The cytomegalovirus (CMV) myc-CREB plasmid was created by B. Lonze (Ginty Lab). msMBD3, ratRBBP4, and ratRBBP7 were subcloned into the CMV-myc expression vector. Mutagenesis to create RBBP7, RBBP4 and MBD3 cystine-to-serine mutants, MBD3 lysine-to-alanine mutants, and mycRBBP7WT and mycRBBP7C166S siRNA-resistant plasmids was carried out using a QuikChange multisite-directed mutagenesis kit (Agilent). HA-tagged ratRBBP7 and HA-tagged msHDAC2 were each cloned into AdEasy pShuttle-CMV vectors, and cysteine and lysine mutants were created using QuikChange multisite-directed mutagenesis kit (Agilent). Flag-mCHD4, hCHD5, and hCHD3 were generated as previously described (21 (link)). siRNAs were generated by Invitrogen. The following sequences were used: siRBBP7, 5′-CCACAUAAUGAAACUAUUCUGGCUU-3′ (forward) and 5′-AAGCCAGAAUAGUUUCAUUAUGUGG-3′ (reverse); siRBBP4, 5′-AAAUCUUUCCCUUCAGGCCUGGUCA-3′ (forward) and 5′-UGACCAGGCCUGAAGGGAAA-GAUUU-3′ (reverse).
Quikchange multi site directed mutagenesis kit
Manufactured by Agilent Technologies
Sourced in United States, United Kingdom
The QuikChange Multi Site-Directed Mutagenesis Kit is a laboratory tool designed for introducing multiple site-specific mutations in plasmid DNA simultaneously. It provides a simple and efficient method for generating variants of a given DNA sequence.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (12 mentions)
Dual luciferase reporter assay system, by Promega (8 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (4 mentions)
Psicheck 2 vector, by Promega (4 mentions)
Dual glo luciferase assay system, by Promega (4 mentions)
Pmirglo dual luciferase mirna target expression vector, by Promega (4 mentions)
Pcdna3, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay kit, by Promega (3 mentions)
Dual luciferase assay system, by Promega (3 mentions)
I gcapture solution, by iNtRON Biotechnology (2 mentions)
Pegfp c1, by Takara Bio (2 mentions)
Gibson assembly master mix, by New England Biolabs (2 mentions)
Q5 high fidelity 2x master mix, by New England Biolabs (2 mentions)
Pselect zeo vector, by InvivoGen (2 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter system, by Promega (2 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Pmirglo, by Promega (2 mentions)
Xl10 gold cells, by Agilent Technologies (2 mentions)
129 protocols using quikchange multi site directed mutagenesis kit
1
Generating Cysteine and Lysine Mutants
2
Site-Directed Mutagenesis Protocol
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All mutants used in this study were generated by using QuikChange Site-directed mutagenesis or QuikChange Multi site-directed mutagenesis kits (Agilent) according to the manufacturer’s instructions.
3
Purification of H6-tagged Ec-RNase III
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Site-directed mutagenesis was accomplished using QuikChange multisite-directed mutagenesis kits (Agilent Technologies) and a single mutagenic oligodeoxynucleotide (sequences available on request). Ec-RNase III and mutant versions were overproduced in H6-tagged form using E. coli BL21(DE3)recA,rnc105 cells41 (link) that carried the respective recombinant pET-15b plasmid, and purified by affinity chromatography essentially as described41 (link). Briefly summarized, cells were grown with vigorous aeration to mid-log phase at 37 °C in LB media containing ampicillin. Approximately 4 hours following addition of IPTG (1 mM final concentration), cells were collected by centrifugation at 4 °C. The pellet was resuspended in column loading buffer, sonicated until lysis was complete, then centrifuged and the clarified supernatant applied to a Ni2+-NTA column. Following repeated column washes, the fractions containing the eluted protein were collected and dialysed against 1 M NaCl, 60 mM Tris-HCl (pH 7.9), 1 mM EDTA, 1 mM DTT for 12 hr at 4 °C (EDTA and DTT can be omitted). An equal volume of glycerol was added and the protein stored at − 20 °C. The H6-tag was removed as needed by treatment with biotinylated thrombin as described24 (link).
