q5 sdm kit  (New England Biolabs)


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    Name:
    Q5 Site Directed Mutagenesis Kit
    Description:
    Q5 Site Directed Mutagenesis Kit 10 rxns
    Catalog Number:
    e0554s
    Price:
    197
    Size:
    10 rxns
    Category:
    PCR Mutagenesis Kits
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    Structured Review

    New England Biolabs q5 sdm kit
    Q5 Site Directed Mutagenesis Kit
    Q5 Site Directed Mutagenesis Kit 10 rxns
    https://www.bioz.com/result/q5 sdm kit/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    q5 sdm kit - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin
    Article Snippet: To the shorter N-terminal MDC1 fragments, a triple nuclear localization signal was added by cloning the fragments into pCMV-myc/nuc (Invitrogen) vector and thereafter subcloning back into pcDNA4/TO by PCR. .. The QuikChange Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutations; deletions were made using the Phusion Site-Directed Mutagenesis kit (New England Biolabs, Inc.) and performed according to the manufacturer's instructions.

    Article Title: OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas
    Article Snippet: Paragraph title: Cloning and mutagenesis of the rat 1.1 kb Ovol1 promoter ... A point mutation in the E-box 1 was created using a mismatch in the mutagenic primer and a kit (Phusion Site-Directed Mutagenesis Kit; New England Biolabs, Ipswich, MA, USA).

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: DNA fragments were cloned into the pGL3-Basic plasmid. .. A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB).

    Article Title: SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated With Tumor Progression
    Article Snippet: Constructs The SMAD4 cDNA sequence was amplified from the cDNA of SAS cells with the primers SMAD4 _Forward and SMAD4 _Reverse, introducing BamHI and EcoRI sites for directional cloning. .. The p.H132Y, p.P296T, and p.A488V mutations were introduced into SMAD4 cDNA with the Q5 Site-Directed Mutagenesis kit (New England BioLabs, Ipswich, MA, USA) with the primers H132Ymut, P296Tmut, and A488Vmut.

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: Plasmid pET20b-6pgdh based on the two DNA fragments was obtained using a Simple Cloning method . .. The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit.

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: Paragraph title: Cloning and site-specific mutagenesis of CBP60g ... Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

    Article Title: MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF
    Article Snippet: In addition, Renilla luciferase (hRLuc) reporter driven by a CMV promoter was cloned into the same vector, serving as the tracking gene and internal control. .. The mutated pEZX-MT01 plasmid containing the mutated VEGF-3′UTR with mutation in the seed region was synthesized using Phusion™ site-directed mutagenesis kit (New England Biolabs.

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: However, the BtgZ1 enzyme site was found inefficient in cloning and was removed by SpeI and EcoR1 enzyme digestion and then replaced with an Arabidopsis U6 promoter‐driven sgRNA cassette (AtU6‐26 promoter/Linker/sgRNA scaffold/AtU6‐26 terminator) derived from pCAMBIA Cas9 + sgRNA (Jiang et al ., ). .. For this dual sgRNA entry vector, one seed RNA is inserted at the BsaI site driven by the FveU6‐2 promoter, and a second seed RNA is inserted into the JH4 vector via Q5 site‐directed mutagenesis kit (NEB, Ipswich, MA) and driven by the AtU6‐26 promoter.

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: Paragraph title: Cloning and mutagenesis ... All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation.

    Luciferase:

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: Paragraph title: Plasmid Construction, RNA Interference, Transfections, and Luciferase Assay. ... A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB).

    Article Title: MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF
    Article Snippet: Firefly and Renilla luciferase activities were measured using a Dual Luciferase Reporter Assay System kit (Promega Corp. WI, USA) and each transfected well was assayed in triplicate as described . .. The mutated pEZX-MT01 plasmid containing the mutated VEGF-3′UTR with mutation in the seed region was synthesized using Phusion™ site-directed mutagenesis kit (New England Biolabs.

    Reporter Assay:

    Article Title: MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF
    Article Snippet: Paragraph title: Dual-Luciferase Reporter Assay ... The mutated pEZX-MT01 plasmid containing the mutated VEGF-3′UTR with mutation in the seed region was synthesized using Phusion™ site-directed mutagenesis kit (New England Biolabs.

    Synthesized:

    Article Title: MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF
    Article Snippet: .. The mutated pEZX-MT01 plasmid containing the mutated VEGF-3′UTR with mutation in the seed region was synthesized using Phusion™ site-directed mutagenesis kit (New England Biolabs. ..

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: This synthesized fragment was inserted into the pENTR2b vector (Invitrogen, Carlsbad, CA) at SalI and EcoRI sites to yield JH2 vector. .. For this dual sgRNA entry vector, one seed RNA is inserted at the BsaI site driven by the FveU6‐2 promoter, and a second seed RNA is inserted into the JH4 vector via Q5 site‐directed mutagenesis kit (NEB, Ipswich, MA) and driven by the AtU6‐26 promoter.

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: The Arthrobacter species FB24 (Arsp-FB24) Arth_1007 intein precursor (locus tag Arth_1007, accession number YP_830503) with an N-terminal His tag was synthesized by DNA2.0, Inc (Menlo Park, CA). .. All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation.

