Pqe 80l

Manufactured by Qiagen

Sourced in Germany

The PQE-80L is a laboratory equipment designed for DNA extraction and purification. It is a fully automated system that can process up to 80 samples simultaneously. The PQE-80L utilizes magnetic bead-based technology to efficiently extract and purify DNA from a variety of sample types, including blood, tissue, and cells. The system is capable of delivering high-quality DNA suitable for downstream applications such as PCR, sequencing, and genomic analysis.

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Lab products found in correlation

Superdex 200 10 300 gl column, by GE Healthcare (6 mentions) Talon resin, by Takara Bio (6 mentions) Ni nta agarose, by Qiagen (3 mentions) Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions) Pfn18a, by Promega (2 mentions) Carbenicillin, by Nacalai Tesque (2 mentions) T4 dna ligase, by Thermo Fisher Scientific (2 mentions) Kapa taq readymix kit, by Roche (2 mentions) Codon optimization tool, by Integrated DNA Technologies (1 mentions) E coli bl21 codonplus de3 ripl, by Agilent Technologies (1 mentions) F proab laciqzδm15 tn10 tetr, by Agilent Technologies (1 mentions) Amicon ultra, by Merck Group (1 mentions) Pjet1.2 blunt, by Thermo Fisher Scientific (1 mentions) Sanger sequencing, by Genomed (1 mentions) Maestro imaging system, by PerkinElmer (1 mentions) Precision plus protein dual color standard, by Bio-Rad (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Anti penta his hrp conjugate, by Qiagen (1 mentions) Primestar hs dna polymerase, by Takara Bio (1 mentions) Dpni, by New England Biolabs (1 mentions)

Close spelling variants of this product

PQE-80L expression vector (4 citations)

51 protocols using pqe 80l

Constructing Plasmid with Kanamycin Resistance

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Example 1

For construction of plasmid pQE-80L-Kana, Kanamycin resistance gene was cleaved from pET-24a(+) (Novagen) by BspHI (BioLab) to generate a 875-bp Kanamycin resistance gene (+3886 to +4760) fragment (SEQ ID NO: 1). The pQE-80L (Qiagen) vector was digested with BspHI to remove Ampicillin resistance gene (+3587 to +4699) fragment (SEQ ID NO:2) and then the Kanamycin resistance gene fragment was ligated into the pQE-80L vector to generate a 4513-bp plasmid (pQE-80L-Kana). (SEQ ID NO:3)

US10836807B2. Recombinant polypeptides and nucleic acid molecules, compositions, and methods of making and uses thereof (2020-11-17). BioGend Therapeutics Co., Ltd. [TW]. Inventors: Da-Wei Sun [TW].

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Cloning of the lacIq gene

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The lacI^q gene was extracted by McsI digestion from the expression vector pQE-80L (Quiagen). The 1610 bp fragment was cloned into the pBBR1MCS5 [31 (link)] previously digested with SmaI. The resulting plasmid pBBRlacI^q was introduced into Sme Tn7pleD*Km strain using E. coli β2163 donor strain as described in [26 (link)].

Romero-Jiménez L., Rodríguez-Carbonell D., Gallegos M.T., Sanjuán J, & Pérez-Mendoza D. (2015). Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria. BMC Microbiology, 15, 190.

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Optimized Recombinant Protein Expression

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Amino acid and DNA sequences chosen for experimental analysis were edited before cloning. Signal peptide was detected using the Phobius Tool [50 (link)] and removed. The coding sequences were optimized for E. coli expression by using the Integrated DNA Technologies (IDT) Codon Optimization Tool (www.idtdna.com). Native restriction sites were eliminated and SacI and SalI restriction sites were added in 5′ and 3′, respectively, to facilitate the cloning in vector pQE-80L (Quiagen).

Talens-Perales D., Sánchez-Torres P., Marín-Navarro J, & Polaina J. (2020). In silico screening and experimental analysis of family GH11 xylanases for applications under conditions of alkaline pH and high temperature. Biotechnology for Biofuels, 13, 198.

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Cloning and Purifying SdeC Protein

Cited 1 time

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Full-length sdeC genes were amplified by PCR and cloned into the bacterial expression vector pQE-80L (Qiagen) with BamHI and SacI restriction sites to generate a 6xHis-SdeC-StrepII fusion construct (Supplemental Table S1). Purification was as previously described^{22 (link)}. Purified aliquots were stored at −80°C in 10% glycerol to minimize or eliminate freeze thaw cycle degradation.

