Quikchange mutagenesis protocol

Manufactured by Agilent Technologies

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The QuikChange mutagenesis protocol is a rapid and efficient method for site-directed mutagenesis. It enables the introduction of specific mutations, insertions, or deletions into double-stranded plasmid DNA.

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Lab products found in correlation

Dcg 4, by Bio-Techne (1 mentions) Ly341495, by Bio-Techne (1 mentions) Snap red, by PerkinElmer (1 mentions) Ly487379, by Bio-Techne (1 mentions) Ly379268, by Bio-Techne (1 mentions) Snap lumi4 tb, by PerkinElmer (1 mentions) Hek293 cell, by American Type Culture Collection (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Mycoalert mycoplasma detection kit, by Lonza (1 mentions) Puc19 plasmid, by New England Biolabs (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Nb bbvci nicking endonuclease, by New England Biolabs (1 mentions) Affinity column, by Qiagen (1 mentions) Shrimp alkaline phosphatase, by New England Biolabs (1 mentions) Phusion polymerase, by Thermo Fisher Scientific (1 mentions) Pgex 6p vector, by GE Healthcare (1 mentions)

Spelling variants of this product

QuikChange (236 citations) QuikChange mutagenesis (94 citations) QuikChange protocol (50 citations) QuikChange site-directed mutagenesis protocol (26 citations) QuikChange II site-directed mutagenesis protocol (7 citations) QuikChange II XL site-directed mutagenesis protocol (3 citations) QuikChange site-directed mutagenesis kit protocol (2 citations) Quikchange II mutagenesis protocol (2 citations)

14 protocols using quikchange mutagenesis protocol

Cloning and Expression of PARL and MPP Skd3

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Codon-optimized DNA fragments encoding _PARLSkd3 and _MPPSkd3 (Twist Biosciences) were cloned into the pET28-His₆-SUMO plasmid behind the SUMO moiety using Gibson assembly. To increase the efficiency of SUMO protease cleavage, a GGS linker sequence was introduced at the N terminus of _MPPSkd3 coding sequence. Site-directed mutagenesis of _PARLSkd3 and _MPPSkd3 was carried out using the QuikChange mutagenesis protocol (Agilent). To express _PARLSkd3_ARD, a stop codon was introduced after R327 using the QuikChange mutagenesis protocol.
The coding sequence for Photinus pyralis luciferase was cloned between the Nco I and Xho I sites in pET28 to generate pET28-Luciferase-His₆. pHis₆-SUMO-Ssa1 and pUlp1-His₆ for expression of Ssa1 and Ulp1 (SUMO protease), respectively, were gifts from E. Deuerling. Expression plasmids for Ydj1 and Sis1 were described in (51 (link)). pTrc-GroEL-D87K was a gift from E. Chapman. pPROEX-Htb-Hsp104 (#1230), pSG25-β-Gal (#63867), and pET-Sac-Abeta(M1-42) (#71875) were from Addgene.

Gupta A., Lentzsch A.M., Siegel A., Yu Z., Chio U.S., Cheng Y, & Shan S.O. (2023). Dodecamer assembly of a metazoan AAA+ chaperone couples substrate extraction to refolding. Science Advances, 9(19), eadf5336.

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Site-Directed Mutagenesis of BFDC Enzyme

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Mutagenesis was performed
on BFDC expression
vector pET24dBFDC-His.^{31 (link),32 (link)} Primers (Table 1 of the Supporting Information) were designed, and site-directed
mutagenesis was performed according to the QuikChange mutagenesis
protocol (Agilent) utilizing Pfu DNA polymerase.
DpnI was used to digest parental DNA prior to its transformation into
chemically competent TOP10 cells. Each plasmid used for protein expression
had the complete mdlC gene sequenced to ensure that
only the desired mutations were present.

Andrews F.H., Rogers M.P., Paul L.N, & McLeish M.J. (2014). Perturbation of the Monomer–Monomer Interfaces of the Benzoylformate Decarboxylase Tetramer. Biochemistry, 53(27), 4358-4367.

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GABA Receptor Subunits Expression

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The pRK5 plasmids encodes either the wild-type rat GB1a, tagged with HA and SNAP inserted just after the signal or the wild-type rat GB2 tagged with Flag and Halo inserted just after the signal peptide (Supplementary Fig. 12). GB1^Ctr was obtained from rat GB1a wild-type sequence by deleting the last 32 amino acids encoding for GB1. GB2^Ctr was obtained from rat GB2 wild-type sequence by adding a GFP-tag at the C-terminal end of GB2. The cysteine substitutions were generated by site-directed mutagenesis using the QuikChange mutagenesis protocol (Agilent Technologies) using the primers described in Supplementary Fig. 13 and Supplementary Fig. 14 for the GB1 and GB2 mutants, respectively.
HEK293 cells (ATCC, CRL-1573) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and transfected by electroporation. Unless stated otherwise, 10⁷ cells were transfected with plasmid DNA containing the coding sequence of the receptor subunits, and completed to a total amount of 10 μg of plasmid DNA with the empty vector pRK5. For the determination of intracellular calcium measurements and inositol phosphate (IP) accumulation, the cells were also transfected with the chimeric G-protein Gq_i9, which allows the coupling of the recombinant GABA_B receptor to the phospholipase C^{52 (link)}.