    Construct:

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: Two guide RNA sequences were inserted into entry vector JH4 by BsaI digestion/ligation and Q5 site‐directed mutagenesis kit (NEB), respectively, through two primer pairs (Fv ARF8‐F/Fv ARF8‐R and Fv ARF8‐F3/Fv ARF8‐R3) (Table ). .. JH19‐FveTAA1 was constructed similarly using two different primer pairs (Fv TAA1‐F/Fv TAA1‐R and Fv TAA1‐F3/Fv TAA1‐R3) (Table ).

    Article Title: SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated With Tumor Progression
    Article Snippet: Paragraph title: Constructs ... The p.H132Y, p.P296T, and p.A488V mutations were introduced into SMAD4 cDNA with the Q5 Site-Directed Mutagenesis kit (New England BioLabs, Ipswich, MA, USA) with the primers H132Ymut, P296Tmut, and A488Vmut.

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: Cloning and site-specific mutagenesis of CBP60g To make the complementation and transgenic site-specific mutagenesis constructs, the genomic coding sequence (with introns) of At5g26920 and an additional 1093 base pairs of DNA sequence upstream of its start codon was first amplified by polymerase chain reaction (PCR) using KOD Hot Start DNA Polymerase (Novagen, CA) and TA-cloned into the pCR8 vector following the manufacturer's protocol (Invitrogen, CA). .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

    Article Title: Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting
    Article Snippet: Expression vectors The vector pEGFP_C1_NaPi2c_WT [ ] for the expression of a fusion protein of N-terminal enhanced green fluorescent protein (eGFP) connected by a linker sequence (SGLRSRAQASNS) to wild-type NaPi2c was constructed by ligation of the human SLC34A3 -sequence (NM_001177316.1) into the pEGFP-C1 vector backbone using EcoRI and SacII restriction sites that were added by mutagenesis [ ]. .. To establish the mutant plasmid pEGFP_C1_NaPi2c_S192L, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Massachusetts, USA) and custom-designed back-to-back primers (NaPi2c_S192L_Fw: 5′-GCTTTCAGCGGCTTGGCGGTGCAC-3′; NaPi2c_S192L_Rv: 5′- CCTCTGAAATTCATCCCGGTCCCCTG-3′) were used to introduce the base exchange 575C > T into pEGFP_C1_NaPi2c_WT according to the manufacturer’s instructions.

    Article Title: High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+
    Article Snippet: .. Construction of mutant libraries by saturation mutagenesis The two-round DNA mutant libraries were constructed by the NEB Phusion site-directed mutagenesis kit. .. In the first round, the single-site saturation mutagenesis library R31 was amplified based on pET28a-Ptac -6pgdh by using a pair of degenerate primers 31_NNK_F/31_NNK_R.

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: .. All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation. .. Expression, purification, and protein characterization Precursors were expressed in either the E. coli NEB Turbo strain or NEB Express strain (New England Biolabs) by induction with 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) at OD600 of 0.4–0.6 in 10 ml LB medium containing 100 µg/ml ampicillin for 2 hours at 37°C or 15°C overnight.

    Amplification:

    Article Title: OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas
    Article Snippet: The 1.1 kb sequence encompassing the 5′ region from −841 to +230 of the rat Ovol1 gene containing the E-box 1 was amplified by PCR using rat genomic DNA as template and cloned into the pGL2-Basic vector. .. A point mutation in the E-box 1 was created using a mismatch in the mutagenic primer and a kit (Phusion Site-Directed Mutagenesis Kit; New England Biolabs, Ipswich, MA, USA).

    Article Title: SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated With Tumor Progression
    Article Snippet: Constructs The SMAD4 cDNA sequence was amplified from the cDNA of SAS cells with the primers SMAD4 _Forward and SMAD4 _Reverse, introducing BamHI and EcoRI sites for directional cloning. .. The p.H132Y, p.P296T, and p.A488V mutations were introduced into SMAD4 cDNA with the Q5 Site-Directed Mutagenesis kit (New England BioLabs, Ipswich, MA, USA) with the primers H132Ymut, P296Tmut, and A488Vmut.

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: Site directed mutagenesis The Tm 6PGDH gene was amplified from plasmid by a primer pair of 6PGDH_IF and 6PGDH_IR. pET20b vector backbone was amplified with a primer pair of 6PGDH_VF and 6PGDH_VR. .. The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit.

    Article Title: Crystal Structure of Cardiac-specific Histone Methyltransferase SmyD1 Reveals Unusual Active Site Architecture *
    Article Snippet: SmyD1 mutants were prepared using the Phusion Site-directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's instructions. .. The amplified linear PCR product was then circularized in a ligation reaction with Quick T4 DNA Ligase (New England Biolabs).

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: Cloning and site-specific mutagenesis of CBP60g To make the complementation and transgenic site-specific mutagenesis constructs, the genomic coding sequence (with introns) of At5g26920 and an additional 1093 base pairs of DNA sequence upstream of its start codon was first amplified by polymerase chain reaction (PCR) using KOD Hot Start DNA Polymerase (Novagen, CA) and TA-cloned into the pCR8 vector following the manufacturer's protocol (Invitrogen, CA). .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

    Article Title: High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+
    Article Snippet: Construction of mutant libraries by saturation mutagenesis The two-round DNA mutant libraries were constructed by the NEB Phusion site-directed mutagenesis kit. .. In the first round, the single-site saturation mutagenesis library R31 was amplified based on pET28a-Ptac -6pgdh by using a pair of degenerate primers 31_NNK_F/31_NNK_R.

