The mouse lung epithelial cell line MLE-15 was provided by Dr. Jining Lu in the Department of Medicine, Columbia University. The pancreatic beta cell line HIT-T15 was purchased from ATCC. Cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS. The pCMV-Isl1 plasmid was purchased from OriGene. To test whether the Nkx2.1 promoter is regulated by Isl1, a 0.4 kb promoter region of the Nkx2.1 gene was cloned into the pGL3 luciferase report vector, and then co-transfected into cells with pCMV-YFP or pCMV-Isl1 plasmids. The following primers were used: forward, 5’-CGTGAAGGTACCCTCTCTTTGAGACCTAAA-3’; reverse, 5’-GCAAGTCTCGAGACATGATTCGGCGTCGGC-3’. Point mutation of the potential binding sites was generated by site-directed mutagenesis using GeneArt Site-Directed Mutagenesis Kit (Thermo Fisher Scientific) with the following primers: forward, 5’-GTCATCAGCATGTAAGCTAATTATCTCGGGCAAGATGT-3’; reverse, 5’-ACATCTTGCCCGAGATACGGCGCTTACATGCTGATGAC-3’. Luciferase activity was determined 48 hours after transfection using the Dual-Luciferase Reporter Assay Kit (Promega) and the GloMax-Multi Detection System (Promega).
Geneart site directed mutagenesis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneArt Site Directed Mutagenesis Kit is a laboratory tool used to introduce specific genetic modifications into DNA sequences. It provides a method to create targeted mutations, insertions, or deletions in a plasmid or gene of interest.
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Lab products found in correlation
Pcdna gαo1 nluc, by GenScript (2 mentions)
Pcdna gα13 nluc, by GenScript (2 mentions)
Pcdna gα15 nluc, by GenScript (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Accuprime pfx dna polymerase, by Thermo Fisher Scientific (2 mentions)
Pmir report firefly luciferase vector, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Glomax, by Promega (2 mentions)
Hit t15, by American Type Culture Collection (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Snap tag sequence, by New England Biolabs (1 mentions)
Pgex 6p 1 vector, by GE Healthcare (1 mentions)
Hiload 16 60 superdex 200 column, by GE Healthcare (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Ha ubiquitin, by Addgene (1 mentions)
Dna sequencing, by Eurofins (1 mentions)
Oligonucleotide primers, by Integrated DNA Technologies (1 mentions)
Mmessenger mmachine t7 kit, by Thermo Fisher Scientific (1 mentions)
36 protocols using geneart site directed mutagenesis kit
1
Isl1 Regulates Nkx2.1 Promoter Activity
2
Generation and Confirmation of Knockout Mutant
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The knockout mutant was obtained using the pDM4 vector as previously described (29 (link)). Briefly, ∼800-bp upstream and downstream fragments of the target gene were amplified, and the first-round products were used as the templates in the second-round PCR. Next, the PCR product was inserted into the suicide vector pDM4. The recombinant was transformed into E. coli S17-1 λpir and then conjugated with V. parahaemolyticus . The mutant was selected and confirmed by PCR. For obtaining the ΔscvE::pScvE strain, the entire coding region of scvE with an N-terminal ribosome binding site was amplified, inserted into pMMB207, and then transconjugated into the ΔscvE strain. Site-directed mutagenesis was performed using a GeneArt site-directed mutagenesis kit (Thermo) according to the manufacturer’s instructions. The primers used are listed in Table S3 in the supplemental material.
3
Generation of Bioluminescent Fusion Proteins
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To generate HiBiT-SMO and ΔCRD HiBiT-SMO, Nluc sequence in Nluc-SMO or ΔCRD Nluc-SMO (coding mouse SMO, ref. 26 (link)) were replaced with HiBiT sequence (nucleotides sequence: 5′-GTG AGC GGC TGG CGG CTG TTC AAG AAG ATT AGC-3′; amino acids sequence: VSGWRLFKKIS). To generate FLAG-SNAP-SMO, mouse SMO sequence from SMO-Rluc8 was inserted into an empty FLAG-SNAP-tagged pcDNA3.1 vector between BamHI and HindIII sites. SMO-Rluc8, HiBiT-FZD6, SNAP-FZD4, SNAP-FZD5, SNAP-FZD6, SNAP-FZD7, FZD4-Nluc, FZD6-Nluc, β2AR, Venus-KRas, and Nluc-DVL2 were generated and validated in our previous studies16 (link),29 (link). SMO-Nluc and the Gs BRET sensor were generated using prolonged overlap extension PCR techniques. The plasmid encoding CD86-Nluc was provided by Dr. Ulrike Zabel (University of Wuerzburg, Wuerzburg, Germany). Plasmids encoding cPKA-YFP and NbSmo2-YFP were a kind gift from Benjamin Myers (University of Utah, Salt Lake City, USA)44 . Plasmid encoding Venus-mGsi was from Nevin Lambert (Augusta University, Georgia, USA75 (link)). Wild-type human H3R DNA vector was purchased from cDNA.org. The desired mutations were generated using GeneArt site-directed mutagenesis kit (Thermo Fisher Scientific). All constructs were validated by sequencing (Eurofins GATC, Konstanz, Germany). Utilized primers are listed in Supplementary Table 1 .
