ecorv hf  (New England Biolabs)


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    Name:
    EcoRV HF
    Description:
    EcoRV HF 20 000 units
    Catalog Number:
    r3195l
    Price:
    244
    Size:
    20 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs ecorv hf
    EcoRV HF
    EcoRV HF 20 000 units
    https://www.bioz.com/result/ecorv hf/product/New England Biolabs
    Average 90 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    ecorv hf - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing"

    Article Title: The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2016.00070

    (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.
    Figure Legend Snippet: (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Techniques Used: Plasmid Preparation, Expressing, Immunocytochemistry, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Transfection, Isolation, Purification, Clone Assay

    Related Articles

    Clone Assay:

    Article Title: A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae) 1
    Article Snippet: .. For the CESA5 entry clones, 2.5 units each of EcoRI-HF and EcoRV-HF were used in 20-μL reactions according to the manufacturer’s instructions (New England Biolabs). .. Sequence a plasmid with the expected restriction fragment pattern to verify the mutation and the absence of PCR errors.

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: Cloning reactions were performed by Gibson assembly using GeneArt Seamless Cloning and Assembly Kit (Life Technologies). .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs).

    Article Title: Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting
    Article Snippet: All synthetic clones were ordered as minigenes in the pIDTBlue vector (IDT), which incorporates a T7 promoter to allow for in vitro transcription. .. The minigenes were linearized with EcoRV-HF (NEB), purified using a PCR cleanup protocol with EconoSpin columns (Epoch Life Sciences), and in vitro transcribed using a T7 High Yield RNA Synthesis Kit (NEB E2040S) according to the manufacturer’s instructions, with subsequent deoxyribonuclease I treatment.

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant CBF3−CEN3 complex ... CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight.

    Amplification:

    Article Title: Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase
    Article Snippet: To construct the pGRA-ROP17-3xHA plasmid, pGRA1plus -HPT-3xHA plasmid ( ) was first digested using EcoRV-HF and NcoI (New England Biolabs) for 4 h at 37°C to remove the GRA1 promoter. .. The empty vector backbone was amplified by PCR using Herculase II polymerase (Agilent) and primers 5′-CACATTTGTGTCACCCCAAATGAGAATTCGATATCAAGCTTGATCAGCAC-3′ and 5′-GAGGCGGCTTTATTACAGAAGGAGCCATGGTACCCGTACGACGTCCCG-3′ with each having 23- and 24-bp overhangs to ROP17 , respectively.

    Article Title: Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting
    Article Snippet: Synthetic antibody RNA spike-ins Sixteen synthetic spike-in clones were designed to incorporate a 5′ constant region (for singleplex PCR amplification), 7 different V-genes, 16 unique CDR3s (amino acid sequences), a J-gene, a partial IgG constant region (with a synthetic portion for a separate ddPCR probe and bioinformatic identification), a 3′ synthetic spacer (to make all clones identical in RNA length), and a final 3′ Eco RV site (to enable efficient run-off transcription) (fig. S2 and table S8). .. The minigenes were linearized with EcoRV-HF (NEB), purified using a PCR cleanup protocol with EconoSpin columns (Epoch Life Sciences), and in vitro transcribed using a T7 High Yield RNA Synthesis Kit (NEB E2040S) according to the manufacturer’s instructions, with subsequent deoxyribonuclease I treatment.

    Article Title: Genomic changes in the biological control agent Cryptolaemus montrouzieri associated with introduction. Genomic changes in the biological control agent Cryptolaemus montrouzieri associated with introduction
    Article Snippet: 2.2 Reduced‐representation genome sequencing Approximately 500 ng of genomic DNA from each sample was processed for reduced‐representation genome sequencing using the specific‐locus amplified fragment sequencing (SLAF‐seq) technique (Sun et al., ), with slight modifications. .. Briefly, in a pilot SLAF experiment, the restriction enzymes Hpy166II and EcoRV‐HF (NEB, USA) were selected because they generate abundant and high‐quality nonrepetitive SLAFs that are relatively evenly distributed across the genome of the model beetle species Tribolium castaneum (Richards et al., ).

    Synthesized:

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs). .. To obtain final constructs, inserts were either obtained by PCR, or synthesized and then assembled into vector backbones linearized by restriction digests.

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight. .. For CEN3 CDEIII-end biotin-avidin labeling, two PCR primers were synthesized (Sigma) with a 5’-biotin modification on the reverse primer.

    Construct:

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: Vector backbones were constructed in silico then assembled from compatible devices as described previously . .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs).

    Article Title: Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase
    Article Snippet: .. To construct the pGRA-ROP17-3xHA plasmid, pGRA1plus -HPT-3xHA plasmid ( ) was first digested using EcoRV-HF and NcoI (New England Biolabs) for 4 h at 37°C to remove the GRA1 promoter. .. Product was incubated with Antarctic phosphatase (New England Biolabs) and gel extracted.

