Qiaquick purification kit

Manufactured by Qiagen

Sourced in Germany, United States, Netherlands, United Kingdom, China

The QIAquick purification kit is a commercial product offered by Qiagen for the purification of DNA fragments. The kit utilizes spin columns and buffers to efficiently extract and concentrate DNA samples from various sources.

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QIAquick PCR Purification Kit (4582 citations) QIAquick kit (80 citations) QIAquick gel purification kit (61 citations) QIAquick 96 PCR Purification Kit (30 citations) QIAquick MinElute PCR Purification kit (15 citations) QIAquick Spin PCR Purification Kit (14 citations) QIAquick DNA purification kit (12 citations) QIAquick polymerase chain reaction (PCR) purification kit (11 citations) QIAquick PCR Purification Kit (250) (10 citations) QIAquick 96-well PCR Purification Kit (9 citations)

136 protocols using qiaquick purification kit

Chromatin Immunoprecipitation and DNA Methylation

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HCC827 and A549 cells were fixed with 1% formaldehyde. Chromatin was prepared by sonication of cell lysate and preclearing with protein A beads. Aliquots of precleared chromatin solution, named IP fractions, were incubated with 2 μg of specific antibody or preimmune rabbit IgG on a rotation platform at 4 °C overnight. One percent of the IP fraction served as the ChIP input control. The antibody-enriched protein-DNA complexes were precipitated with protein A beads from the IP fractions. DNA fragments were released by reverse crosslinking and purified by using a QIAquick purification kit (QIAGEN, Valencia CA, USA). Immunoprecipitated DNA fractions were analyzed by qPCR.
Genomic DNA was extracted from HCC827 and A549 cells and prepared by sonication. The sonicated DNA was then immunoprecipitated with a monoclonal antibody against 5-methylcytidine (5mC) at 4 °C overnight. A portion of the sonicated DNA was left untreated to serve as an input control. DNA fragments were released by reverse crosslinking and purified by using a QIAquick purification kit (QIAGEN, Valencia CA, USA). Immunoprecipitated DNA fractions were analyzed by qPCR.

Lin S., Ruan H., Qin L., Zhao C., Gu M., Wang Z., Liu B., Wang H, & Wang J. (2023). Acquired resistance to EGFR-TKIs in NSCLC mediates epigenetic downregulation of MUC17 by facilitating NF-κB activity via UHRF1/DNMT1 complex. International Journal of Biological Sciences, 19(3), 832-851.

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Plasmid Isolation and Protein Purification

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Standard methods were used for restriction enzyme digestion and molecular recombination. E. coli cell transformation was done as previously described (Sambrook et al., 1989 ). Plasmids were isolated using the QIAprep^® Spin Miniprep Kit (Qiagen, Valencia, CA, United States), and PCR products were purified using QIAquick^® purification kits (Qiagen). Proteins were expressed with a His_6X-tag in the strain E. coli BL21 (DE3) and purified as previously described (Loto et al., 2017 (link)).

Merli M.L., Padgett-Pagliai K.A., Cuaycal A.E., Garcia L., Marano M.R., Lorca G.L, & Gonzalez C.F. (2021). ‘Candidatus Liberibacter asiaticus’ Multimeric LotP Mediates Citrus sinensis Defense Response Activation. Frontiers in Microbiology, 12, 661547.

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Site-Directed Mutagenesis of NinaB

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Mutagenesis of NinaB was performed using the QuikChange XL site-directed mutagenesis kit (Agilent). Mutants were verified by sequence analysis of DNA maxipreps and validated plasmids purified by QIAquick purification kits (Qiagen). The primers used were F106L: 5'-GTTGTCACAGAGTTAGGTACGCGGGCCGTG-3' and 5'-CACGGCCCGCGTACCTAACTCTGTGACAAC-3'; T151S: 5'-GGAGATGAAATTTATGCTTTCTCTGAAGGTCCTG-3' and 5'-CAGGACCTTCAGAGAAAGCATAAATTTCATCTCC-3'; F309L: 5'-CGGAGCCGCTCTTGTACTTGCACATC-3' and 5'-GATGTGCAAGTACAAGAGCGGCTCCG-3'; Y330L: 5'-GACCTCTGCGCTTTAAAGGACGCCAAGGC-3' and 5'-GCCTTGGCGTCCTTTAAAGCGCAGAGGTC-3'.