4
Generation of TSPYL5, ALDH1A1, ALDH1A3, AKT1, and PTEN Expression Vectors
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Complementary DNAs encoding full-length human TSPYL5, ALDH1A1, ALDH1A3, AKT1, and PTEN were prepared by RT-PCR using total RNA isolated from H460 or A549 cells. The PCR primers that were used are listed in Supplementary Table 2 . The i-GCapture solution (iNtRON Biotechnology, Seongnam, Korea) was added to the PCR mixture to amplify the GC-rich TSPYL5 template. Each cDNA insert was cloned into the HindIII and EcoRI sites of pcDNA3.1 (Invitrogen). The alanine-substituted TSPYL5 mutant (T120A-TSPYL5, T120D-TSPYL5, T177A-TSPYL5, T326A-TSPYL5, and T409A-TSPYL5) expression vectors were generated using QuikChange Multi Site-Directed Mutagenesis Kits (Agilent Technologies, Santa Clara, CA, USA; mutagenesis primer sequences are listed in Supplementary Table 3 ).
5
Mutagenesis of NCX1 Intracellular Cysteine Residues
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Mutagenesis reactions were performed with the Quikchange Lightning and Quikchange Multi Site-Directed Mutagenesis kits (Agilent, Santa Clara, CA, USA). Individual cysteines were mutated to alanines using the Quikchange Lightning Site-Directed Mutagenesis Kit (Agilent). The mutagenesis primer pairs used were as follows: C383A: forward, CAAGGGACCTATCAGGCTCTGGAGAACTGTGGG, reverse, CCCACAGTTCTCCAGAGCCTGATAGCTCCCTTG; C387A: forward, CAGTGTCTGGAGAACGCTGGGACTGTAGCCC, reverse, GGGCTACAGTCCCAGCGTTCTCCAGACACTG; C383A/C387A: forward, GGGACCTATCAGGCTCTGGAGAACGCTGGGACTGTAGCC, reverse, GGCTACAGTCCCAGCGTTCTCCAGAGCCTGATAGGTCCC; C485A: forward, GTTTCTGCGCTCGCTGCCCTGGGATCTCCC, reverse, GGGAGATCCCAGGGCAGCGAGCGCAGAAAC; C557A: forward, GGACTTTGAGGACACTGCTGGAGAGCTCGAATTCC, reverse, GGAATTCGAGCTCTCCAGCAGTGTCCTCAAAGTCC; C731A: forward, GATGATGACGACGATGAAGCTGGAGAGGAGAAGCTG, reverse, CAGCTTCTCCTCTCCAGCTTCATCGTCGTCATCATC; C739A: forward, GGAGAAGCTGCCCTCCGCTTTCGATTATGTGATGC, reverse, GCATCACATAATCGAAAGCGGAGGGCAGCTTCTCC; C914A: forward, CAAGCTCCTCACATCCGCCCTCTTCGTGCTCCTATG, reverse, CATAGGAGCACGAAGAGGGCGGATGTGAGGAGCTTGG.
A cys-less NCX1 intracellular loop (cysteines 383, 387, 485, 557, 731, 739 all mutated to alanine) was created using the Quikchange Multi Site-Directed Mutagenesis Kit, which requires forward primers only. The same mutagenesis reaction also yielded all other mutants described.
A cys-less NCX1 intracellular loop (cysteines 383, 387, 485, 557, 731, 739 all mutated to alanine) was created using the Quikchange Multi Site-Directed Mutagenesis Kit, which requires forward primers only. The same mutagenesis reaction also yielded all other mutants described.
6
Cloning and Mutagenesis of Human Tumor Suppressors
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Complementary DNAs encoding full-length human TSPYL5, ALDH1A1, ALDH1A3, AKT1, and PTEN were prepared by RT-PCR using total RNA isolated from H460 or A549 cells. The PCR primers that were used are listed in Supplementary Table 2. The i-GCapture solution (iNtRON Biotechnology, Seongnam, Korea) was added to the PCR mixture to amplify the GC-rich TSPYL5 template. Each cDNA insert was cloned into the HindIII and EcoRI sites of pcDNA3.1 (Invitrogen). The alanine-substituted TSPYL5 mutant (T120A-TSPYL5, T120D-TSPYL5, T177A-TSPYL5, T326A-TSPYL5, and T409A-TSPYL5) expression vectors were generated using QuikChange Multi Site-Directed Mutagenesis Kits (Agilent Technologies, Santa Clara, CA, USA; mutagenesis primer sequences are listed in Supplementary Table 3).