    Activity Assay:

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB). .. Luciferase activity was measured with the Dual-Glo Luciferase Assay system (Promega) and normalized to the Renilla internal control.

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: Mutations were made in the homing nuclease domain active site to block endonuclease activity (Asp123 Ala and Asp219 Ala). .. All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation.

    Cell Culture:

    Article Title: CRY1/2 selectively repress PPARδ and limit exercise capacity
    Article Snippet: Paragraph title: Cell culture and transfection ... Transfections in HEK 293T cells were carried out using calcium phosphate or polyethylenimine (PEI; Polysciences Inc #23966-2) following standard protocols. pcDNA3-2xFlag-mCRY1 and pcDNA3-2xFlag-mCRY2 are described in ( ). pcDNA3-Myc-mCRY1 and pcDNA3- Myc-mCRY2 are described in ( ). pcDNA3.2-mPPARα-V5, pcDNA3.2-mPPARδ-V5, pcDNA3.2-mPPARγ-V5 and pcDNA3-2xFlag-PPARδ-FL, pcDNA3-2xFlag-PPARδ-DBD, pcDNA3- 2xFlag-PPARδ-LBD are described in ( ; ). pcDNA3.2- mPPARδΔH12-V5 was generated using Q5 Site-Directed Mutagenesis kit and protocol (NEB #E0554S).

    Expressing:

    Article Title: Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting
    Article Snippet: Paragraph title: Expression vectors ... To establish the mutant plasmid pEGFP_C1_NaPi2c_S192L, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Massachusetts, USA) and custom-designed back-to-back primers (NaPi2c_S192L_Fw: 5′-GCTTTCAGCGGCTTGGCGGTGCAC-3′; NaPi2c_S192L_Rv: 5′- CCTCTGAAATTCATCCCGGTCCCCTG-3′) were used to introduce the base exchange 575C > T into pEGFP_C1_NaPi2c_WT according to the manufacturer’s instructions.

    Transformation Assay:

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit. .. The PCR product was digested by DpnI followed by the purification of gel electrophoresis using a Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Irvine, CA) and then transformed into E. coli BL21 (DE3).

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA). .. Arabidopsis transformation was carried out using Agrobacterium tumefaciens stain C58C1 as described by .

    Derivative Assay:

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: However, the BtgZ1 enzyme site was found inefficient in cloning and was removed by SpeI and EcoR1 enzyme digestion and then replaced with an Arabidopsis U6 promoter‐driven sgRNA cassette (AtU6‐26 promoter/Linker/sgRNA scaffold/AtU6‐26 terminator) derived from pCAMBIA Cas9 + sgRNA (Jiang et al ., ). .. For this dual sgRNA entry vector, one seed RNA is inserted at the BsaI site driven by the FveU6‐2 promoter, and a second seed RNA is inserted into the JH4 vector via Q5 site‐directed mutagenesis kit (NEB, Ipswich, MA) and driven by the AtU6‐26 promoter.

    Transfection:

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: Paragraph title: Plasmid Construction, RNA Interference, Transfections, and Luciferase Assay. ... A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB).

    Article Title: MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF
    Article Snippet: Firefly and Renilla luciferase activities were measured using a Dual Luciferase Reporter Assay System kit (Promega Corp. WI, USA) and each transfected well was assayed in triplicate as described . .. The mutated pEZX-MT01 plasmid containing the mutated VEGF-3′UTR with mutation in the seed region was synthesized using Phusion™ site-directed mutagenesis kit (New England Biolabs.

    Article Title: CRY1/2 selectively repress PPARδ and limit exercise capacity
    Article Snippet: .. Transfections in HEK 293T cells were carried out using calcium phosphate or polyethylenimine (PEI; Polysciences Inc #23966-2) following standard protocols. pcDNA3-2xFlag-mCRY1 and pcDNA3-2xFlag-mCRY2 are described in ( ). pcDNA3-Myc-mCRY1 and pcDNA3- Myc-mCRY2 are described in ( ). pcDNA3.2-mPPARα-V5, pcDNA3.2-mPPARδ-V5, pcDNA3.2-mPPARγ-V5 and pcDNA3-2xFlag-PPARδ-FL, pcDNA3-2xFlag-PPARδ-DBD, pcDNA3- 2xFlag-PPARδ-LBD are described in ( ; ). pcDNA3.2- mPPARδΔH12-V5 was generated using Q5 Site-Directed Mutagenesis kit and protocol (NEB #E0554S). .. In experiments shown in , HEK 293T cells were co-treated with 10μM MG-132 (Millipore #474790-10MG) and isoform-specific PPAR ligand for 6–8 hours using the following concentrations: GW1516 (Enzo #ALX-420-032-M001) 0.1μM and 1μM for PPARδ, WY-14643 (Enzo #GR-200) 1 μM and 10μM for PPARα, and Rosiglitazone (Cayman #71742) 10μM and 100μM for PPARγ.