Kotewicz K.M., Zhang M., Kim S., Martin M.S., Chowdhury A.R., Tai A., Scheck R.A, & Isberg R.R. (2024). Sde Proteins Coordinate Ubiquitin Utilization and Phosphoribosylation to Promote Establishment and Maintenance of the Legionella Replication Vacuole. bioRxiv.

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Recombinant Multi-Domain Protein Purification

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The (Azu-I27)4, (AzuC112A-I27)₄, (AzuC26A-I27)₄ and (Plasto-I27)₄ polyproteins were subcloned using the BamHI, BglII and KpnI restriction sites⁵⁹. Synthetic gene for plastocyanin was produced by Life Technologies (Paisley, UK). The multidomain proteins were cloned into the pQE80L (Qiagen) expression vector, and transformed into the BLR(DE3) Escherichia coli expression strain. All mutations were introduced by using the QuikChange Site-Directed Mutagenesis kit (Agilent). The cells were grown in LB broth supplemented with 100 μg/mL ampicillin at 37°C. After reaching an OD₆₀₀ of ~0.6 the cultures were induced with Isopropyl β-D-1-thiogalactopyranoside (1 mM) and incubated overnight at 20°C (constructs containing Azurin) or 16°C (Plastocyanin construct). For some constructs, CuSO₄ (0.5 mM) was supplemented to the cultures right before induction to increase copper intake. After harvesting the cells, disruption using a French Press was performed. The polyproteins from the lysate were purified by metal affinity chromatography on Talon resin (Clontech) followed by gel-filtration using a Superdex 200 10/300 GL column (GE Biosciences). Protein concentration in the samples was estimated using the Bradford protein assay.
Details on the biochemistry characterization assays regarding circular dichroism, Ellman’s assays and ICP-OES measurements are detailed in the Supplementary Methods Section.

Beedle A.E., Lezamiz A., Stirnemann G, & Garcia-Manyes S. (2015). The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins. Nature Communications, 6, 7894.

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Multimeric Protein Constructs with Disulfide I27

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All multimeric constructs were created from multiple rounds of digestion and ligation using BamHI/BglII restriction sites as described previously(Wiita et al., 2006 (link)). The sequence of the disulfide containing I27 domain has been reported previously: two cysteine residues were introduced via QuickChange at positions G32 and A75, while the native cysteine residues at positions C47 and C63 were mutated to alanines (Ainavarapu et al., 2007 (link)). The N-terminal SpyCatcher and C-terminal Avi expression construct was designed in the pQE-80L (QIAGEN) plasmid for these experiments. The N-terminal HaloTag and C-terminal SpyTag was designed in the pFN18a plasmid (Promega).

Eckels E.C., Haldar S., Tapia-Rojo R., Rivas-Pardo J.A, & Fernández J.M. (2019). The Mechanical Power of Titin Folding. Cell reports, 27(6), 1836-1847.e4.

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Recombinant HSP Protein Purification

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Full-length HSP1 (CmsHSP1) and HSP22F (CreHSP22F) genes were amplified and cloned in pQE-80 L (Qiagen, Venlo, The Netherlands). The vectors were transformed into Escherichia coli BL21 and selected on Luria–Bertani (LB) agar medium containing 50 µg/ml Carbenicillin (Nacalai Tesque, Kyoto, Japan). The cultures were grown in LB medium at 37 °C for several hours and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at OD 600 0.7–1 to a final concentration of 1 mM. Proteins were purified using Ni-NTA agarose (Qiagen), following the manufacturer’s instructions. The isolated CmsHSP1 and CreHSP22F fusion proteins were injected into mice.

Kobayashi Y., Harada N., Nishimura Y., Saito T., Nakamura M., Fujiwara T., Kuroiwa T, & Misumi O. (2014). Algae Sense Exact Temperatures: Small Heat Shock Proteins Are Expressed at the Survival Threshold Temperature in Cyanidioschyzon merolae and Chlamydomonas reinhardtii. Genome Biology and Evolution, 6(10), 2731-2740.

