Phusion mutagenesis kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Phusion Mutagenesis kit is a tool used for site-directed mutagenesis. It allows for the introduction of specific mutations into DNA sequences.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (4 mentions) Pgl4 luciferase reporter vector, by Promega (2 mentions) Jellyop, by Thermo Fisher Scientific (1 mentions) Psicheck luciferase reporter vector, by Promega (1 mentions) Reporter lysis buffer, by Promega (1 mentions) Bac2bac system, by Thermo Fisher Scientific (1 mentions) Psicheck 2 vector, by Promega (1 mentions)

Close spelling variants of this product

Phusion Site-Directed Mutagenesis Kit (180 citations) Phusion mutagenesis (5 citations) Phusion site-directed mutagenesis kit protocol (2 citations)

8 protocols using phusion mutagenesis kit

Generation of JellyOp Fusion Proteins

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JellyOp (obtained from Addgene) and human OPN1MW amplified from retinal cDNA was cloned in front of the IRES site of a pIRES2-TurboFP635 vector or in the position of the IRES site of a pIRES2-mKate vector to create fusion proteins^{22 (link)}. The JellyOp(K72T) mutation was introduced to the pIRES2-JellyOp-TurboFP635 vector using a Phusion mutagenesis kit (ThermoFisher).
For viral production, JellyOp was cloned into a pAAV-770En_454P(hGRM6)-JellyOp-IRES2-TurboFP635-WPRE-BGHpA plasmid^{20 (link)}. Viral vectors were produced in HEK293 cells by the triple plasmid co-transfection method using the pXX80 helper plasmid and the rep-cap plasmid encoding AAV(7m8)^{45 (link)} as described in detail elsewhere^{46 (link)}. The viral titre was 4 × 10¹³ GC/ml. The virus was stored in aliquots at −80 °C until the day of use.

van Wyk M, & Kleinlogel S. (2023). A visual opsin from jellyfish enables precise temporal control of G protein signalling. Nature Communications, 14, 2450.

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Mutational Analysis of SIRT1 3'UTR

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The Phusion Mutagenesis kit (Thermo Fisher, MA, USA) was used to mutate the binding sites as the manufacturer’s protocol described. cDNAs that included the wild-type sequences (WT-SIRT1) or mutated binding sequences (MUT-SIRT1) with miR-19a/b-3p in SIRT1 3′ UTR were cloned into the pGL4 luciferase reporter vector (Promega, WI, USA). Neuroblastoma cells were co-transfected with the recombinant plasmid together with miR-19a/b-3p mimics or mimics negative control (NC) (synthesized from Genepharma, Shanghai, China). Then, 24 h after, the co-transfected cells were harvested in the Reporter Lysis Buffer. The luciferase activity of each sample was measured using the Dual-Luciferase Reporter Assay System (Promega, WI, USA).

Zhou F., Wang Y.K., Zhang C.G, & Wu B.Y. (2021). miR-19a/b-3p promotes inflammation during cerebral ischemia/reperfusion injury via SIRT1/FoxO3/SPHK1 pathway. Journal of Neuroinflammation, 18, 122.

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Mutational Analysis of miR-378 Binding Sites

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The Phusion Mutagenesis kit (ThermoFisher, Waltham, MA, USA) was used to mutate the binding sites according to the manufactures' instructions. cDNAs that included the wild‐type sequences or mutated binding sequences with miR‐378 in circATRNL1 and Smad4 3′ UTR were cloned into the psiCHECK luciferase reporter vector (Promega, Madison, WI, USA). Ovarian cancer cells were co‐transfected with the recombinant plasmid together with miR‐378 mimics or NC. Twenty‐four hours after the co‐transfected cells were harvested in the Reporter Lysis Buffer, the luciferase activity of each sample was measured using the Dual‐Luciferase Reporter Assay System (Promega).

Wang J., Li Y., Zhou J., Shen F., Shi X, & Chen Y. (2021). CircATRNL1 activates Smad4 signaling to inhibit angiogenesis and ovarian cancer metastasis via miR‐378. Molecular Oncology, 15(4), 1217-1233.

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Mutagenesis and Luciferase Assay for miRNA-3'UTR Interaction

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The Phusion Mutagenesis kit (F541, Thermo Fisher, USA) was used to mutate the binding sites. cDNAs that contained the wild-type sequences WT-FOXO₃ or mutated binding sequencesor MUT-FOXO₃ with miR-211-5p in FOXO₃ 3′ UTR were cloned into the pGL4 luciferase reporter vector (Promega, USA). HEK-293 cells were co-transfected with the recombinant plasmid together with miR-211-5p mimics or mimics NC (synthesized from Genepharma, China). Then, the co-transfected cells were obtained in the Reporter Lysis Buffer after transfection for 24 h. The luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega, USA).

