Qiaquick gel extraction kit

Manufactured by Qiagen

Sourced in Germany, United States, Netherlands, United Kingdom, Japan, Canada, France, Spain, China, Italy, India, Switzerland, Austria, Lithuania, Sweden, Australia

The QIAquick Gel Extraction Kit is a product designed for the purification of DNA fragments from agarose gels. It efficiently extracts and purifies DNA from gel slices after electrophoresis.

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Gel extraction kit (1089 citations) QIAquick kit (80 citations) Gel extraction (53 citations) QIAquick Gel Extraction (25 citations) QIAquick Extraction Kit (9 citations) QIAquick PCR purification and gel extraction kits (7 citations)

3 307 protocols using qiaquick gel extraction kit

Viral RNA Extraction and Sequencing Protocol

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For each sample, 50 μl plasma was used to extract viral RNA using the MagMAX-96 viral RNA isolation kit (Ambion, Austin, TX, United States) according to the manufacturer’s instructions. The cDNA was generated with the reverse primers using the TaKaRa RNA PCR Kit (AMV) Ver.3.0 (Takara Bio, Shiga, Japan). The PCR conditions were as follows: denaturation at 94°C for 2 min; 32 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 30 s, and extension at 68°C for 30 s; and a final elongation step at 68°C for 10 min. The PCR products were run on 1% agarose gels and extracted using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). The products were quantified using a spectrophotometer (NanoDropND-1000, Thermo Fisher Scientific, Waltham, MA, United States).
The barcode-tagged products of the same fragment were pooled and purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The DNA was end-repaired, A-tailed, and PE-adapter ligated. After ligation of the adapters, each sample was purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) and sequenced using the PE250 strategy on an Illumina MiSeq according to the manufacturer’s instructions. A base-calling pipeline (Sequencing Control Software, SCS; Illumina) was used to process the raw fluorescent images and the called sequences.

Dong X., Meng F., Hu T., Ju S., Li Y., Sun P., Wang Y., Chen W., Zhang F., Su H., Li S., Cui H., Chen J., Xu S., Fang L., Luan H., Zhang Z., Chang S., Li J., Wang L., Zhao P., Shi W, & Cui Z. (2017). Dynamic Co-evolution and Interaction of Avian Leukosis Virus Genetic Variants and Host Immune Responses. Frontiers in Microbiology, 8, 1168.

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Preparation of CasRx Pre-gRNA Cloning Backbone

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Digest and dephosphorylate 2 μg of the CasRx pre-gRNA cloning backbone plasmid with BbsI for 2 h at 37°C.
After BbsI digestion, 5′-phosphate groups were removed from the digested plasmid by calf intestinal alkaline phosphatase (CIP) to prevent self-ligation.
DNA electrophoresis was performed in 0.8% agarose gel to ensure plasmid DNA was completely digested. The CIP-digested plasmid was then purified using QIAquick Gel Extraction kit (QIAGEN) and eluted in 10 mM Tris–HCl, pH 8 (or the elution buffer from kit) (Supplementary Method—QIAquick Gel Extraction kit).
It must be noted that an ∼3-kb digested band should be present on 0.8% agarose gel.

Chuang Y.F., Wang P.Y., Kumar S., Lama S., Lin F.L, & Liu G.S. (2021). Methods for in vitro CRISPR/CasRx-Mediated RNA Editing. Frontiers in Cell and Developmental Biology, 9, 667879.

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Hi-C Library Construction for Plant Genomics

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A Hi-C sequencing library was constructed according to protocols described by Servant et al.⁶⁸ and Burton et al.^{69 (link)}. Briefly, the cells of young leaves of the ‘Big Five-pointed Star’ cultivar were fixed with formaldehyde and then dissociated, and the cross-linked products were treated with restriction endonucleases to produce cohesive ends. A biotin marker was introduced at the cohesive ends, which were repaired to produce blunt ends. The blunt ends were ligated; the cross-links were released to separate DNA from proteins; the DNA was extracted; a Covaris E220 instrument (Covaris, Brighton, UK) was used to fragment DNA to the correct size and then repair the ends; the fragmented DNA segments were purified by gel electrophoresis and recycled with a QIAquick Gel Extraction Kit (Qiagen Inc., Germany); those DNA segments without a biotin marker were removed; Poly(A) sequences were added to the remaining DNA segments including biotin markers; PCR adaptors were added; PCR was performed; and the PCR products were purified by gel electrophoresis and recycled by using a QIAquick Gel Extraction Kit (Qiagen Inc.).

, & Wang Y. (2021). A draft genome, resequencing, and metabolomes reveal the genetic background and molecular basis of the nutritional and medicinal properties of loquat (Eriobotrya japonica (Thunb.) Lindl). Horticulture Research, 8, 231.