4
Recombinant Expression and Purification of Mouse Junctophilin-2
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Mouse Junctophilin-2 cDNA (NP_001192005.1) was amplified by PCR and cloned into pGEX-6P-1 vector (GE Healthcare). Site-directed mutagenesis of Δ162-167, Δ479-486, Δ563-568 and Δ644-649 was carried out using the GeneArt™ site-directed mutagenesis kit (Thermo Fisher Scientific). For specific dye labeling, the SNAP-tag sequence (New England BioLabs) was amplified by PCR and cloned into the vector immediately downstream of the JP2 sequence, while the stop codon of JP2 was removed by site-directed mutagenesis. Sequences were confirmed by DNA sequencing. Recombinant JP2 was expressed as N-terminally GST-tagged fusion protein in Escherichia coli BL21 induced by addition of 1.0 mM IPTG at OD 0.6 followed by incubation at 30 °C for 4 h. After cell lysis and centrifugation, the protein in the supernatant was affinity purified on Gluthatione Sepharose 4B prepacked columns (GE Healthcare). The tag was removed by on-column cleavage using PreScission Protease (GE Healthcare) resulting in the elution of untagged JP2. Finally, JP2 was purified by size-exclusion chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare) in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM DTT, 1% Triton X-100.
5
ALDOA Gene Mutagenesis in Lentiviral Vector
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The human ALDOA gene was cloned into the plenti6.3 lentiviral vector. Site-directed mutagenesis of the D33A, K293A and Y361S residues was performed using a GeneArt Site-Directed Mutagenesis kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions.
6
Site-Directed Mutagenesis of CDC42 and BEM4 Plasmids
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To insert point mutations in plasmids carrying the CDC42 or BEM4 genes, the GeneArt Site-Directed Mutagenesis Kit was used (Thermo Fisher Cat #A13282, Grand Island, NY) according to the manufacturer’s protocol. Briefly, an overlapping ∼40 nucleotide primer set was designed with the desired point mutations designed in the center of the primers. The template was amplified using AccuPrime Pfx DNA polymerase (Thermo Fisher Cat #12344024, Grand Island, NY). The template was methylated by adding DNA methylase and S-adenosyl methionine in the PCR, which was provided by the manufacturer. Following PCR, the free ends of the PCR product were recombined in vitro by the addition of recombinase enzyme mix. The recombined PCR product was transformed into E. coli cells (One Shot MAX Efficiency DH5α-T1R). The specially designed host E. coli strain that contains McrBc endonuclease digested the methylated template, leaving the nonmethylated PCR amplified mutated product intact.
7
Generating Site-Directed Mutants of GFP-FANCL
8
Constructing pEGFP-C3-Rbx1 Plasmid
9
Efficient β-catenin Manipulation in L6-G4-myc Cells
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For siRNA knockdown experiments, L6-G4-myc were transfected at 50% confluence with validated specific siRNA (Ctnnb1RSS331357; 5′-CCCAGAAUGCCGUUCGCCUUCAUUA-3′) or with control siRNA (Stealth™ RNAi siRNA Negative Control, Med GC). Transfections were performed in OptiMEM Reduced Serum Medium using Lipofectamine® 2000 and contained siRNA to a final concentration of 30 nM. For overexpression of β-catenin, L6-G4-myc or parental L6 cells were transfected with BcatWTdEGFP cDNA expression plasmid at 70% confluence using Lipofectamine® 2000 at a ratio of 1 ug DNA to 5 uL reagent. Prior to transfection, mutagenic primers were used to introduce the S552A amino acid change into the BcatWTdEGFP plasmid. Primer sequences were: 5′-AAGACATCACTGAGCCTGCC-3′ (forward) and 5′-GGCACGAGAGACTTGAGCTT-3′ (reverse). Mutagenesis PCR was performed using the GENEART® Site-Directed Mutagenesis Kit (ThermoFisher, Waltham, MA, USA) according to manufacturer's instructions, and the integrity of all vectors was confirmed by sequencing of the β-catenin and GFP cDNA regions. All reagents for transfection experiments were from Life Technologies and used according to the manufacturer's instructions. BcatWTdEGFP was a gift from Dr. Lucia Alonso-Gonzalez (University of Otago, Christchurch, New Zealand).
10
Mutagenesis of Human GRIN2A cDNA
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Mutagenesis was performed on the cDNA of human GRIN2A in the pCI-Neo vector (pCMV GluN2A). GluN2A mutations with respect to the amino acid numbering in NP_000824.1 were C436R, T531M, R518H, K669N, L812M. Oligonucleotide primers (Integrated DNA Technologies) were designed to incorporate single-base substitutions using the GeneArt® Site-Directed Mutagenesis Kit (Thermofisher Scientific) (Table S3 ). The presence of each mutation was confirmed by DNA sequencing (Eurofins Genomics) and the whole coding sequence was also examined to ensure that no other mutations were introduced by polymerase chain reaction (PCR) errors. Plasmid DNA was amplified and purified using EZNA endo-free plasmid maxi kit (VWR International).
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