Zhang M., Xing J., Zhao S., Lu M., Liu Y., Lin L., Gao W., Chen L., Li W., Shang J., Zhou J., Yin X, & Zhu X. (2024). Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO3 axis. Cell Biology and Toxicology, 40(1), 29.

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Cloning and Expression of Human Gelsolin Domains

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Synthetic gene coding for the second domain of human gelsolin (G2l, residues 133-266) was purchased from Eurofins Genomics and subcloned into a pET-28 plasmid at the Nde I/Xho I sites (Novagen) carrying a 6xHis-tag at the N-terminus.
G2l mutants (D187N and N184K) were produced by site-directed mutagenesis using G2l wt as template and the phusion mutagenesis kit (Thermofisher). The shorter construct of N184K (G2s, res 151-266) used for crystallographic studies was also obtained by site-directed mutagenesis according to the two-step protocol described in ref. 20 (link).
Recombinant plasmids (G2l_wt/pET28, G2l_D187N/pET28, G2l_N184K/pET28 and G2s_N184K/pET28) were transformed in the E. coli strain BL21 pLys (DE3) (InvitrogenTM). Cell cultures were grown in LB Broth at 37 °C. Expression of G2l and G2s was induced by addition of 1 mM IPTG.

Bonì F., Milani M., Porcari R., Barbiroli A., Ricagno S, & de Rosa M. (2016). Molecular basis of a novel renal amyloidosis due to N184K gelsolin variant. Scientific Reports, 6, 33463.

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Investigating HMGA1 mRNA-let-7d-5p Interaction

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The WT sequences or mutated binding sites of let-7d-5p in 3′ untranslated region (UTR) of HMGA1mRNA and the full length of circBANP were cloned into the luciferase report vector (psiCHECK2). Commercial kit (the Phusion Mutagenesis kit, Thermo Fisher Scientific) was utilized to mutate the predicted binding sites as the protocol described. CRC cells were seeded in individual wells of the 24-well plates first overnight and then recombinant constructs were transfected into CRC cells together with let-7d-5p mimics or miR-NC by using the Lipofectamine 3000. 48 h after transfection, the cells were harvested in the Reporter Lysis Buffer from the commercial kit (Promega, P.R. China) and relative luciferase activities were measured.

Ni Y., Lu C., Wang W., Gao W, & Yu C. (2021). circBANP promotes colorectal cancer growth and metastasis via sponging let-7d-5p to modulate HMGA1/Wnt/β-catenin signaling. Molecular Therapy Oncolytics, 21, 119-133.

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Codon-Optimized Dbr1 and TTDN1 Expression

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A codon-optimized human Dbr1 expression plasmid was synthesized by DNA2.0. Truncated hDbr1 expression constructs (1–502, 1–443, 1–399, 1–332, 1–314, 1–253, Δ511–528) were generated with PCR-based approaches (Phusion mutagenesis kit, Thermo). The Drn1 cDNA was purchased from Genecopoeia (Product ID: V1400; Cat #GC-OG08504) and subcloned into a modified pET vector along with a C-terminal 8x-HIS tag. A codon-optimized TTDN1 expression plasmids containing a C-terminal 8x-HIS tag was ordered from GenScript. All E. coli expression plasmids contained T7 promoters. For insect cell expression, the native human Dbr1 cDNA sequence was subcloned into the pFastBac vector along with a C-terminal 8x-HIS tag (Invitrogen Bac2Bac system).

Clark N.E., Katolik A., Gallant P., Welch A., Murphy E., Buerer L., Schorl C., Naik N., Naik M.T., Holloway S.P., Cano K., Weintraub S.T., Howard K.M., Hart P.J., Jogl G., Damha M.J, & Fairbrother W.G. (2023). Activation of human RNA lariat debranching enzyme Dbr1 by binding protein TTDN1 occurs though an intrinsically disordered C-terminal domain. The Journal of Biological Chemistry, 299(9), 105100.

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Mutagenesis of miRNA Binding Sites

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The Phusion Mutagenesis kit (ThermoFisher, MA, USA) was employed to mutate the binding sites following the manufactures' instructions. cDNAs that included the wild type sequences (HCP5-WT, IGF1-WT) or mutated binding sequences (HCP5-MUT, IGF1-MUT) with miR-497 were cloned into the psiCHECK2 vector (Promega, WI, USA). HCMs were co-transfected with the recombinant plasmid together with miR-497 mimics or NC. The co-transfected cells were harvested after 48 h with the Reporter Lysis Buffer. The luciferase activity of each sample was measured using the Dual-Luciferase Reporter Assay System (Promega, WI, USA).

Li K.S., Bai Y., Li J., Li S.L., Pan J., Cheng Y.Q., Li K., Wang Z.G., Ji W.J., Zhou Q, & Wang D.J. (2021). LncRNA HCP5 in hBMSC-derived exosomes alleviates myocardial ischemia reperfusion injury by sponging miR-497 to activate IGF1/PI3K/AKT pathway. International journal of cardiology, 342.

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