    Incubation:

    Article Title: Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials
    Article Snippet: .. Prior to rheology measurements, 1624 ng sPX (final concentration of 0.24 × 10−6 m in 175 μL final sample volume; R = 0.01) were incubated with 5 μL of EcoRV-HF (20 000 units per milliliter, New England Biolabs Inc., USA) and 1.75 μL of 10x CutSmart buffer in a final volume of 20 μL for 1 h at 37 °C. .. Corresponding PAGE analysis showed a digestion rate of at least 80% after 1 h of incubation (Figure S2, ).

    Article Title: Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials
    Article Snippet: .. Three different samples were prepared similar to as described above and 150 μL of final sample solutions was pipetted into a small cuvette (first sample: 24 × 10−6 m actin; second sample: 24 × 10−6 m actin with synthetic crosslinkers featuring two phalloidin binding domains (sPX) at R = 0.4; third sample: 24 × 10−6 m actin with sPX at R = 0.4, which were incubated for 1 h at 37 °C with 300 units of EcoRV-HF (New England Biolabs Inc., USA)) prior to the experiments. ..

    Article Title: A Requirement for Global Transcription Factor Lrp in Licensing Replication of Vibrio cholerae Chromosome 2
    Article Snippet: Plates were incubated for 16 h at 37°C and colonies were monitored for development of red color. .. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, CA, United States), digested with EcoRV-HF (NEB, MA, United States), and ligated overnight at 16°C with T4 DNA ligase (NEB).

    Article Title: Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase
    Article Snippet: To construct the pGRA-ROP17-3xHA plasmid, pGRA1plus -HPT-3xHA plasmid ( ) was first digested using EcoRV-HF and NcoI (New England Biolabs) for 4 h at 37°C to remove the GRA1 promoter. .. Product was incubated with Antarctic phosphatase (New England Biolabs) and gel extracted.

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight. .. The PCR product of CEN3biotin was incubated in PBS with avidin protein (Sigma) on ice for 30 min, resulting in CEN3biotin-avidin .

    Luciferase:

    Article Title: The delivery of mRNA to colon inflammatory lesions by lipid-nano-particles containing environmentally-sensitive lipid-like materials with oleic acid scaffolds
    Article Snippet: 2.10 In vitro transcription (IVT) of mRNA pDNA containing Luciferase cassette under a T7 promoter (pcDNA3.1-(+)-Luc(0)) was used as a template for the in vitro transcription. .. The pDNA was linearized using EcoRV-HF (New England Biolab).

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

    In Silico:

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: Vector backbones were constructed in silico then assembled from compatible devices as described previously . .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs).

    Expressing:

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs). ..

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant CBF3−CEN3 complex ... CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight.

    Modification:

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: The C-termini of Ndc10 and Ndc10DBD were modified with a TEV-His tag for Ndc10 and Ndc10DBD purification. .. CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight.

    Transformation Assay:

    Article Title: A Requirement for Global Transcription Factor Lrp in Licensing Replication of Vibrio cholerae Chromosome 2
    Article Snippet: The transformation mixtures were spread first on LB plates with appropriate antibiotics and incubated overnight at 37°C. .. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, CA, United States), digested with EcoRV-HF (NEB, MA, United States), and ligated overnight at 16°C with T4 DNA ligase (NEB).

    Genetically Modified:

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: Construction of Mammalian Plasmid pVITRO-Luc2 The manipulation of genetic material and generation of genetically modified organisms were approved by the UTS Biosafety Committee (2001-19-R-GC; 2009-02-R-GC). .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ).

    Chromatography:

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: .. CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight. .. For CEN3 CDEIII-end biotin-avidin labeling, two PCR primers were synthesized (Sigma) with a 5’-biotin modification on the reverse primer.

    Ligation:

    Article Title: A Requirement for Global Transcription Factor Lrp in Licensing Replication of Vibrio cholerae Chromosome 2
    Article Snippet: Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, CA, United States), digested with EcoRV-HF (NEB, MA, United States), and ligated overnight at 16°C with T4 DNA ligase (NEB). .. The ligation product was used to transform DH5α(λpir ) cells to recover transposon containing circularized genomic DNA, which were replication competent by virtue of the presence of R6Kγori within the transposon.

    Generated:

    Article Title: Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting
    Article Snippet: The minigenes were linearized with EcoRV-HF (NEB), purified using a PCR cleanup protocol with EconoSpin columns (Epoch Life Sciences), and in vitro transcribed using a T7 High Yield RNA Synthesis Kit (NEB E2040S) according to the manufacturer’s instructions, with subsequent deoxyribonuclease I treatment. .. A master pool of spike-in cDNA mixture was generated by reverse transcription (as described earlier), but with 500 ng of RNA mixture input per 20 μl of reaction.