Babino D., Golczak M., Kiser P.D., Wyss A., Palczewski K, & von Lintig J. (2016). The biochemical basis of vitamin A3 production in arthropod vision. ACS chemical biology, 11(4), 1049-1057.

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Molecular Techniques for Liberibacter DNA

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Standard methods were used for chromosomal DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation and transformation (Sambrook et al., 1989). Plasmids were isolated using QIAprep^® Spin Miniprep Kit (Qiagen, Valencia, CA, USA), and PCR products were purified using QIAquick^® purification kits (Qiagen). The primers utilized are described in Table 4. Liberibacter asiaticus DNA was isolated from leaf tissues of infected plants using the Isolate II Plant DNA kit (Bioline, Taunton, Massachusetts, USA).

Loto F., Coyle J.F., Padgett K.A., Pagliai F.A., Gardner C.L., Lorca G.L, & Gonzalez C.F. (2017). Functional characterization of LotP from Liberibacter asiaticus. Microbial Biotechnology, 10(3), 642-656.

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Site-Directed Mutagenesis of citO Gene

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Standard methods were used for chromosomal DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transformation (Sambrook and Russell, 2001 ). Plasmids were isolated using spin miniprep kits (Qiagen, Valencia, CA) and PCR products were purified using QIAquick purification kits (Qiagen). All the primers used for this study are described in Table 2. The plasmids for protein purification, protein expression in E. faecalis, and transcriptional fusions are previously described in Blancato et al. (2008 (link)).
Site-directed mutagenesis was performed using the QuikChange Site-directed Mutagenesis kit (Stratagene) according to the manufacturer's protocol. Plasmids pET-citO or pCitO were used as the templates. All the selected amino acids were changed to alanine. The mutations were verified by DNA sequencing using T7 universal primers.

Blancato V.S., Pagliai F.A., Magni C., Gonzalez C.F, & Lorca G.L. (2016). Functional Analysis of the Citrate Activator CitO from Enterococcus faecalis Implicates a Divalent Metal in Ligand Binding. Frontiers in Microbiology, 7, 101.

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Labeling and Hybridizing DNA Microarray

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DNA was labeled with Dye Cy-5 by a random priming method as previously described (Yang et al., 2017 (link)) and the labeled DNA was purified with QIA quick purification kits (Qiagen, Valencia, CA, United States). SpeedVac (ThermoSavant, Milford, MA, United States) was used to dry DNA at 45°C for 45 min. We hybridized DNA with GeoChip 4.0 on a MAUI hybridization station (BioMicro, Salt Lake City, UT, United States) at 42°C for 16 h, as described previously (Yue et al., 2015 (link)). The slides were scanned using a NimbleGen MS200 scanner (Roche, Madison, WI, United States) at 633 nm using 100% laser power and 75% photomultiplier tube gain. Signal intensities were quantified by scanned images.

Qi Q., Zhao M., Wang S., Ma X., Wang Y., Gao Y., Lin Q., Li X., Gu B., Li G., Zhou J, & Yang Y. (2017). The Biogeographic Pattern of Microbial Functional Genes along an Altitudinal Gradient of the Tibetan Pasture. Frontiers in Microbiology, 8, 976.

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Yeast DNA Extraction and ITS Sequencing

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Genomic DNA was extracted from purified yeast cells using the method described by Looke et al. [12 (link)] with some modifications. The universal primers, ITS1 (5′-TCCGTAGGTGAACCTGCG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), were used to amplify the ITS regions of the selected species [13 (link)]. PCR amplification was conducted in a total reaction mixture volume of 25 mL using 1x PCR buffer (DreamTaq™) in a C1000TM Thermo Cycler (Bio-Rad, Germany). PCR products were purified using QIAquick purification kits (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. The DNA amplicons were sequenced using the Gene Analyzer 3121 sequencer with the same primers ITS1 and ITS4 (Macrogen Co., Seoul, South Korea). The ITS sequences were analyzed using BioEdit version 7.2.5. A total of 32 isolates, including 18 C. albicans, 11 non-Candida species, and 3 Saccharomyces cerevisiae, were examined. Isolates were identified by comparing the sequencing data against databases using the BLAST of the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Table 1 shows the GenBank accession numbers of the ITS1 and ITS4 regions of type (or reference) isolates of the 32 species sequenced.

