Quick change site directed mutagenesis kit

Manufactured by Qiagen

Sourced in United States, Germany

The Quick Change Site-Directed Mutagenesis Kit is a laboratory tool used to introduce specific mutations into double-stranded plasmid or viral DNA. The kit provides a simple and efficient method to create desired changes in the DNA sequence.

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Lab products found in correlation

In fusion advantage pcr cloning kit, by Takara Bio (1 mentions) Ngc medium pressure chromatography system, by Bio-Rad (1 mentions) Pfuultra hf, by Agilent Technologies (1 mentions)

7 protocols using quick change site directed mutagenesis kit

Engineered Cytochrome P450 Mutants

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The CYP102 (also called P450 BM3
or CYP102(A1)) C62A/C156S/K97C triple mutant has been reported.^{29 (link)} The His-containing mutant C62A/C156S/K97C/W96H
and CYP119 mutants S81C/H86 and S81C/H76W were prepared by the same
procedure. The plasmid for CYP119 was obtained from Prof. Paul Ortiz
de Montellano (UCSF). All mutations were made using a QuickChange
Site-Directed Mutagenesis kit (Qiagen). All primers were obtained
from Operon. The proteins were expressed with an N-terminal His₆ tag in Escherichia coli BL21(DE3)
cells as described previously.^{29 (link)} All proteins
were purified according to a literature procedure^{29 (link)} and characterized by ESI mass spectrometry.

Pospíšil P., Luxem K.E., Ener M., Sýkora J., Kocábová J., Gray H.B., Vlček A J.r, & Hof M. (2014). Fluorescence Quenching of (Dimethylamino)naphthalene Dyes Badan and Prodan by Tryptophan in Cytochromes P450 and Micelles. The Journal of Physical Chemistry. B, 118(34), 10085-10091.

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RANKL 3'UTR Luciferase Assay

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RANKL 3′-UTR constructs were PCR amplified using cDNA encoding RANKL (NM_033012.3) as templates, and subcloned into pGL3-Basic vector using In-Fusion™ advantage PCR cloning Kit (Clontech, USA) for Luciferase reporter gene assay. The construction of the RANKL 3′-UTR mutant was done by PCR using the Quickchange™ site directed mutagenesis kit from Qiagen (Qiagen, USA).

Wang T., Yin H., Wang J., Li Z., Wei H., Liu Z., Wu Z., Yan W., Liu T., Song D., Yang X., Huang Q., Zhou W, & Xiao J. (2015). MicroRNA-106b inhibits osteoclastogenesis and osteolysis by targeting RANKL in giant cell tumor of bone. Oncotarget, 6(22), 18980-18996.

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Identifying CjPhoX Disulfide Bonds

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To identify the cysteine residues of CjPhoX linked by disulfide bonds, a set of recombinant plasmids was constructed from pMA1 carrying the cjphoX gene with its own promoter. The Cys-to-Ala point mutations were generated using the Quick Change Site-Directed Mutagenesis Kit (Qiagen) according to the manufacturer's instructions, starting with 100 ng of pMA1 template and 125 ng of each primer (primer pairs: C1Afor – C1Arev, C2Afor – C2Arev, C3Afor – C3Arev, C4Afor – C4Arev, C5Afor – C5Arev). The resulting plasmids were designated pAG101-pAG105, respectively and their correct construction was confirmed by sequencing. Subsequently the plasmids pAG101-pAG105 were introduced into C. jejuni 81116 lacking cjphoX, and the resulting strains were used for the PhoX activity assays.

Grabowska A.D., Wywiał E., Dunin-Horkawicz S., Łasica A.M., Wösten M.M., Nagy-Staroń A., Godlewska R., Bocian-Ostrzycka K., Pieńkowska K., Łaniewski P., Bujnicki J.M., van Putten J.P, & Jagusztyn-Krynicka E.K. (2014). Functional and Bioinformatics Analysis of Two Campylobacter jejuni Homologs of the Thiol-Disulfide Oxidoreductase, DsbA. PLoS ONE, 9(9), e106247.

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Recombinant Production of Mutant HP0377 Proteins

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To obtain mutated HP0377 proteins for biochemical experiments, a set of recombinant plasmids were constructed. pUWM544 [21 (link)], which carries the hp0377 gene (amino acids 25–221) without its promoter and signal sequence, was used as a starting plasmid for all constructs. Point mutations were generated using the Quick Change Site-Directed Mutagenesis Kit (Qiagen) according to the manufacturer’s instructions, starting with pUWM544 as a template. Correct construction of the plasmids was verified by sequencing. Next, the hp0377-modified nucleotide sequences from pUWM544-derived recombinant plasmids were inserted into pET28a. All plasmids carried the HP0377-His₆ translation fusion. HP0377 and its mutated forms were overexpressed by autoinduction from an E. coli Rosetta strain and purified using NGC Medium-Pressure Chromatography Systems by Bio-Rad as previously described [21 (link)]. HP1227 was overexpressed by IPTG induction and purified from an E. coli Rosetta strain as previously described [21 (link)]. EcDsbC E. coli protein was overexpressed from E.coli BL21harboring pET28a/EcDsbC using autoinduction [30 (link)].