    Ligation:

    Article Title: Crystal Structure of Cardiac-specific Histone Methyltransferase SmyD1 Reveals Unusual Active Site Architecture *
    Article Snippet: SmyD1 mutants were prepared using the Phusion Site-directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's instructions. .. The amplified linear PCR product was then circularized in a ligation reaction with Quick T4 DNA Ligase (New England Biolabs).

    Article Title: Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting
    Article Snippet: Expression vectors The vector pEGFP_C1_NaPi2c_WT [ ] for the expression of a fusion protein of N-terminal enhanced green fluorescent protein (eGFP) connected by a linker sequence (SGLRSRAQASNS) to wild-type NaPi2c was constructed by ligation of the human SLC34A3 -sequence (NM_001177316.1) into the pEGFP-C1 vector backbone using EcoRI and SacII restriction sites that were added by mutagenesis [ ]. .. To establish the mutant plasmid pEGFP_C1_NaPi2c_S192L, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Massachusetts, USA) and custom-designed back-to-back primers (NaPi2c_S192L_Fw: 5′-GCTTTCAGCGGCTTGGCGGTGCAC-3′; NaPi2c_S192L_Rv: 5′- CCTCTGAAATTCATCCCGGTCCCCTG-3′) were used to introduce the base exchange 575C > T into pEGFP_C1_NaPi2c_WT according to the manufacturer’s instructions.

    Introduce:

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: .. The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit. .. The mutant N32D, N32D/R33I/T34I, N32D/R33L/T34S, and N32E/R33I/T34I were prepared by their respective primer pairs ( ).

    Article Title: Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting
    Article Snippet: .. To establish the mutant plasmid pEGFP_C1_NaPi2c_S192L, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Massachusetts, USA) and custom-designed back-to-back primers (NaPi2c_S192L_Fw: 5′-GCTTTCAGCGGCTTGGCGGTGCAC-3′; NaPi2c_S192L_Rv: 5′- CCTCTGAAATTCATCCCGGTCCCCTG-3′) were used to introduce the base exchange 575C > T into pEGFP_C1_NaPi2c_WT according to the manufacturer’s instructions. .. Correctness of all sequences was verified by Sanger sequencing.

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: .. All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation. .. Expression, purification, and protein characterization Precursors were expressed in either the E. coli NEB Turbo strain or NEB Express strain (New England Biolabs) by induction with 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) at OD600 of 0.4–0.6 in 10 ml LB medium containing 100 µg/ml ampicillin for 2 hours at 37°C or 15°C overnight.

    Generated:

    Article Title: Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin
    Article Snippet: HA-MDC1 fragments were generated by PCR and subcloned into a pcDNA4/TO (Invitrogen) plasmid. .. The QuikChange Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutations; deletions were made using the Phusion Site-Directed Mutagenesis kit (New England Biolabs, Inc.) and performed according to the manufacturer's instructions.

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: .. A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB). .. The FOXO Reporter and Negative Control Reporter were from BPS Bioscience.

    Article Title: CRY1/2 selectively repress PPARδ and limit exercise capacity
    Article Snippet: .. Transfections in HEK 293T cells were carried out using calcium phosphate or polyethylenimine (PEI; Polysciences Inc #23966-2) following standard protocols. pcDNA3-2xFlag-mCRY1 and pcDNA3-2xFlag-mCRY2 are described in ( ). pcDNA3-Myc-mCRY1 and pcDNA3- Myc-mCRY2 are described in ( ). pcDNA3.2-mPPARα-V5, pcDNA3.2-mPPARδ-V5, pcDNA3.2-mPPARγ-V5 and pcDNA3-2xFlag-PPARδ-FL, pcDNA3-2xFlag-PPARδ-DBD, pcDNA3- 2xFlag-PPARδ-LBD are described in ( ; ). pcDNA3.2- mPPARδΔH12-V5 was generated using Q5 Site-Directed Mutagenesis kit and protocol (NEB #E0554S). .. In experiments shown in , HEK 293T cells were co-treated with 10μM MG-132 (Millipore #474790-10MG) and isoform-specific PPAR ligand for 6–8 hours using the following concentrations: GW1516 (Enzo #ALX-420-032-M001) 0.1μM and 1μM for PPARδ, WY-14643 (Enzo #GR-200) 1 μM and 10μM for PPARα, and Rosiglitazone (Cayman #71742) 10μM and 100μM for PPARγ.

    DNA Sequencing:

    Article Title: Crystal Structure of Cardiac-specific Histone Methyltransferase SmyD1 Reveals Unusual Active Site Architecture *
    Article Snippet: SmyD1 mutants were prepared using the Phusion Site-directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's instructions. .. After transforming into competent E. coli DH5α cells, the plasmid sequences were verified by DNA sequencing.

    Sequencing:

    Article Title: OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas
    Article Snippet: The 1.1 kb sequence encompassing the 5′ region from −841 to +230 of the rat Ovol1 gene containing the E-box 1 was amplified by PCR using rat genomic DNA as template and cloned into the pGL2-Basic vector. .. A point mutation in the E-box 1 was created using a mismatch in the mutagenic primer and a kit (Phusion Site-Directed Mutagenesis Kit; New England Biolabs, Ipswich, MA, USA).

