Quickchange kit

Manufactured by Qiagen

The QuickChange kit is a laboratory tool designed for site-directed mutagenesis. It provides a simple and efficient method to introduce specific mutations into double-stranded DNA plasmids.

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Quickchange II XL Mutagenesis Kit (2 citations) QuickChange II-Gold site-directed mutagenesis kit (2 citations) QuickChange II site-directed mutagenesis kit (2 citations) QuickChange mutagenesis kit (2 citations)

4 protocols using quickchange kit

Preparation and Purification of Cell Extracts and Proteins for DNA Repair Assays

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Whole-cell extracts were prepared from confluence-arrested XLF-deficient Bustel
fibroblasts [12 (link)] as described
[13 (link),14 (link)]. HCT116 cell extracts were made similarly, except that that growth
medium was replaced with medium containing 0.5% serum when the cells reached
50% confluence, and cells were harvested 5 days later. Recombinant His6-tagged XLF
and tyrosyl-DNA phosphodiesterase (TDP1) were expressed in E. coli and purified by
nickel-affinity chromatography and monoQ FPLC [14 (link)]. Mutations were introduced using QuickChange kit (Qiagen). Artemis
nuclease [15 (link)], X4L4 complex
[16 (link)] and Ku [17 (link)] were produced in baculovirus-infected
insect cells. Recombinant human endoIII homologue (hNTH) [18 (link)] was kindly provided by Dr. David Pederson, University
of Vermont and was stored in small aliquots at −80°C. Tg-containing
oligonucleotides were obtained from Midland Certified Reagents, with structural
verification by mass spectrometry. Unmodified oligomers were from Integrated DNA
Technologies and other enzymes were from New England Biolabs.

Almohaini M., Chalasani S.L., Bafail D., Akopiants K., Zhou T., Yannone S.M., Ramsden D.A., Hartman M.C, & Povirk L.F. (2016). Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair. DNA repair, 41, 16-26.

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Purification and Characterization of Lipoxygenases

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WT MoLOX was expressed in and purified from P. pastoris X-33 cells as previously described.^{28 (link)}G. graminis lipoxygenase, GgLOX (gene synthesized by Genscript), was expressed and purified in a similar manner as MoLOX. The final purification for all variants was carried out using a HiPrep 26/60 Sephacryl S-200 column on an AKTA FPLC system with 50 mM HEPES (pH 7.5), 150 mM NaCl, and 10% glycerol. The fractions that corresponded to the peak with lipoxygenase activity were concentrated to 100 μM, frozen in aliquots with N₂ (l), and stored at −80°C. Mn content was determined using ICP-MS. Site-directed mutants were generated with the Qiagen QuickChange kit.

Sharma A., Whittington C., Jabed M., Hill S.G., Kostenko A., Yu T., Li P., Doan P.E., Hoffman B.M, & Offenbacher A.R. (2023). 13C ENDOR Spectroscopy-Guided MD Computations Reveals the Structure of the Enzyme-Substrate Complex of an Active, N-linked Glycosylated Lipoxygenase. Biochemistry, 62(10), 1531-1543.

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Recombinant protein expression constructs

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The cDNA corresponding to the IQ domain of Na_V1.1 (IQ, amino acids 1909–1936) was generated by PCR using the cDNA of human Na_V1.1 as the template. The primers were designed using VectorNTI software. The human CaM and its truncated proteins—N-lobe (a.a. 2–80) and C-lobe (a.a. 76–148)—were subcloned from HEK293 cells [21 (link),34 (link),35 (link)]. The CaMKII mutant T286D (CaMKIIT286D), which is a constitutively active type of CaMKII in the absence of CaM and Ca²⁺, was created with rat CaMKIIα cDNA as a template [36 (link)]. Mutants including IQ mutant (I1922E, Q1923E), potential CaMKII phosphorylation sites mutant (T1909A + S1918A + T1934A), CaM₁₂ (E31A + E67A), CaM₃₄ (S101F + E140A), and CaM₁₂₃₄ (E31A + E67A + S101F + E140A) were constructed by site-directed mutagenesis using a Quickchange™ kit (QIAGEN) [37 (link),38 (link)]. The above DNAs were individually ligated into pGEX-6P-1 H320 expression vectors (GE Biosciences, New York, NY, USA).

Li J., Yu Z., Xu J., Feng R., Gao Q., Boczek T., Liu J., Li Z., Wang Q., Lei M., Gong J., Hu H., Minobe E., Ji H.L., Kameyama M, & Guo F. (2018). The Effect of Ca2+, Lobe-Specificity, and CaMKII on CaM Binding to NaV1.1. International Journal of Molecular Sciences, 19(9), 2495.

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Purification and Characterization of Lipoxygenases

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WT MoLOX was expressed in and purified from P. pastoris X-33 cells as previously described. 28 G. graminis lipoxygenase, GgLOX (gene synthesized by Genscript), was expressed and purified in a similar manner as MoLOX. The final purification for all variants was carried out using a HiPrep 26/60 Sephacryl S-200 column on an AKTA FPLC system with 50 mM HEPES (pH 7.5), 150 mM NaCl, and 10% glycerol. The fractions that corresponded to the peak with lipoxygenase activity were concentrated to 100 μM, frozen in aliquots with N 2 (l), and stored at -80°C. Mn content was determined using ICP-MS. Site-directed mutants were generated with the Qiagen QuickChange kit.

Sharma A., Whittington C., Jabed M., Hill S.G., Kostenko A., Yu T., Li P., Doan P.E., Hoffman B.M, & Offenbacher A.R. (2023). ¹³C Electron Nuclear Double Resonance Spectroscopy-Guided Molecular Dynamics Computations Reveal the Structure of the Enzyme-Substrate Complex of an Active, N-Linked Glycosylated Lipoxygenase. Biochemistry, 62(10).

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