, USA). Following qPCR qualification, DNA libraries were sequenced on the Illumina HiSeq 2000 (Illumina, CA, USA) to generate paired-end 50-bp reads by the Beijing Genomics Institute (BGI, China).
Subsequently, the two samples were sonicated to produce DNA fragments ranging from 100 to 500 bp. After end repairing, phosphorylating and A-tailing with Paired-End DNA Sample Prep kit (Illumina, USA), the DNA was ligated to an Illumina sequencing primer adaptor. Then the fragments were used for methylated DNA immunoprecipitation (MeDIP) enrichment using a Magnetic Methylated DNA Immunoprecipitation kit (Diagenod, Belgium) following the manufacturer’s recommendations and the qualifying DNA was used for PCR amplification. Then bands between 220 and 320 bp were excised from the gel and purified with a QIAquick Gel Extraction Kit (Qiagen, Germany). The products were quantified with a Quant-iT™ dsDNA HS Assay Kit (Invitrogen, USA) using an Agilent 2100 Analyzer (Agilent Technologies, USA). Following qPCR qualification, DNA libraries were sequenced on the Illumina HiSeq 2000 (Illumina, CA, USA) to generate paired-end 50-bp reads by the Beijing Genomics Institute (BGI, China).
7
Quantitative Methylation Analysis of Wnt Genes
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Following ultrasonic sonication (P-141008; Diagenode SA, Seraing, Belgium) of mouse and human spinal cord tissues, genomic DNA was extracted from the homogenates using a Genomic DNA Miniprep kit (Axygen Scientific, Inc., Union City, CA, USA). A total of 500 ng genomic DNA from each sample was bisulfite-treated using an EZ DNA Methylation-Gold™ kit (cat. no. D5005; Zymo Research, Irvine, CA, USA). The Sequenom MassARRAY platform (CapitalBio Corporation, Beijing, China) was used to perform quantitative methylation analysis of Wnt2b and Wnt7b (13 (link)). The primers used to detect DNA methylation levels of the target regions are presented in Table III .
8
Mitochondrial DNA Quantification in Macrophages
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Mitochondria were extracted from macrophage by using the Mitochondria Isolation Kit (Beyotime, China) according to the manufacturer's instructions, to allow the extraction of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) by using the genomic DNA miniprep kit (Axygen, USA). The short regions of the tRNA-LeuUUR and β2-microglobulin genes were used to quantify mtDNA and nDNA respectively, from which the mtDNA/nDNA ratio was calculated to determine the mtDNA copy number as previously described [31 (link)].
9
Mitochondrial DNA Copy Number Determination
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The cytosol of mDPC6T cells was extracted with the Mitochondria Isolation Kit (Beyotime) according to the manufacturer's protocol. The cytosolic supernatant was collected without mitochondria. The genomic DNA miniprep kit (Axygen) was used to isolate the DNA from the collected cytoplasm supernatant. DNA (10 ng) was subjected to real-time PCR on a CFX96 system (Bio-Rad) with SYBR qPCR master mix (Vazyme). The short regions of the tRNA-LeuUUR and β2-microglobulin genes were amplified to determine mitochondrial DNA and nuclear DNA, respectively, as previously described [26 ]. The mtDNA/nDNA ratio was calculated to determine the mtDNA copy number. All primers are listed in the Additional file.
10
CRISPR-Cas9 Genome Editing of IPEC-J2 Cells
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Guide RNAs were designed using the online CRISPR design tool (http://crispr.mit.edu/ ) and then cloned into the BbsI‐digested plasmids (pSpCas9n) containing the entire guide RNA scaffold. After transfection, the genomic DNA of IPEC‐J2 cells were extracted using Genomic DNA Miniprep kit (Axygen, AZ, USA). The genomic region flanking the MyD88 target site was amplified using polymerase chain reaction (PCR). The products underwent a re‐annealing process to facilitate heteroduplex formation. After re‐annealing, the products were treated with T7 Endonuclease I (T7E I) following the manufacturer's recommended protocol.
Isolation of clonal cell lines that were confirmed by T7E I were performed by serial dilutions. Detection of genomic microdeletions was performed by PCR sequencing. Off‐target activity was analysed by deep sequencing and the Blast search (http://blast.ncbi.nlm.nih.gov/Blast.cgi ).
Isolation of clonal cell lines that were confirmed by T7E I were performed by serial dilutions. Detection of genomic microdeletions was performed by PCR sequencing. Off‐target activity was analysed by deep sequencing and the Blast search (
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