    Article Title: SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated With Tumor Progression
    Article Snippet: Constructs The SMAD4 cDNA sequence was amplified from the cDNA of SAS cells with the primers SMAD4 _Forward and SMAD4 _Reverse, introducing BamHI and EcoRI sites for directional cloning. .. The p.H132Y, p.P296T, and p.A488V mutations were introduced into SMAD4 cDNA with the Q5 Site-Directed Mutagenesis kit (New England BioLabs, Ipswich, MA, USA) with the primers H132Ymut, P296Tmut, and A488Vmut.

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: .. The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit. .. The mutant N32D, N32D/R33I/T34I, N32D/R33L/T34S, and N32E/R33I/T34I were prepared by their respective primer pairs ( ).

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: Cloning and site-specific mutagenesis of CBP60g To make the complementation and transgenic site-specific mutagenesis constructs, the genomic coding sequence (with introns) of At5g26920 and an additional 1093 base pairs of DNA sequence upstream of its start codon was first amplified by polymerase chain reaction (PCR) using KOD Hot Start DNA Polymerase (Novagen, CA) and TA-cloned into the pCR8 vector following the manufacturer's protocol (Invitrogen, CA). .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

    Article Title: Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting
    Article Snippet: Expression vectors The vector pEGFP_C1_NaPi2c_WT [ ] for the expression of a fusion protein of N-terminal enhanced green fluorescent protein (eGFP) connected by a linker sequence (SGLRSRAQASNS) to wild-type NaPi2c was constructed by ligation of the human SLC34A3 -sequence (NM_001177316.1) into the pEGFP-C1 vector backbone using EcoRI and SacII restriction sites that were added by mutagenesis [ ]. .. To establish the mutant plasmid pEGFP_C1_NaPi2c_S192L, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Massachusetts, USA) and custom-designed back-to-back primers (NaPi2c_S192L_Fw: 5′-GCTTTCAGCGGCTTGGCGGTGCAC-3′; NaPi2c_S192L_Rv: 5′- CCTCTGAAATTCATCCCGGTCCCCTG-3′) were used to introduce the base exchange 575C > T into pEGFP_C1_NaPi2c_WT according to the manufacturer’s instructions.

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: For this dual sgRNA entry vector, one seed RNA is inserted at the BsaI site driven by the FveU6‐2 promoter, and a second seed RNA is inserted into the JH4 vector via Q5 site‐directed mutagenesis kit (NEB, Ipswich, MA) and driven by the AtU6‐26 promoter. .. To insert the target seed sequence, 5′‐gctc G[19 bp target sequence]‐3′ is annealed to a reverse‐strand oligo (5′‐aaac G[19 bp in reverse complement]‐3′) to create the double‐stranded target sequence, which is inserted into JH1 at the Bsa1 sites (Figure a).

    Binding Assay:

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: For testing CaM binding, mapping the CBP60g CBD, and identifying crucial amino acids of the CBP60g CBD, full length and various partial cDNA sequences of CBP60g (without the promoter or introns) was cloned into the pDEST15 vector (Invitrogen, CA) and expressed in E. coli . .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: The Arsp-FB24 Arth_1007 intein with 5 native extein residues on both sides was cloned by PCR into a model precursor termed MIP, with the intein flanked by the E. coli Maltose Binding Protein (M) and the ΔSal fragment of D. immitis paramyosin (P) as previously described , , , . .. All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation.

    Nucleic Acid Electrophoresis:

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit. .. The PCR product was digested by DpnI followed by the purification of gel electrophoresis using a Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Irvine, CA) and then transformed into E. coli BL21 (DE3).

    Mutagenesis:

    Article Title: Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin
    Article Snippet: .. The QuikChange Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutations; deletions were made using the Phusion Site-Directed Mutagenesis kit (New England Biolabs, Inc.) and performed according to the manufacturer's instructions. ..

    Article Title: OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas
    Article Snippet: .. A point mutation in the E-box 1 was created using a mismatch in the mutagenic primer and a kit (Phusion Site-Directed Mutagenesis Kit; New England Biolabs, Ipswich, MA, USA). .. Primers for quantitative PCR and RT-PCR experiments to detect pulled-down DNA fragments from ChIP experiments are in .

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: .. A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB). .. The FOXO Reporter and Negative Control Reporter were from BPS Bioscience.

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: .. Two guide RNA sequences were inserted into entry vector JH4 by BsaI digestion/ligation and Q5 site‐directed mutagenesis kit (NEB), respectively, through two primer pairs (Fv ARF8‐F/Fv ARF8‐R and Fv ARF8‐F3/Fv ARF8‐R3) (Table ). ..

    Article Title: SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated With Tumor Progression
    Article Snippet: .. The p.H132Y, p.P296T, and p.A488V mutations were introduced into SMAD4 cDNA with the Q5 Site-Directed Mutagenesis kit (New England BioLabs, Ipswich, MA, USA) with the primers H132Ymut, P296Tmut, and A488Vmut. ..

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: .. The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit. .. The mutant N32D, N32D/R33I/T34I, N32D/R33L/T34S, and N32E/R33I/T34I were prepared by their respective primer pairs ( ).

    Article Title: Crystal Structure of Cardiac-specific Histone Methyltransferase SmyD1 Reveals Unusual Active Site Architecture *
    Article Snippet: .. SmyD1 mutants were prepared using the Phusion Site-directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's instructions. .. Briefly, point mutations were introduced with the phosphorylated primers using PCR, in which pSUMO-SmyD1 (see above) containing the wild-type SmyD1 gene was used as PCR template.