Grzeszczuk M.J., Bąk A., Banaś A.M., Urbanowicz P., Dunin-Horkawicz S., Gieldon A., Czaplewski C., Liwo A, & Jagusztyn-Krynicka E.K. (2018). Impact of selected amino acids of HP0377 (Helicobacter pylori thiol oxidoreductase) on its functioning as a CcmG (cytochrome c maturation) protein and Dsb (disulfide bond) isomerase. PLoS ONE, 13(4), e0195358.

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Generating Recombinant H-1PV Mutants

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Point mutations (see Table 1) were introduced into the infectious molecular clone of wild-type H-1PV (pH1) [24 (link)] using the Quick Change Site-Directed Mutagenesis Kit (Qiagen, Hilden, Germany) to generate pH1-PMI, pH1-PMII, and pH1-PMIII. Specific PCR amplifications were performed in 50 µL reaction buffer containing 50 ng of pH1 template plasmid DNA, 125 ng of each forward and reverse primer (GATC Biotech, Konstanz, Germany), 200 µM of each dNTP, and 2.5 units of high fidelity DNA polymerase (PfuUltra HF, Agilent Technologies, Santa Clara, CA, USA). The PCR cycles were performed as following: 95 °C for 55 s for initial denaturation; 12 cycles of 95 °C for 30 s and 55 °C for 60 s for annealing; and 68 °C for 8 min for extension. To generate the double mutant, the PMI primers were used to introduce the PMI mutation into pH1-PMII. All constructs were verified by sequencing (GATC Biotech, Konstanz, Germany).

Hashemi H., Condurat A.L., Stroh-Dege A., Weiss N., Geiss C., Pilet J., Cornet Bartolomé C., Rommelaere J., Salomé N, & Dinsart C. (2018). Mutations in the Non-Structural Protein-Coding Sequence of Protoparvovirus H-1PV Enhance the Fitness of the Virus and Show Key Benefits Regarding the Transduction Efficiency of Derived Vectors. Viruses, 10(4), 150.

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Mutagenesis of HP0377 Proteins

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To obtain mutated HP0377 proteins, a set of recombinant plasmids was constructed from pUWM544, which carries the hp0377 gene without its promoter and signal sequence. Cys-to-Ser point mutations were generated using the Quick Change Site-Directed Mutagenesis Kit (Qiagen) according to the manufacturer’s instructions, starting with 100 ng of pUWM544 template and 125 ng of each primer (primer pairs: N6_377_C89S_for- N6_377_C89S_rev, hp377_C92SII- hp377_C92SII_rev).
To obtain C25A mutated HP0377 protein, a recombinant plasmid was constructed from pUWM399 carrying the hp0377 gene with its promoter and signal sequence. The Cys-to-Ala point mutation was generated as described above, using primers hp377_C25A_for and hp377_C25A_rev.

Roszczenko P., Grzeszczuk M., Kobierecka P., Wywial E., Urbanowicz P., Wincek P., Nowak E, & Jagusztyn-Krynicka E.K. (2015). Helicobacter pylori HP0377, a member of the Dsb family, is an untypical multifunctional CcmG that cooperates with dimeric thioldisulfide oxidase HP0231. BMC Microbiology, 15, 135.

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Investigating Transcriptional Regulation of Heavy Metal Response Genes

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To further assess the possible binding activation of the MRE motifs in HsMT1 and HsMT2 promoters with HsMTF-like, series of recombinant plasmids were constructed and employed as follows. Six kinds of recombinant plasmids, which included wild-type HsMT1 and HsMT2 promoter-luciferase reporter vectors (referred to pMT1-1121 and pMT2-1121), HsMTs promoter-luciferase reporter vectors with mutated MREs (named as pMT1-1121-MREmut, pMT2-1121-MREamut, pMT2-1121-MREbmut and pMT2-1121-MREabmut), and an eukaryotic expression vector pcDNA-HsMTFlike were prepared.
The whole HsMTF-like ORF cDNA sequence was digested with the restriction enzymes Nhe I and Hind Ш, and inserted into the eukaryotic expression vector pcDNA3.1 at the same sites. Positive clones containing the pcDNA3.1-HsMTF-like were subjected to sequence confirmation.
In order to construct the plasmid of pMT1-1121-MREmut, pMT2-1121-MREamut, pMT2-1121-MREbmut and pMT2-1121-MREabmut, MRE motifs were mutated by fusion PCR with a pair of complementary primers containing the desired nucleotides. The mutations were generated using a QuickChange Site-Directed Mutagenesis kit (Qiagen) and the primers used for mutagenesis were listed in Table 1. Finally, the obtained PCR fragments were inserted into pGL3-Basic vector and verified by DNA sequencing at Sangon Biotech (Shanghai, China).

Wang C., Shu F., Hong Y., Wang J., Peng K., Sheng J., Wu D., Hu B., Shi J, & Jian S. (2018). Analysis of the structure and activity of the promoter regions of the metallothionein genes of the freshwater pearl mussel Hyriopsis schlegelii. Bioscience, biotechnology, and biochemistry, 82(5).

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