Xue L., Sun Q., Zhao H., Rovira X., Gai S., He Q., Pin J.P., Liu J, & Rondard P. (2019). Rearrangement of the transmembrane domain interfaces associated with the activation of a GPCR hetero-oligomer. Nature Communications, 10, 2765.

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GPCR Expression and Characterization

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HEK293 cells (ATCC, CRL-1573, lot: 3449904) were cultivated in DMEM (Thermo Fischer Scientific, Courtaboeuf, France) complemented with 10% (v/v) fetal bovine serum. Absence of mycoplasma was routinely checked using the MycoAlert Mycoplasma detection kit (LT07-318, Lonza, Amboise, France), according to the manufacturer protocol. All drugs (DCG-IV, LY341495, LY379268, and LY487379) were from Tocris Bioscience (Bristol, UK). LSP4-2022 was provided by Dr. F. Acher (Paris, France). All HTRF reagents, labeled monoclonal antibodies anti-c-Myc-d2 (61MYCDAA, Cisbio Bioassays, Codolet, France), and anti-6His-d2 (61HISDLA, Cisbio Bioassays), labeled ligands (SNAP-Lumi4-Tb, SNAP-Red, CLIP-Red, and BG-Alexa Fluor 488) were a kind gift from Cisbio Bioassays. The pRK5 plasmids encoding wild-type rat mGluR subunits, with a HA-tag and with SNAP or CLIP inserted just after the signal peptide, were previously described^{46 (link)}. pEGFP-C2 plasmid encoding EGFP was from Clontech (Mountain View, CA, USA). Point mutations were introduced in the SNAP-tag mGlu2 or mGlu3 plasmids according to the QuikChange mutagenesis protocol (Agilent Technologies, Santa Clara, CA, USA).

Scholler P., Nevoltris D., de Bundel D., Bossi S., Moreno-Delgado D., Rovira X., Møller T.C., El Moustaine D., Mathieu M., Blanc E., McLean H., Dupuis E., Mathis G., Trinquet E., Daniel H., Valjent E., Baty D., Chames P., Rondard P, & Pin J.P. (2017). Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation. Nature Communications, 8, 1967.

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Constructs for Tsa1 Protein Expression

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Constructs for expression of wt Tsa1 in yeast (p416-GPD) and bacteria (pET45b) have been reported previously.^{32 (link)} For yeast experiments, genes encoding wild-type Tsa1 or Tsa1 decamer interface variants (all with an N-terminal FLAG tag) were placed under the control of the GPD promoter, which allows for constitutive, high-level expression in yeast.^{31 (link),32 (link)} For bacterial over-expression, the Tsa1 gene was tagged at the N-terminus with a vector-encoded 6X-His tag. Codons for aromatic residues at the decamer interface in Tsa1 were mutated to Ala or Leu using the QuikChange mutagenesis protocol (Agilent) with the primers reported in Table S1 in the Supporting Information, as described previously.^{31 (link),32 (link)} All constructs containing mutations were verified by DNA sequencing.

Loberg M.A., Hurtig J.E., Graff A.H., Allan K.M., Buchan J.A., Spencer M.K., Kelly J.E., Clodfelter J.E., Morano K.A., Lowther W.T, & West J.D. (2019). Aromatic Residues at the Dimer–Dimer Interface in the Peroxiredoxin Tsa1 Facilitate Decamer Formation and Biological Function. Chemical research in toxicology, 32(3), 474-483.

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Bioluminescent Receptor Protein Constructs

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The pRK5 plasmids encoding wild-type human PAFR and human β₂AR were tagged with a double tag, either Flag-SNAP or Flag-Halo, inserted immediately after the signal peptide^{57 (link)} (Supplementary Figs. 20a, 21). Mutations in the pRK5 plasmid were generated by site-directed mutagenesis using the QuikChange mutagenesis protocol (Agilent Technologies). The probes (full-length mVenus, mCherry, or Rluc) were fused to the C terminus of PAFR or β₂AR, respectively, with the AflII restriction site as a linker (Supplementary Figs. 20b, 22). Plasmids encoding human βarr1 and βarr2 tagged with Rluc have been described previously^{97 (link)}. The plasmids encoding membrane-targeted Venus (Venus-CAAX) were obtained from Addgene^{102 (link)}. The EPAC1 cDNA was obtained from Dr. Lily Jiang (University of Texas Southwestern, Dallas). cDNA for the human GRK2 and GRK3 in pcDNA3.1 was fused to Renilla luciferase (provided by ARPEGE platform, IGF).