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA). .. For determination of CaM binding and production of transgenic plants carrying mutated versions of CBP60g , site-specific mutagenesis was carried out beginning with a full-length cDNA clone or a genomic clone, respectively, in pCR8.

    Article Title: MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF
    Article Snippet: .. The mutated pEZX-MT01 plasmid containing the mutated VEGF-3′UTR with mutation in the seed region was synthesized using Phusion™ site-directed mutagenesis kit (New England Biolabs. ..

    Article Title: Bacterial encapsulins as orthogonal compartments for mammalian cell engineering
    Article Snippet: .. In order to generate encapsulin derivatives featuring C-terminal acidic peptides of magnetotactic bacteria Mms proteins that are implicated in mediation of magnetite formation either the C-terminal peptide of Mms6 (YAYMKSRDIESAQSDEEVELRDALA) or Mms7 (YVWARRRHGTPDLSDDALLAAAGEE) of Magnetospirillum magneticum were fused either to the inward-facing N-terminus of MxEncAFLAG or to the C-terminus of either the MxEncB or C using Q5® Site-Directed Mutagenesis. .. Low passage number HEK293T (ECACC: 12022001, obtained via Sigma-Aldrich) and CHO (ECACC: 85050302, obtained via Sigma-Aldrich) cells were cultured in advanced DMEM with 10 % FBS and penicillin–streptomycin at 100 µg/ml at 37 °C and 5% CO2 .

    Article Title: Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting
    Article Snippet: .. To establish the mutant plasmid pEGFP_C1_NaPi2c_S192L, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Massachusetts, USA) and custom-designed back-to-back primers (NaPi2c_S192L_Fw: 5′-GCTTTCAGCGGCTTGGCGGTGCAC-3′; NaPi2c_S192L_Rv: 5′- CCTCTGAAATTCATCCCGGTCCCCTG-3′) were used to introduce the base exchange 575C > T into pEGFP_C1_NaPi2c_WT according to the manufacturer’s instructions. .. Correctness of all sequences was verified by Sanger sequencing.

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: .. For this dual sgRNA entry vector, one seed RNA is inserted at the BsaI site driven by the FveU6‐2 promoter, and a second seed RNA is inserted into the JH4 vector via Q5 site‐directed mutagenesis kit (NEB, Ipswich, MA) and driven by the AtU6‐26 promoter. ..

    Article Title: High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+
    Article Snippet: .. Construction of mutant libraries by saturation mutagenesis The two-round DNA mutant libraries were constructed by the NEB Phusion site-directed mutagenesis kit. .. In the first round, the single-site saturation mutagenesis library R31 was amplified based on pET28a-Ptac -6pgdh by using a pair of degenerate primers 31_NNK_F/31_NNK_R.

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: .. All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation. .. Expression, purification, and protein characterization Precursors were expressed in either the E. coli NEB Turbo strain or NEB Express strain (New England Biolabs) by induction with 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) at OD600 of 0.4–0.6 in 10 ml LB medium containing 100 µg/ml ampicillin for 2 hours at 37°C or 15°C overnight.

    Article Title: CRY1/2 selectively repress PPARδ and limit exercise capacity
    Article Snippet: .. Transfections in HEK 293T cells were carried out using calcium phosphate or polyethylenimine (PEI; Polysciences Inc #23966-2) following standard protocols. pcDNA3-2xFlag-mCRY1 and pcDNA3-2xFlag-mCRY2 are described in ( ). pcDNA3-Myc-mCRY1 and pcDNA3- Myc-mCRY2 are described in ( ). pcDNA3.2-mPPARα-V5, pcDNA3.2-mPPARδ-V5, pcDNA3.2-mPPARγ-V5 and pcDNA3-2xFlag-PPARδ-FL, pcDNA3-2xFlag-PPARδ-DBD, pcDNA3- 2xFlag-PPARδ-LBD are described in ( ; ). pcDNA3.2- mPPARδΔH12-V5 was generated using Q5 Site-Directed Mutagenesis kit and protocol (NEB #E0554S). .. In experiments shown in , HEK 293T cells were co-treated with 10μM MG-132 (Millipore #474790-10MG) and isoform-specific PPAR ligand for 6–8 hours using the following concentrations: GW1516 (Enzo #ALX-420-032-M001) 0.1μM and 1μM for PPARδ, WY-14643 (Enzo #GR-200) 1 μM and 10μM for PPARα, and Rosiglitazone (Cayman #71742) 10μM and 100μM for PPARγ.

    Subcloning:

    Article Title: Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin
    Article Snippet: To the shorter N-terminal MDC1 fragments, a triple nuclear localization signal was added by cloning the fragments into pCMV-myc/nuc (Invitrogen) vector and thereafter subcloning back into pcDNA4/TO by PCR. .. The QuikChange Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutations; deletions were made using the Phusion Site-Directed Mutagenesis kit (New England Biolabs, Inc.) and performed according to the manufacturer's instructions.

    Purification:

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit. .. The PCR product was digested by DpnI followed by the purification of gel electrophoresis using a Zymoclean™ Gel DNA Recovery Kit (Zymo Research, Irvine, CA) and then transformed into E. coli BL21 (DE3).

