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Quikchange mutagenesis method

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The QuikChange mutagenesis method is a technique used to introduce site-directed mutations into double-stranded plasmid DNA. It allows for the rapid and efficient generation of desired mutations without the need for subcloning or ligation steps.

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Ni nta, by Macherey-Nagel (2 mentions)

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QuikChange (236 citations) QuikChange mutagenesis (94 citations) QuikChange method (57 citations) QuikChange site-directed mutagenesis method (25 citations)

4 protocols using quikchange mutagenesis method

1

Recombinant Expression and Purification of Galectin Proteins

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Using a standard PCR-based cloning strategy, the coding region of the full-length hGal1 (residues 1–135), 3 (residues 1–250), 7 (residues 1–136) and CRD domain of hGal3 (residues 113–250) were generated and inserted into modified pET-15b (hGal1, hGal3 and hGal3-CRD) or pET-28a (hGal7) vector (Novagen) with in-frame N- and C-terminal 6xHis-tag, respectively. QuikChange mutagenesis method (Agilent Technologies) was applied to replace the corresponding codons of residues Glu165 and Arg186 in full-length hGal3 with Ala codon to allow the expression of mutant proteins hGal3-E165A and hGal3-R186A. All the wild type and mutated proteins were produced in Escherichia coli BL21 (DE3), with 0.5 mM IPTG induction for 16 h at 20 °C. Recombinant proteins were purified by Ni2+-affinity and size-exclusion chromatography to homogeneity. Purified proteins were then stored in gel-filtration buffer (25 mM Tris-HCl pH8.0, 300 mM NaCl and 5 mM β-mercaptoethanol) and concentrated to ~ 4, 20 and 9 mg/ml as determined by the method of Bradford for both Biolayer interferometry experiments and crystallization trials, respectively.
Hsieh T.J., Lin H.Y., Tu Z., Huang B.S., Wu S.C, & Lin C.H. (2015). Structural Basis Underlying the Binding Preference of Human Galectins-1, -3 and -7 for Galβ1-3/4GlcNAc. PLoS ONE, 10(5), e0125946.
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2

Cloning and Mutagenesis of NmClpX and ClpP

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Codon-optimized genes encoding NmClpX (Uniprot entry: Q9JYY3) bearing an N-terminal His6-TEV affinity tag and ClpP (Uniprot entry: Q9JZ38) with an N-terminal His6-SUMO tag were synthesized by GenScript (Piscataway, NJ) and cloned into the NdeI and BamHI sites of pET28a+ (Novagen, Madison, WI). Point mutations were introduced with the Quikchange mutagenesis method (Agilent, Santa Clara, CA).
Ripstein Z.A., Vahidi S., Houry W.A., Rubinstein J.L, & Kay L.E. (2020). A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery. eLife, 9, e52158.
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3

Generating MstE Point Mutants

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To generate point mutants of MstE, pET-29b MstE was used from the previous study21 . Point mutations were introduced with the QuikChange mutagenesis method (Agilent Technologies) and confirmed by sequencing. Primers were designed using the PrimerX tool (http://www.bioinformatics.org/primerx/). The generated plasmids containing mutant MstE were transformed into E. coli BL21 tuner (DE3) cells (Novagen). For expressions, cells were grown in terrific broth supplemented with 50 μg/mL kanamycin to an OD600 of approximately 0.8 at 30 °C, induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 16 °C for another 20 h. Cells were harvested by centrifugation (20 min, 5000 × g, 4 °C), resuspended in buffer (50 mM phosphate buffer pH 7.8, 300 mM NaCl, 10 mM imidazole) and lysed by sonication (10 × 10 s pulses with 10 s breaks, on ice). After centrifugation at 15000 × g and 4 °C for 30 min, MstE was purified from the supernatant with Ni-NTA (Macherey-Nagel). The column was washed with buffer containing 20 mM imidazole and the protein was eluted stepwise with buffer containing 100 mM imidazole followed by 200 mM imidazole. Elution fractions were run on an SDS-PAGE and used for enzymatic assays directly.
Moosmann P., Ecker F., Leopold-Messer S., Cahn J.K., Dieterich C.L., Groll M, & Piel J. (2020). A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds. Nature chemistry, 12(10), 968-972.
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4

Generating MstE Point Mutants

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To generate point mutants of MstE, pET-29b MstE was used from the previous study21 . Point mutations were introduced with the QuikChange mutagenesis method (Agilent Technologies) and confirmed by sequencing. Primers were designed using the PrimerX tool (http://www.bioinformatics.org/primerx/). The generated plasmids containing mutant MstE were transformed into E. coli BL21 tuner (DE3) cells (Novagen). For expressions, cells were grown in terrific broth supplemented with 50 μg/mL kanamycin to an OD600 of approximately 0.8 at 30 °C, induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 16 °C for another 20 h. Cells were harvested by centrifugation (20 min, 5000 × g, 4 °C), resuspended in buffer (50 mM phosphate buffer pH 7.8, 300 mM NaCl, 10 mM imidazole) and lysed by sonication (10 × 10 s pulses with 10 s breaks, on ice). After centrifugation at 15000 × g and 4 °C for 30 min, MstE was purified from the supernatant with Ni-NTA (Macherey-Nagel). The column was washed with buffer containing 20 mM imidazole and the protein was eluted stepwise with buffer containing 100 mM imidazole followed by 200 mM imidazole. Elution fractions were run on an SDS-PAGE and used for enzymatic assays directly.
Moosmann P., Ecker F., Leopold-Messer S., Cahn J.K., Dieterich C.L., Groll M, & Piel J. (2020). A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds. Nature chemistry, 12(10), 968-972.
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