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Genomic Integration and Verification

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Following the insertion of each DNA substrate, colony PCR was performed to amplify the integrated section. The size of the inserted fragment was verified with agarose gel electrophoresis, the product band was purified using the QIAquick Gel Extractionkit (Qiagen), and the sequence was verified by Sanger sequencing (MacroGen). After genomic insertion of the whole 1,4-BDO biosynthetic pathway, the genome sequence was verified by Illumina sequencing. Briefly, genomic DNA was prepared using a Genomic DNA Extraction Kit (Qiagen), followed by shearing to 300 bp (Covaris M220). After shearing, DNA was purified and concentrated to 40 μl final volume using a QIAquick PCR purification kit (Qiagen). Gel electrophoresis was performed to check the size of the bands, and only those in the range of 300–600 bp were excised and purified using QIAquick Gel Extraction kit (Qiagen). Samples for Illumina sequencing were prepared by NEBNext modules (NEBNext End Repair Module®, NEBNext dA-Tailing Module®, NEBNext Quick Ligation Module®, NEBNext Multiplex Oligo for Illumina®, New England Biolab, USA). The purified sample was then sent for Illumina sequencing (MacroGen), and the data were mapped to reference sequence by CLC Genomics Workbench (version 6.5.1).

Jeong J., Seo H.N., Jung Y.K., Lee J., Ryu G., Lee W., Kwon E., Ryoo K., Kim J., Cho H.Y., Cho K.M., Park J.H, & Bang D. (2015). Repetitive genomic insertion of gene-sized dsDNAs by targeting the promoter region of a counter-selectable marker. Scientific Reports, 5, 8712.

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DNA Assembly and Sequencing Protocol

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PCR amplifications were performed in 25 or 50 μl reaction mixtures using Phusion DNA polymerase (Thermo Scientific) or Dream Taq master mix kit (Thermo Scientific) according to the supplier’s instructions. Oligonucleotide primers were synthesized by Sigma-Aldrich. PCR amplified DNA fragments were assembled in a 5.2 μl final reaction volume using the modified Gibson Assembly procedure [20 (link), 23 (link), 24 (link)]. DNA assemblies were confirmed by PCR amplification and sequencing. DNA sequencing was performed by Source Bioscience. Qiaquick Gel Extraction kit (Qiagen) and Qiaprep Spin Miniprep kit (Qiagen) were used for DNA extraction and purification and plasmid isolation, respectively. BACs were isolated with Qiaquick Gel Extraction kit (Qiagen) or PhasePrep BAC DNA kit (Sigma-Aldrich), according to the manufacturer’s instructions. B. subtilis genomic DNA was obtained using the GeneJET genomic DNA purification kit (Thermo Scientific).

Juhas M., Wong C, & Ajioka J.W. (2016). Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis. PLoS ONE, 11(10), e0165778.

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ADAR1d Cysteine Residue Profiling

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hADAR1d sequence containing each Cys residue (159–201 nt) was amplified from the plasmid libraries isolated from yeast cells by two-step PCR using Phusion hot-start DNA polymerase (Thermo Fisher Scientific) to prepare Illumina Miseq sequencing samples (Primers are listed in Supplementary Table 5). The first PCR used primers containing overhang adapter sequence and amplicon-specific sequence. The PCR products were purified by agarose gel and QIAquick Gel Extraction Kit (Qiagen) and used for a second PCR with primers containing a leader sequence (P5, P7), an 8 bp barcode specific to each sample, and a sequence overlapping with the adapter sequence in the primers used in the previous PCR. Final PCR products were purified by agarose gel and QIAquick Gel Extraction Kit (Qiagen), pooled in an equal amount, and sequenced by Illumina Miseq PE250.

Park S., Doherty E.E., Xie Y., Padyana A.K., Fang F., Zhang Y., Karki A., Lebrilla C.B., Siegel J.B, & Beal P.A. (2020). High-throughput mutagenesis reveals unique structural features of human ADAR1. Nature Communications, 11, 5130.

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Genomic DNA Extraction and PCR Amplification of M. bovirhinis

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M. bovirhinis genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The primers were developed and commercially synthesized (Sigma, Rehovot, Israel) based on the nucleotide sequence of the M. bovirhinis strain HAZ141_2 genome (Accession number AP018135.1; [1 (link)]). The principal primers used in PCR experiments are listed in Table S1. PCRs were carried out in 50 μL volumes containing 50–100 ng of template DNA, 0.5 μL of Phire Hot Start II DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA), x5 Phire reaction buffer, 1 μL of 10 mM dNTPs, and 0.4 μM each primer. PCR amplifications were carried out in a C1000 Touch™ Thermal Cycler (Bio-Rad, Hercules, CA, USA). PCR amplicons were purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). Sequencing was performed at Hylab (Rehovot, Israel). PCR amplicons used for cloning were extracted from agarose gels and purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany).