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Generating Bacterial Strain Variants

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All strains, plasmids and primers used in this study are listed in Tables S5, S6 and S7, respectively. All plasmids used for in vivo experiments are derivatives of pJN105 (Newman & Fuqua, 1999 (link)). Amino acid substitutions in the DPBB domain of RlpA were generated using a multistep PCR procedure involving megapriming (Kwok et al., 1994 (link)). Plasmids for overproducing hexahistidine (His₆-) tagged RlpA variants are derivatives of pQE-80L (Qiagen). All strains used for in vivo experiments are derivatives of P. aeruginosa strain UCBPP- PA14. All deletion constructs were generated using plasmid pEXG2 as previously described (Rietsch et al., 2005 (link)). pEXG2 derivatives were transferred from derivatives of E. coli strain SM10 to wild type PA14 by conjugation as described (Schweizer, 1992 (link)) except that Irg was used to counter select against the E. coli donor strain because a ΔrlpA mutant is not viable on the (low osmolarity) minimal-citrate media often used to counter select E. coli after such matings. Resolution of the co-integrant was selected for on LB0N plates containing 5% sucrose (~150 mM, which allows for growth of the ΔrlpA mutant in the absence of NaCl).

Jorgenson M.A., Chen Y., Yahashiri A., Popham D.L, & Weiss D.S. (2014). The Bacterial Septal Ring Protein RlpA is a Lytic Transglycosylase that Contributes to Rod Shape and Daughter Cell Separation in Pseudomonas aeruginosa. Molecular microbiology, 93(1), 113-128.

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Protein Expression in E. coli

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Escherichia coli SURE 2 Supercompetent Cells [e14(McrA-) Δ(mcrCB-hsdSMR-mrr) 171 endA1 gyrA96 thi-1 supE44 relA1 lac recB recJ sbsC umuC::Tn5 (Kanr) uvrC [F’ proAB lacIqZΔM15 Tn10 (Tetr)]] (Agilent Technologies, Santa Clara, CA, USA) was used as a host strain in order to verify the gene sequences. For protein expression, recombinant pQE-80L (Qiagen, Hilden, Deutschland) vectors were transformed into E. coli BL21-CodonPlus(DE3)-RIPL [E. coli B F^–ompT hsdS(rB^– mB^–) dcm⁺ Tet^r gal λ(DE3) endA Hte [argU proL Camr] [argU ileY leuW Strep/Specr]] (Agilent Technologies). Bacteria were cultivated at 37 °C and 18 °C on Luria-Bertani solid medium (0.1% peptone, 0.05% yeast extract, 0.1% sodium chloride, 0.1% agar) or Luria-Bertani liquid medium (0.1% peptone, 0.05% yeast extract, 0.1% sodium chloride).

Martinelli L., Redou V., Cochereau B., Delage L., Hymery N., Poirier E., Le Meur C., Le Foch G., Cladiere L., Mehiri M., Demont-Caulet N, & Meslet-Cladiere L. (2020). Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast, Naganishia uzbekistanensis. Marine Drugs, 18(12), 637.

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Codon-Optimized Recombinant Human TRAIL and DR5 Expression

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The human TRAIL cDNA fragment (coding amino acids 94–281) was codon optimized, synthesized by IDT (Integrated DNA Technologies, Inc. Coralville, Iowa) and inserted into a bacterial expression vector, pQE80L (Qiagen) to generate a His-tagged human recombinant TRAIL, which was purified per a protocol published previously (21 (link)). The human DR5 cDNA fragment (coding amino acids from 1 to 334) was codon optimized and synthesized by IDT, fused with EGFP at the C-terminus, and cloned into the pBabe-puro retroviral vector. The ORF of galectin-3 was codon optimized and synthesized by IDT and cloned into thepLVEF-1a lentivirus expression vector (kindly provided by Dr. Guihua Sun, Diabetes & Metabolism Research Institute at City of Hope) fused with mCherry at the N-terminus. For retrovirus production, the pBabe/DR5-EGFP plasmid was transfected into Phoenix cells for packaging. Virus containing supernatant was harvested 48–72 h post-transfection for target cell infection in the presence of polybrene at 10 μg/ml. The lentivirus vector pLVEF-1a-mCherry-galection-3 was co-transfected into 293T cells with packaging plasmids for lentivirus production. Virus containing supernatant was harvested 48–72 h post-transfection and purified for target cell infection in the presence of polybrene at 10 μg/ml.

Liang R., Yao Y., Wang G., Yue E., Yang G., Qi X., Wang Y., Zhao L., Zheng T., Zhang Y, & Wenge Wang E. (2020). Repositioning Quinacrine Toward Treatment of Ovarian Cancer by Rational Combination With TRAIL. Frontiers in Oncology, 10, 1118.

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