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Geneart site directed mutagenesis plus kit

Manufactured by Thermo Fisher Scientific
Sourced in United States

The GeneArt® Site-Directed Mutagenesis PLUS Kit is a tool designed to efficiently introduce specific mutations into DNA sequences. It provides a simple and reliable method for generating site-directed mutations in plasmid DNA.

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11 protocols using geneart site directed mutagenesis plus kit

1

Site-Directed Mutagenesis of F13A Plasmid

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We used the pcDNA5/FRT expression plasmid containing the human wild-type F13A cDNA sequence (pcDNA5/FRT-WT-F13A) we had cloned earlier9 (link). The mutant F13A expression plasmid with a mutation in exon 2 at codon 36 (c.109C>T, Pro36Ser) was constructed by site-directed mutagenesis of the wild-type expression plasmid (pcDNA5/FRT-WT-F13A) using the GeneArt® Site-Directed Mutagenesis PLUS Kit (Invitrogen). The mutagenic primers were designed using the web-based GeneArt® Primer and Construct Design Tool (http://www.thermofisher.com/order/oligoDesigner). The following primers were synthesised by Microsynth: forward primer 5’-CTTCAGGGCGTGGTGTCCCGGGGCGTCAACC-3’, and reverse primer 5’-GGTTGACGCCCCGGGACACCACGCCCTGAAG-3’. The mutagenesis reaction was performed according to the manufacturer’s instruction using One Shot® MAX Efficiency® DH5α™-T1R competent cells (Invitrogen). Plasmids were purified using the PureLink™ HiPure Plasmid Midiprep Kit (Invitrogen) and then sequenced by Microsynth using their standard CMV-forward and BGH-reverse primers. The plasmids with correct wild-type (pcDNA5/FRT-WT-F13A) and mutant (pcDNA5/FRT-MU-F13A) sequences were used for transfection.
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2

Spastin Phosphorylation Site Mutagenesis

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The spastin gene (NCBI: NM_014946.3; human spastin WT) was obtained from the NCBI database. GFP-spastin was ligated by spastin WT cDNA and pEGFP-C1 (Clontech Laboratories, Inc., Mountain View, CA, USA; cat. no. #P0134) as described previously. 22 Eleven phosphorylation sites (S207, Y212, S245, S261, S268, Y269, T292, S302, T303, T305, T306) that were identified by proteomics as being present in 3 or more records were selected for mutation from the records in the PhosphositePlus database (https://www.phosphosite.org).20 (link) Specific recombinant primers were designed by CE Design (Version 1.04) (Table 1). The spastin mutant (abbreviated as spastin 11A) was constructed by the GeneArt Site-Directed Mutagenesis PLUS Kit (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA; cat. no. #A14606) in which all 11 phosphorylation sites were mutated into alanine.
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3

P53 Mutant Plasmid Transfection

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The Sk-Ov-3 cell line was transfected with 4 different types of plasmids, including pBABE-neo, Flag-p53/pRK5 and pCMV-Neo-Bam p53 R249S plasmids, which were purchased from AddGene (USA), and a fourth plasmid was constructed using the GeneArt Site-Directed Mutagenesis PLUS kit (Invitrogen, life technologies) according to the manufacturer’s protocol. The Flag-p53/pRK5 plasmid was used as a template, and a 25 bp set of primers (Additional file 1: Table S1) was designed containing the point mutation in the middle.
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4

Construction of FXIII-A Variant Plasmids

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The WT human F13A gene was obtained as Ultimate ORF Clone ID IOH11901 (Invitrogen, Carlsbad, CA, USA), and subcloned into the pcDNA5/FRT plasmid (Invitrogen) between the NheI and Acc65I restriction sites. pcDNA5/FRT plasmids encoding delN-FXIII-A variants were constructed either by direct mutagenesis by PCR amplification of the pcDNA5/FRT-WT-F13A plasmid with specific primers (Microsynth, Balgach, Switzerland), or by subcloning PCR-amplified F13A with the desired deletion into the plasmid with HindIII and XhoI restrictases. Phusion DNA polymerase, all restrictases, T4 polynucleotide kinase and T4 DNA ligase were from Fermentas (Thermo Fisher Scientific, Waltham, MA, USA). The R11Q and R12Q variants were created by site-directed mutagenesis of the WT expression plasmid (GeneArt sitedirected mutagenesis PLUS Kit; Invitrogen).
One Shot TOP10 competent cells (Invitrogen) were used to expand plasmids. Bacterial media and ampicillin for selection were from Sigma-Aldrich (St Louis, MO, USA). Kits for DNA and plasmid purification were from Invitrogen and Qiagen (Hilden, Germany). Microsynth provided sequencing services.
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5