Lysnyansky I, & Borovok I. (2021). The aadE*-sat4-aphA-3 Gene Cluster of Mycoplasma bovirhinis HAZ141_2 Undergoes Genomic Rearrangements Influencing the Primary Promoter Sequence. Antibiotics, 10(11), 1335.

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Validation of MCDA Assay Using PCR-Sequencing

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To further confirm the reliability and specificity of the MCDA assay, a MCDA-PCR-sequencing strategy was applied to analyze the MCDA products. The MCDA products corresponding to the level of 62.5 fg template DNA were verified by agarose gel electrophoresis, and the ladders between 100 bps and 1500 bps were extracted and purified by using QIAquick Gel Extraction Kit (QIAGEN, Hidlen, Germany) to obtain the template DNA for PCR. A set of two primers (P1 and P2) was used (Table 1), and PCR was performed in a final volume of 20 μl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.001% gelatin, 0.2 μM each of P1 and P2 primers, 0.2 mM each of dNTPs, 0.5 μl DNA template, and 0.5 units of Taq DNA polymerase (ExTaq; Takara). The program consisted of the initial denaturation of 5 min at 95 °C, 32 cycles of 30 s at 95 °C, 30 s at 58 °C and 1 min at 72 °C, plus a final 5 min extension at 72 °C. The PCR products also were verified by electrophoresis, and then extracted and purified by using QIAquick Gel Extraction Kit (QIAGEN) again to obtain the template for sequencing. Sequencing was performed by Tsingke (Beijing, China) and the sequence data were compared with the target gene sequences in GenBank database.

Wang Y., Wang Y., Ma A.J., Li D.X., Luo L.J., Liu D.X., Jin D., Liu K, & Ye C.Y. (2015). Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification. Scientific Reports, 5, 11902.

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Genotyping of Floxed Alleles in Mice

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RNA isolation from E9.5 embryos or E16.5 tissues with subsequent cDNA synthesis was performed as described above. Primers located in exon 2 and exon 7 (Ex2Ex7; see Suppl. Table 1 for primer sequences) together with either the Platinum® Taq DNA Polymerase High Fidelity or the AmpliTaq Gold® DNA Polymerase (both Invitrogen) were used to amplify the loxP-flanked region ranging from exon 4 through exon 6. The resulting PCR products were separated on an agarose gel and photographed using the Gel Doc 2000 imaging system (BioRad). PCR products from E9.5 embryos were extracted from the gel and purified applying the QIAquick Gel Extraction Kit (Qiagen), followed by sequencing using the Ex2Ex7 primers. Alternatively, PCR fragments were cloned into the pCR^™4-TOPO® TA sequencing vector using the TOPO TA Cloning® Kit (Invitrogen) and sequenced with the included M13 primer.
Yolk sac DNA from E9.5 embryos was amplified using Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) with the primer pairs Intron3F2 and Intron6R3, or MSF5-6 F (located in intron 4) and MSF5-6 R (located in intron 6) (Suppl. Table 1). The resulting PCR product was loaded on an agarose gel, purified using QIAquick Gel Extraction Kit (Qiagen) and sent for sequencing (Eurofins Genomics).

Hoelzl M.A., Heby-Henricson K., Gerling M., Dias J.M., Kuiper R.V., Trünkle C., Bergström Å., Ericson J., Toftgård R, & Teglund S. (2017). Differential requirement of SUFU in tissue development discovered in a hypomorphic mouse model. Developmental biology, 429(1), 132-146.

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Genetic Analysis of FEVR Genes

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Peripheral blood was collected in EDTA tubes from patients and parents with FEVR and normal control subjects and was preserved in -20 ˚C prior to use. Genomic DNA was isolated using the salt precipitation method, including lysis, precipitation, wash and resuspension. Genomic DNA was isolated using the salt precipitation method. PCR primers were designed to include flanking intronic sequences of each exon of the four genes (FZD4, LRP5, TSPAN12, and NDP) known to be responsible for FEVR (Supplementary Table). The exons of the four genes were analyzed via the direct sequencing of PCR products. Amplified products were purified using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA) and sequenced with forward and reverse primers by the BigDye® Terminator v3.1 Cycle Sequencing Kit (ABI Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Amplified products were purified using the QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA) and sequenced with forward and reverse primers by the BigDye® Terminator v3.1 Cycle Sequencing Kit (ABI Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions (After the template and the primers were set, prepare the BigDye premix reactions for 96-Well plate. Then perform the cycle sequencing on the system, purify the extension products and analyze the samples on a sequencer).

Fei P., Zhang Q., Huang L., Xu Y., Zhu X., Tai Z., Gong B., Ma S., Yao Q., Li J., Zhao P, & Yang Z. (2014). Identification of two novel LRP5 mutations in families with familial exudative vitreoretinopathy. Molecular Vision, 20, 395-409.

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