Al Halteet S., Abdel-Hadi A., Hassan M, & Awad M. (2020). Prevalence and Antifungal Susceptibility Profile of Clinically Relevant Candida Species in Postmenopausal Women with Diabetes. BioMed Research International, 2020, 7042490.

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Genomic DNA Extraction and Purification

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Standard procedures were used for molecular manipulations⁴⁶. Genomic DNA from L. delbrueckii subsp. lactis CRL 581 was extracted as detailed previously¹⁶. Restriction enzymes, T4 DNA ligase, and DNA polymerases were purchased from Promega (Buenos Aires, Argentina). PCR amplifications were carried out using GoTaq DNA polymerase (Promega). Plasmids were isolated using spin miniprep kits (Qiagen) and PCR products were purified using Qiaquick purification kits (Qiagen).

Brown L., Villegas J.M., Elean M., Fadda S., Mozzi F., Saavedra L, & Hebert E.M. (2017). YebC, a putative transcriptional factor involved in the regulation of the proteolytic system of Lactobacillus. Scientific Reports, 7, 8579.

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Molecular Cloning of ldtR Gene

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Standard methods were used for chromosomal DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, ligation, and transformation [28] . Plasmids were isolated using QIAprep Spin Miniprep Kit (Qiagen, Valencia, CA), and PCR products were purified using Qiaquick purification kits (Qiagen).
For protein expression and purification, ldtR gene was amplified from ‘Ca. L. asiaticus’ str. psy62 or S. meliloti 1021 chromosomal DNA via PCR, and then cloned into the p15TV-L plasmid as described previously [10] (link).

Pagliai F.A., Gardner C.L., Bojilova L., Sarnegrim A., Tamayo C., Potts A.H., Teplitski M., Folimonova S.Y., Gonzalez C.F, & Lorca G.L. (2014). The Transcriptional Activator LdtR from ‘Candidatus Liberibacter asiaticus’ Mediates Osmotic Stress Tolerance. PLoS Pathogens, 10(4), e1004101.

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Plastome Sequencing of Two Tropical Tree Species

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Distemonanthus benthamianus is a large, deciduous, wind-dispersed, light-demanding pioneer tree distributed from UG to LG. Anthonotha macrophylla is an evergreen shrub to medium-sized tree, rarely lianescent, non-pioneer, occurring in lower strata of tropical rain forest and in secondary forest, distributed from UG to Congolia (the Eastern part of Central African forest), whose seeds are consumed but possibly also dispersed by monkeys. We sequenced the plastome of 47 georeferenced individuals of each species sampled across their distribution ranges. The samples were composed of field-collected leaves dried in silica gel (18 individuals of A. macrophylla and 47 of D. benthamianus) and from herbarium specimens (29 individuals of A. macrophylla) collected at the National Herbarium of the Netherlands, Naturalis (WAG) and the Herbarium of the Université Libre de Bruxelles (BRLU) (Table S1 in Appendix S1). Total genomic DNA was extracted for each sample (c. 10 µg) from 20 to 30 mg of dried leaves, using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987) . Extracted DNA was purified with QIAquick purification kits (Qiagen, Venlo, Netherlands) followed by Qubit ® 2.0 Fluorometer DNA quantification (Life Technologies, Invitrogen, Foster City, USA) and by QIAxcel (Qiagen) DNA quality control.

Demenou B.B., Migliore J., Heuertz M., Monthe F.K., Ojeda D.I., Wieringa J.J., Dauby G., Albreht L., Boom A, & Hardy O.J. (2020). Plastome phylogeography in two African rain forest legume trees reveals that Dahomey Gap populations originate from the Cameroon volcanic line. Molecular phylogenetics and evolution, 150.

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