    Polymerase Chain Reaction:

    Article Title: Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin
    Article Snippet: To the shorter N-terminal MDC1 fragments, a triple nuclear localization signal was added by cloning the fragments into pCMV-myc/nuc (Invitrogen) vector and thereafter subcloning back into pcDNA4/TO by PCR. .. The QuikChange Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutations; deletions were made using the Phusion Site-Directed Mutagenesis kit (New England Biolabs, Inc.) and performed according to the manufacturer's instructions.

    Article Title: OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas
    Article Snippet: The 1.1 kb sequence encompassing the 5′ region from −841 to +230 of the rat Ovol1 gene containing the E-box 1 was amplified by PCR using rat genomic DNA as template and cloned into the pGL2-Basic vector. .. A point mutation in the E-box 1 was created using a mismatch in the mutagenic primer and a kit (Phusion Site-Directed Mutagenesis Kit; New England Biolabs, Ipswich, MA, USA).

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: .. A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB). .. The FOXO Reporter and Negative Control Reporter were from BPS Bioscience.

    Article Title: SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated With Tumor Progression
    Article Snippet: The PCR product then was subcloned into the pBabe puro vector. .. The p.H132Y, p.P296T, and p.A488V mutations were introduced into SMAD4 cDNA with the Q5 Site-Directed Mutagenesis kit (New England BioLabs, Ipswich, MA, USA) with the primers H132Ymut, P296Tmut, and A488Vmut.

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit. .. PCR reaction solution (50 μL) containing 1 ng of plasmid template (pET20b-6pgdh ) was conducted as follows: 98 °C denaturation for 1 min; 20 cycles of 98 °C denaturation for 30 s, 60 °C annealing for 30 s and 72 °C extension for 2.5 min; and 72 °C extension for 5 min.

    Article Title: Crystal Structure of Cardiac-specific Histone Methyltransferase SmyD1 Reveals Unusual Active Site Architecture *
    Article Snippet: SmyD1 mutants were prepared using the Phusion Site-directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's instructions. .. Briefly, point mutations were introduced with the phosphorylated primers using PCR, in which pSUMO-SmyD1 (see above) containing the wild-type SmyD1 gene was used as PCR template.

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: Cloning and site-specific mutagenesis of CBP60g To make the complementation and transgenic site-specific mutagenesis constructs, the genomic coding sequence (with introns) of At5g26920 and an additional 1093 base pairs of DNA sequence upstream of its start codon was first amplified by polymerase chain reaction (PCR) using KOD Hot Start DNA Polymerase (Novagen, CA) and TA-cloned into the pCR8 vector following the manufacturer's protocol (Invitrogen, CA). .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

    Article Title: High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+
    Article Snippet: Construction of mutant libraries by saturation mutagenesis The two-round DNA mutant libraries were constructed by the NEB Phusion site-directed mutagenesis kit. .. PCR reaction solution (50 μL) containing 1 ng of plasmid template was conducted as follows: 98 °C denaturation for 1 min; 20 cycles of 98 °C denaturation for 30 s, 60 °C annealing for 30 s and 72 °C extension for 3 min; and 72 °C extension for 5 min.

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: The Arsp-FB24 Arth_1007 intein with 5 native extein residues on both sides was cloned by PCR into a model precursor termed MIP, with the intein flanked by the E. coli Maltose Binding Protein (M) and the ΔSal fragment of D. immitis paramyosin (P) as previously described , , , . .. All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation.

    Blocking Assay:

    Article Title: The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene
    Article Snippet: Mutations were made in the homing nuclease domain active site to block endonuclease activity (Asp123 Ala and Asp219 Ala). .. All mutations were constructed using the Phusion™ site-directed mutagenesis kit (New England Biolabs) with appropriate primers to introduce the desired mutation.

    CRISPR:

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: Paragraph title: CRISPR/Cas9 editing of ARF8 and TAA1 ... Two guide RNA sequences were inserted into entry vector JH4 by BsaI digestion/ligation and Q5 site‐directed mutagenesis kit (NEB), respectively, through two primer pairs (Fv ARF8‐F/Fv ARF8‐R and Fv ARF8‐F3/Fv ARF8‐R3) (Table ).

    Plasmid Preparation:

    Article Title: Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage–modified chromatin
    Article Snippet: To the shorter N-terminal MDC1 fragments, a triple nuclear localization signal was added by cloning the fragments into pCMV-myc/nuc (Invitrogen) vector and thereafter subcloning back into pcDNA4/TO by PCR. .. The QuikChange Site-Directed Mutagenesis kit (Stratagene) was used to generate point mutations; deletions were made using the Phusion Site-Directed Mutagenesis kit (New England Biolabs, Inc.) and performed according to the manufacturer's instructions.

    Article Title: OVO homologue-like 1 (Ovol1) transcription factor: a novel target of neurogenin-3 in rodent pancreas
    Article Snippet: The 1.1 kb sequence encompassing the 5′ region from −841 to +230 of the rat Ovol1 gene containing the E-box 1 was amplified by PCR using rat genomic DNA as template and cloned into the pGL2-Basic vector. .. A point mutation in the E-box 1 was created using a mismatch in the mutagenic primer and a kit (Phusion Site-Directed Mutagenesis Kit; New England Biolabs, Ipswich, MA, USA).