Liu J., Tang H., Xu C., Zhou S., Zhu X., Li Y., Prézeau L., Xu T., Pin J.P., Rondard P., Ji W, & Liu J. (2022). Biased signaling due to oligomerization of the G protein-coupled platelet-activating factor receptor. Nature Communications, 13, 6365.

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Site-Directed Mutagenesis Protocol

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Site-directed mutagenesis was carried out based on either the original QuikChange mutagenesis protocol (Agilent Technologies, Santa Clara, CA, USA) or on a modified version using only partially overlapping primers [31 (link)]. The sequence of the primers used in our polymerase chain reactions (PCRs) is listed in Table 1.

Benedek A., Temesváry-Kis F., Khatanbaatar T., Leveles I., Surányi É.V., Szabó J.E., Wunderlich L, & Vértessy B.G. (2019). The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition. Biomolecules, 9(6), 221.

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Topological Alteration of DNA by GyrA Fragments

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The ability of C-terminal fragments of GyrA to introduce writhe into DNA was tested in a similar way to that previously reported [31 (link)]. In order to emulate the DNA substrate pSG483 described therein, a single recognition site of Nb.BbvCI nicking endonuclease was introduced into pUC19 plasmid (New England Biolabs) using the Quikchange^® mutagenesis protocol (Agilent). The plasmid was treated by Nb.BbvCI nicking endonuclease (New England Biolabs) to create a topologically free, relaxed DNA (S1 Fig). Proteins at various molar excesses over the nicked plasmid (300 ng, 0.08 pmol) were incubated at 37°C for 30 minutes in 30 μl reactions. The nick was sealed using 60 units of T4 DNA ligase (New England Biolabs) at 37°C for 1 hour. The protein bound to the DNA was removed by adding EDTA (10 mM final concentration), SDS (1% final concentration) and proteinase K (50 μg/ml final concentration) prior to subjecting the samples to gel electrophoresis using 1% agarose gel and 1X TAE buffer for 1 hour.

Nagano S., Seki E., Lin T.Y., Shirouzu M., Yokoyama S, & Heddle J.G. (2015). Investigating the Roles of the C-Terminal Domain of Plasmodium falciparum GyrA. PLoS ONE, 10(11), e0142313.

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RNA Preparation and Labeling for DMS Footprinting

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RNAs were prepared by in vitro transcription essentially as described [59 (link)]. The DNA template was plasmid DNA for E^ΔP5abc or a PCR product assembled from oligonucleotides for P5abc. P5abc constructs for DMS footprinting included 5′ and 3′ flanking hairpins for normalization and single-stranded regions to serve as markers or to anneal with a primer for reverse transcription and RNA isolation [42 (link)]. E^ΔP5abc mutants were generated by a Quikchange mutagenesis protocol (Agilent). Oligonucleotides for PCR assembly and mutagenesis were from IDT (San Diego, CA). DNA templates and RNA were isolated by affinity column (Qiagen). P5abc RNA was 5′-end labeled by treating it with shrimp alkaline phosphatase (New England Biolabs) followed by polynucleotide kinase and [γ-³²P]-ATP. Labeled RNA was purified by polyacrylamide gel electrophoresis, eluted into TE buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and stored at −20 °C.

Gracia B., Xue Y., Bisaria N., Herschlag D., Al-Hashimi H.M, & Russell R. (2016). RNA Structural Modules Control the Rate and Pathway of RNA Folding and Assembly. Journal of molecular biology, 428(20), 3972-3985.

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Cloning and Mutagenesis of ROS1 Construct

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The retroviral construct pCX4 ROS1 was cloned as described previously (Davare et al, 2013 (link)). Briefly, full‐length ROS1 cDNA was cloned using the SalI and XhoI sites in the multiple cloning site of pENTR4‐No ccDB (696‐1) vector (Addgene Plasmid #17424) via In‐Fusion™ cloning using PCR amplification that included the addition of C‐terminal Flag tag. Simultaneously, pCX4‐puro was converted into a Gateway™ Destination vector (pCX4‐DEST‐Puro) by inserting the Gateway™ Reading Frame Cassette A (ThermoFisher) into the multiple cloning site using EcoRI and XhoI restriction sites. ROS1‐FLAG cDNA was subcloned into pCX4‐DEST‐Puro via LR clonase reaction. ROS1 mutants were generated using site‐directed mutagenesis (QuikChange™ Mutagenesis Protocol, Agilent Technologies Inc., Santa Clara, CA, USA) of the pENTR4‐ROS1‐FLAG construct and subsequently subcloned into pCX4‐DEST‐Puro.

Iyer S.R., Nusser K., Jones K., Shinde P., Keddy C., Beach C.Z., Aguero E., Force J., Shinde U, & Davare M.A. (2023). Discovery of oncogenic ROS1 missense mutations with sensitivity to tyrosine kinase inhibitors. EMBO Molecular Medicine, 15(10), e17367.

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