7
Construction of NAP1 Gene Deletion Mutants
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Strain BWP17 (43 (link)) was used to create all the gene deletion mutants in this study. A gene deletion cassette contained a selection marker gene, URA3, HIS1, or ARG4, flanked with DNA fragments corresponding to the 5′ and 3′ untranslated region of the gene. The two copies of NAP1 were deleted with the recyclable hisG-URA3-hisG cassette, so that all three selection markers (HIS1, ARG4, and URA3) can be used for subsequent genetic manipulations. All gene deletion mutants were verified by PCR. To reintegrate the WT or mutated NAP1 gene into the nap1Δ/Δ mutant, the full-length NAP1 open reading frame (ORF) together with the 971-bp upstream promoter region sequence was PCR amplified with KpnI and XhoI sites added to the 5′ and 3′ ends, respectively. The amplified DNA fragment was cloned in frame with a C-terminal Myc, HA, or GFP tag in the CIP10 vector (44 (link)). A PmeI site was generated in the promoter region for linearization by site-directed mutagenesis using the QuikChange multi-site-directed mutagenesis kit (Agilent Technologies). The same mutagenesis procedure was used to generate all NAP1 mutants in this study. Mutated constructs were integrated into the HZX01 strain at the endogenous NAP1 promoter region.
8
Characterization of Cx36 Mutant Variants
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Electrophysiological measurements were performed using HeLa (human cervix carcinoma cells, ATCC CCL2) cells transfected with wild type mouse Cx36 fused with enhanced green fluorescent protein (EGFP). Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum. Cells were maintained in a 5% CO2 incubator in a moist atmosphere at 37 OC. Media and culture reagents were obtained from Sigma-Aldrich, Germany. Single point mutations, Cx36*E8Q-EGFP and Cx36*E12Q-EGFP, and double point mutation, Cx36*E8Q-E12Q-EGFP were generated using the QuikChange Multi Site-directed mutagenesis kit (Agilent, USA). Mutants were subcloned into pIRESPuro2 vector (Clontech, USA). Transfection procedures were performed using Lipofectamine 2000 (Life technologies, USA) following the manufacturer's protocol.
9
Yeast Strain Engineering Protocols
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Yeast strains (Table S2) are based on the S288C background, and genetic modifications were introduced using standard procedures. Bim1-YE/AA-FLAG, HA-Kog1-FL, and HA-kog1ΔYF strains were generated by subcloning the 3′ UTR (500 bp), 5′ UTR (500 bp), and the respective ORFs and tags into a pRS306 plasmid (for plasmids, see Table S1). Mutations were introduced using the Phusion Site-Directed Mutagenesis kit (Thermo Fisher Scientific) or QuikChange Multi Site-Directed Mutagenesis kit (Agilent Technologies). Linearized vector constructs were integrated, replacing the endogenous locus. Stu2-*NES, stu2-2A, and stu2-2D mutants were generated by overlapping PCR with primers containing the required mutations and transforming the PCR product into cells. The stu2-NLS-GFP strain was generated by introducing the NLS coding sequence of SV40 large T antigen (PKKKRKV) into the forward PCR primer.
10
Generation and Characterization of p62, DAXX Mutants
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Plasmids generated in this study are listed in Supplementary Table 1 . Flag-DAXX (#27974), pCE-BiFC-VC155 (#22020), pCE-BiFC-VN173 (#22019) and LentiCRISPRv2 (#52961) were from Addgene. HA-DAXX was a kind gift from Dr Hsiu-Ming Shih. pMXs-puro-GFP-p62 K7A D69A was from Addgene (Plasmid #38281) gifted by N. Mizushima. p62 or DAXX point mutants were generated with the Quikchange Multi Site-directed Mutagenesis kit according to manufacturing instruction (Agilent Technologies, #200514).
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