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: Paragraph title: Plasmid Construction, RNA Interference, Transfections, and Luciferase Assay. ... A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB).

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: .. Two guide RNA sequences were inserted into entry vector JH4 by BsaI digestion/ligation and Q5 site‐directed mutagenesis kit (NEB), respectively, through two primer pairs (Fv ARF8‐F/Fv ARF8‐R and Fv ARF8‐F3/Fv ARF8‐R3) (Table ). ..

    Article Title: SMAD4 Somatic Mutations in Head and Neck Carcinoma Are Associated With Tumor Progression
    Article Snippet: The PCR product then was subcloned into the pBabe puro vector. .. The p.H132Y, p.P296T, and p.A488V mutations were introduced into SMAD4 cDNA with the Q5 Site-Directed Mutagenesis kit (New England BioLabs, Ipswich, MA, USA) with the primers H132Ymut, P296Tmut, and A488Vmut.

    Article Title: Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries
    Article Snippet: Plasmid pET20b-6pgdh based on the two DNA fragments was obtained using a Simple Cloning method . .. The QuickChangeTM site-directed mutagenesis method (Stratagene, La Jolla, CA) was used to introduce point mutations into the Tm 6PGDH sequence according to the protocol of the NEB Phusion site-directed mutagenesis kit.

    Article Title: Crystal Structure of Cardiac-specific Histone Methyltransferase SmyD1 Reveals Unusual Active Site Architecture *
    Article Snippet: SmyD1 mutants were prepared using the Phusion Site-directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's instructions. .. After transforming into competent E. coli DH5α cells, the plasmid sequences were verified by DNA sequencing.

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: For testing CaM binding, mapping the CBP60g CBD, and identifying crucial amino acids of the CBP60g CBD, full length and various partial cDNA sequences of CBP60g (without the promoter or introns) was cloned into the pDEST15 vector (Invitrogen, CA) and expressed in E. coli . .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

    Article Title: MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF
    Article Snippet: .. The mutated pEZX-MT01 plasmid containing the mutated VEGF-3′UTR with mutation in the seed region was synthesized using Phusion™ site-directed mutagenesis kit (New England Biolabs. ..

    Article Title: Evaluating pathogenicity of SLC34A3-Ser192Leu, a frequent European missense variant in disorders of renal phosphate wasting
    Article Snippet: .. To establish the mutant plasmid pEGFP_C1_NaPi2c_S192L, the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, Massachusetts, USA) and custom-designed back-to-back primers (NaPi2c_S192L_Fw: 5′-GCTTTCAGCGGCTTGGCGGTGCAC-3′; NaPi2c_S192L_Rv: 5′- CCTCTGAAATTCATCCCGGTCCCCTG-3′) were used to introduce the base exchange 575C > T into pEGFP_C1_NaPi2c_WT according to the manufacturer’s instructions. .. Correctness of all sequences was verified by Sanger sequencing.

    Article Title: Efficient genome editing of wild strawberry genes, vector development and validation
    Article Snippet: .. For this dual sgRNA entry vector, one seed RNA is inserted at the BsaI site driven by the FveU6‐2 promoter, and a second seed RNA is inserted into the JH4 vector via Q5 site‐directed mutagenesis kit (NEB, Ipswich, MA) and driven by the AtU6‐26 promoter. ..

    Article Title: High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+
    Article Snippet: Construction of mutant libraries by saturation mutagenesis The two-round DNA mutant libraries were constructed by the NEB Phusion site-directed mutagenesis kit. .. The two-site saturation mutagenesis library A30/T32 was amplified from plasmid of pET28a-Ptac -6pgdh (R31I) by using a pair of degenerate primers 30_32_NNK_F/30_32_NNK_R.

    Negative Control:

    Article Title: Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis
    Article Snippet: A series of ZEB2 promoter fragments containing mutations were generated by PCR mutagenesis (Q5 Site-Directed Mutagenesis Kit from NEB). .. The FOXO Reporter and Negative Control Reporter were from BPS Bioscience.

    Transgenic Assay:

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: Cloning and site-specific mutagenesis of CBP60g To make the complementation and transgenic site-specific mutagenesis constructs, the genomic coding sequence (with introns) of At5g26920 and an additional 1093 base pairs of DNA sequence upstream of its start codon was first amplified by polymerase chain reaction (PCR) using KOD Hot Start DNA Polymerase (Novagen, CA) and TA-cloned into the pCR8 vector following the manufacturer's protocol (Invitrogen, CA). .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

    Staining:

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA). .. Arabidopsis transformation was carried out using Agrobacterium tumefaciens stain C58C1 as described by .

    Chick Chorioallantoic Membrane Assay:

    Article Title: Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae
    Article Snippet: For testing CaM binding, mapping the CBP60g CBD, and identifying crucial amino acids of the CBP60g CBD, full length and various partial cDNA sequences of CBP60g (without the promoter or introns) was cloned into the pDEST15 vector (Invitrogen, CA) and expressed in E. coli . .. Site-specific mutagenesis of CBP60g was performed using the Phusion™ Site-Directed Mutagenesis Kit (New England Biolabs Inc., MA USA).

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    New England Biolabs q5 sdm kit
    Q5 Sdm Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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