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Mutagenic oligonucleotides

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Mutagenic oligonucleotides are synthetic DNA molecules designed to introduce specific mutations into target DNA sequences. These oligonucleotides are used in various molecular biology applications, including site-directed mutagenesis, gene editing, and the study of gene function.

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Lab products found in correlation

Imidazole, by Thermo Fisher Scientific (2 mentions) Wizard plus sv miniprep dna purification system, by Promega (2 mentions) Nickel ida agarose, by GoldBio (2 mentions) Luria broth powder, by Merck Group (2 mentions) Nickel 2 chloride hexahydrate, by Thermo Fisher Scientific (2 mentions) Kanamycin monosulfate, by GoldBio (2 mentions) Dpni, by New England Biolabs (2 mentions) Dntps, by New England Biolabs (2 mentions) Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions) Bradford protein assay kit, by Bio-Rad (1 mentions) 4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by MP Biomedicals (1 mentions) Isopropyl β d thiogalactopyranoside iptg, by GoldBio (1 mentions)

5 protocols using mutagenic oligonucleotides

1

Bt Cry51Aa2 Protein Expression and Purification

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Mutagenic oligonucleotides (Integrated DNA Technologies, IDT, Coralville, Iowa) were designed as sense and antisense pairs with a 15–20 bp overlap, and no significant secondary structure at the 3′ ends, to enable whole-plasmid PCR (Supplementary Data 1). Plasmid pMON106128 served as the base vector for the Cry51Aa2 and Cry51Aa2.834 coding region templates (Supplementary Data 2). Variant clones generated by site-directed mutagenesis were confirmed by sequencing and transformed into B. thuringiensis using electroporation. Expressed crystal proteins recovered from sporulated and lysed cultures were solubilized in 25 mM sodium carbonate (pH 10.5) buffer, analysed by SDS–PAGE to confirm the presence of a single band, and quantified by densitometry using bovine serum albumin as a standard8 (link). For high-throughput screening of variant proteins, crystals were solubilized in carbonate buffer and supernatant total protein was quantified using the modified Bradford protein assay kit from Bio-Rad Laboratories (Hercules, CA). Total protein recovery from a B. thuringiensis strain containing the base vector pMON106128 was used to establish background protein concentration in test samples.
Gowda A., Rydel T.J., Wollacott A.M., Brown R.S., Akbar W., Clark T.L., Flasinski S., Nageotte J.R., Read A.C., Shi X., Werner B.J., Pleau M.J, & Baum J.A. (2016). A transgenic approach for controlling Lygus in cotton. Nature Communications, 7, 12213.
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2

Phage-Displayed UbV Library UtU Construction

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The phage-displayed UbV library UtU was constructed by combinatorial site-directed mutagenesis of a phagemid designed for the display of UbV.v27.1 on phage. Two regions (region 1, positions 5–14; region 2, positions 62–74) were simultaneously mutated with a soft randomization strategy 65 , in which the nucleotide ratios of diversified positions in the Mutagenic oligonucleotides were adjusted to 70% of the wt nucleotide and 10% of each of the other three nucleotides (Table S2). Mutagenic oligonucleotides were purchased from Integrated DNA Technologies. The constructed library UtU contained 1.7 × 1010 unique members.
Veggiani G., Yates B.P., Martyn G.D., Manczyk N., Singer A.U., Kurinov I., Sicheri F, & Sidhu S.S. (2022). A Panel of Engineered Ubiquitin Variants Targeting the Family of Human Ubiquitin Interacting Motifs. ACS chemical biology, 17(4), 941-956.
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3

Production and Purification of Recombinant Elastase

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Mutagenic oligonucleotides were purchased from Integrated DNA Technologies. Phusion High-Fidelity DNA Polymerase, dNTPs and Dpn I were purchased from New England Biolabs. Kanamycin Monosulfate, isopropyl-β-D-thiogalactopyranoside (IPTG), tris(hydroxymethyl)aminomethane (TRIS) and Nickel-IDA agarose were purchased from Gold Biotechnology. Human neutrophil elastase was purchased from Elastin Products Company. Methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide (MSA2PV-pNA), Triton X-100, dimethylsulfoxide (DMSO), succinic acid and Luria Broth powder were purchased from Sigma-Aldrich. Imidazole was purchased from Alfa Aesar. Nickel (II) Chloride-hexahydrate was purchased from Acros Organics. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from MP Biomedical, LLC. Plasmid DNA was purified using the Wizard Plus SV Miniprep DNA Purification System (Promega).
Herdendorf T.J, & Geisbrecht B.V. (2018). Investigation of human neutrophil elastase inhibition by Staphylococcus aureus EapH1: The key role played by Arginine 89. Biochemistry, 57(50), 6888-6896.
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4

Recombinant Protein Purification Protocol

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Kanamycin monosulfate, isopropyl-β-D-thiogalactopyranoside (IPTG), tris(hydroxymethyl)aminomethane (TRIS) and Nickel-IDA agarose were purchased from Gold Biotechnology. Methoxysuccinyl-Ala-Ala-Pro-Val-P-nitroanilide (MSA2PV-pNA), Triton X-100, dimethylsulfoxide (DMSO), succinic acid and Luria Broth powder were purchased from Sigma-Aldrich. Nickel (II) Chloride-hexahydrate was purchased from Acros Organics. Imidazole was purchased from Alfa Aesar. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from MP Biomedical, LLC. Human neutrophil elastase was purchased from Elastin Products Company. Mutagenic oligonucleotides were purchased from Integrated DNA Technologies. Phusion High-Fidelity DNA Polymerase, dNTPs and Dpn I were purchased from New England Biolabs. Plasmid DNA was purified using the Wizard Plus SV Miniprep DNA Purification System (Promega).
Herdendorf T.J, & Geisbrecht B.V. (2019). Staphylococcus aureus Evasion Proteins EapH1 and EapH2: Residue-Level Investigation of an Alternative Binding Motif for Human Neutrophil Elastase. Archives of biochemistry and biophysics, 676, 108140.
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5

Molecular Cloning Using CloneJET

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All chemicals were purchased from Sigma-Aldrich unless otherwise noted. The CloneJET PCR cloning system was purchased from Thermo Fisher Scientific. Mutagenic oligonucleotides and sequencing primers were purchased from Integrated DNA Technologies. All enzymes were purchased from New England BioLabs.
Liu L., Huang Y, & Wang H.H. (2023). Fast and efficient template-mediated synthesis of genetic variants. Nature methods, 20(6).
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