Generating Zebrafish IKK1 Fusion Constructs

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Zebrafish Ikk1 (Chuk) cDNA was purchased from Open Biosystems (Clone ID: 5914247, GE Dharmacon). Full-length (FL)-Ikk1 was amplified with Advantage 2 Taq Polymerase (Clontech) using forward primer 5′-GCTAGCTGTCAATATGGAGAAACCCCCT-3′ and reverse primer 5′-TAATGGATTTGGTACCGACAAACGCGCTGATTTA-3′. The amplified PCR products were cloned into pCR Blunt II-TOPO using the Zero Blunt® TOPO® PCR Cloning Kit (Life Technologies). To generate tdTomato fusion constructs, pCRTOPO-FL-Ikk1 and ptdTomato-N1 vector (Clontech) were digested with KpnI and NheI, and ligated. The Ikk1(C179A)–tdTomato variant was generated from FL-Ikk1–tdTomato using site-directed mutagenesis (GeneArt® Site-Directed Mutagenesis PLUS Kit, LifeTechnologies). The constructs were subsequently cloned into a plasmid containing the zebrafish tp63 promoter (pT2KXIG_tp63:AcGFP, courtesy of Gromoslav Smolen, Harvard University, Cambridge, MA). First, GFP was removed via digestion with BamHI and NotI, and both sites were Klenow blunt-ended. The Ikk1–tdTomato plasmids were digested with SnaBI and SfoI and ligated into pT2KXIG_tp63 to generate tp63:FL-ikk1–tdTomato and tp63:ikk1(C179A)–tdTomato. HEK001 were transfected using Nanofectin (PAA, Q050-005) when 60% confluent in 8-chamber vessels. It was determined that 0.5 µg of DNA per 0.6 µl of transfection reagent was optimal.
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6

Expression and Characterization of Tubulin Mutants

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Human KL1-expression vector was sourced commercially (RC220944; OriGene Technologies, Rockville, MD, USA) and the KL1 protein was expressed in a pEGFP-C1 vector (Clontech, Mountain View, CA, USA) (GFP-KL1) as previously reported [20 (link)]. Mouse α-TAT1 expression vector was also purchased commercially (MR206707; OriGene Technologies) and flag-tagged α-TAT1 was expressed from a pCMV6-entry vector. The expression vector for rat katanin (pEGFP-C1-p60 katanin) was kind gift of Dr. PW. Baas, Drexel University, Philadelphia, PA. The human α-tubulin construct [20 (link)] was N-terminally GFP-tagged and expressed in pEGFP-C1 (GFP-tubulin(wt)). The tubulin K40R mutation was introduced using a GeneArt Site-Directed Mutagenesis PLUS kit (Life Technologies, Carlsbad, CA, USA) (GFP-tubulin(K40R)) and the structure of the construct was confirmed by sequencing.
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7

Site-Directed Mutagenesis of Hoxb13 Phosphorylation Sites

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Phospho-mimetic (S204A) and non-phosphorylated (S204D, S204E) Hoxb13 mutants were constructed from pCMV-murine Hoxb13-EGFP using GeneArt Site-Directed Mutagenesis Plus Kit (Life Technologies, cat# A14604). The point mutations were produced by overlap extension PCR with primers bearing mutations as follows:

Hoxb13_S204A_F: GCGTTTGCAGAGCCCGCCGTCCAGCACCCTCCT

Hoxb13_S204A_R: AGGAGGGTGCTGGACGGCGGGCTCTGCAAACGC

Hoxb13_S204D_F: GCGTTTGCAGAGCCCGACGTCCAGCACCCTCCT

Hoxb13_S204D_R: AGGAGGGTGCTGGACGTCGGGCTCTGCAAACGC

Hoxb13_S204E_F: GCGTTTGCAGAGCCCGAGGTCCAGCACCCTCCT

Hoxb13_S204E_R: AGGAGGGTGCTGGACCTCGGGCTCTGCAAACGC

All mutations were verified by DNA sequencing.
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8

Site-Directed Mutagenesis of Hoxb13 Phosphorylation Sites

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Phospho-mimetic (S204A) and non-phosphorylated (S204D, S204E) Hoxb13 mutants were constructed from pCMV-murine Hoxb13-EGFP using GeneArt Site-Directed Mutagenesis Plus Kit (Life Technologies, cat# A14604). The point mutations were produced by overlap extension PCR with primers bearing mutations as follows:

Hoxb13_S204A_F: GCGTTTGCAGAGCCCGCCGTCCAGCACCCTCCT

Hoxb13_S204A_R: AGGAGGGTGCTGGACGGCGGGCTCTGCAAACGC

Hoxb13_S204D_F: GCGTTTGCAGAGCCCGACGTCCAGCACCCTCCT

Hoxb13_S204D_R: AGGAGGGTGCTGGACGTCGGGCTCTGCAAACGC

Hoxb13_S204E_F: GCGTTTGCAGAGCCCGAGGTCCAGCACCCTCCT

Hoxb13_S204E_R: AGGAGGGTGCTGGACCTCGGGCTCTGCAAACGC

All mutations were verified by DNA sequencing.
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9

Engineered HEV-3ra Mutants for Functional Studies

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Using rabbit HEV-3ra infectious clone LR or indicator replicon LRG as the backbone, the HEV-3ra mutants containing a single-site mutation, Y1320H, K1383N, K1634G, or K1634R, were constructed with the GeneArt site-directed mutagenesis system (Invitrogen-Thermo Fischer, Waltham, MA, USA) according to the manufacturer’s instructions. The LRG double mutant Y1320H/K1383N was constructed with GeneArt site-directed mutagenesis PLUS kit (Thermo Scientific, Waltham, MA, USA). As recommended for the mutagenesis system, PCRs were performed with AccuPrime Pfx DNA polymerase (Invitrogen-Thermo Scientific, Waltham, MA, USA). All primers used in this study for viral genomic sequencing and introduction of mutations are commercially synthesized (Integrated DNA Technologies, Coralville, IA, USA) and listed in Table S2.
One Shot MAX Efficiency DH5α-T1R competent cells (Invitrogen-Thermo Fischer, Waltham, MA, USA) were transformed with each of the HEV-3ra constructs containing specific mutation(s). The transformed cells were cultured in 50 mL lysogeny broth medium with ampicillin, and plasmid midipreps were performed with Qiagen Plasmid Plus midikit (Qiagen, Germantown, MD, USA). The entire viral genome in each of the HEV-3ra constructs was confirmed through Sanger sequencing to ensure that no additional nucleotide substitution had been introduced.
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10

Validation of miR-29b-1/a Binding Site

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Mutation of the predicted 3′ UTR binding site of miR-29b-1/a on pEZX-MT06 miRNA 3′ UTR target dual luciferase expression vectors was performed using the GeneArt® Site-Directed Mutagenesis PLUS Kit (ThermoFisher Scientific) according to manufacturer’s instructions. Where indicated, HEK-293 cells were plated in 24 well plates and co-transfected with 200 ng of wild type (wt) or mutant (mut) vectors plus a 20 nM final concentration of miR-29b-1/a mimic, anti-miR-29a or negative control mimic using FuGENE HD (Promega) according to manufacturer’s protocol. Twenty-four h post transfection, a dual luciferase assay (Promega) was performed. Luciferase expression was determined relative to negative control mimic and statistical evaluation performed using GraphPad Prism Software (Graph Pad Software, San Diego, CA).
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