    Sequencing:

    Article Title: A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae) 1
    Article Snippet: For the CESA5 entry clones, 2.5 units each of EcoRI-HF and EcoRV-HF were used in 20-μL reactions according to the manufacturer’s instructions (New England Biolabs). .. Sequence a plasmid with the expected restriction fragment pattern to verify the mutation and the absence of PCR errors.

    Article Title: Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials
    Article Snippet: Prior to rheology measurements, 1624 ng sPX (final concentration of 0.24 × 10−6 m in 175 μL final sample volume; R = 0.01) were incubated with 5 μL of EcoRV-HF (20 000 units per milliliter, New England Biolabs Inc., USA) and 1.75 μL of 10x CutSmart buffer in a final volume of 20 μL for 1 h at 37 °C. .. No detectable digestion of control DNA was observed due to sequence specificity of the enzyme (data not shown).

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: Each spike-in sequence contained a T7 promoter for in vitro transcription. .. Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter).

    Article Title: Genomic changes in the biological control agent Cryptolaemus montrouzieri associated with introduction. Genomic changes in the biological control agent Cryptolaemus montrouzieri associated with introduction
    Article Snippet: Paragraph title: Reduced‐representation genome sequencing ... Briefly, in a pilot SLAF experiment, the restriction enzymes Hpy166II and EcoRV‐HF (NEB, USA) were selected because they generate abundant and high‐quality nonrepetitive SLAFs that are relatively evenly distributed across the genome of the model beetle species Tribolium castaneum (Richards et al., ).

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: To prepare CEN3 DNA, 24 copies of the 147 base pairs of CEN3 (sequence: ATC AAA TAG TAC AAA TAA GTC ACA TGA TGA TAT TTG ATT TTA TTA TAT TTT TAA AAA AAG TAA AAA ATA AAA AGT AGT TTA TTT TTA AAA AAT AAA ATT TAA AAT ATT AGT GTA TTT GAT TTC CGA AAG TTA AAA AAG AAA TAG TAA GAA GAT: red indicates the CEN3 sequence, CDEI and CDEIII, underlined) were assembly into a pUC18 plasmid, separated by EcoRV sites. .. CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight.

    Binding Assay:

    Article Title: Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials
    Article Snippet: To investigate the necessity of two actin binding domains on one crosslinker for the stiffening of reconstituted actin networks, bulk rheology measurements were performed on actin with functional, undigested, as well as EcoRV-HF-digested sPX (incubated for 1 h at 37 °C). .. Prior to rheology measurements, 1624 ng sPX (final concentration of 0.24 × 10−6 m in 175 μL final sample volume; R = 0.01) were incubated with 5 μL of EcoRV-HF (20 000 units per milliliter, New England Biolabs Inc., USA) and 1.75 μL of 10x CutSmart buffer in a final volume of 20 μL for 1 h at 37 °C.

    Article Title: Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials
    Article Snippet: .. Three different samples were prepared similar to as described above and 150 μL of final sample solutions was pipetted into a small cuvette (first sample: 24 × 10−6 m actin; second sample: 24 × 10−6 m actin with synthetic crosslinkers featuring two phalloidin binding domains (sPX) at R = 0.4; third sample: 24 × 10−6 m actin with sPX at R = 0.4, which were incubated for 1 h at 37 °C with 300 units of EcoRV-HF (New England Biolabs Inc., USA)) prior to the experiments. ..

    DNA Extraction:

    Article Title: A Requirement for Global Transcription Factor Lrp in Licensing Replication of Vibrio cholerae Chromosome 2
    Article Snippet: Candidate colonies were grown in LB overnight for genomic DNA isolation. .. Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, CA, United States), digested with EcoRV-HF (NEB, MA, United States), and ligated overnight at 16°C with T4 DNA ligase (NEB).

    Magnetic Beads:

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: .. Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter). .. Approximately, 1 µg of the digested plasmid was then used for in vitro transcription (MEGAscript T7 Transcription Kit, ThermoFisher Scientific).

    Mutagenesis:

    Article Title: A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae) 1
    Article Snippet: For the CESA5 entry clones, 2.5 units each of EcoRI-HF and EcoRV-HF were used in 20-μL reactions according to the manufacturer’s instructions (New England Biolabs). .. Sequence a plasmid with the expected restriction fragment pattern to verify the mutation and the absence of PCR errors.

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: The Ctf13I76A-Y79A and Ctf13I76R-Y79R mutations were made and combined with Cep3 , Ndc10 and Skp1 in pU2 for the mutant CBF3 complexes. .. CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight.

    Isolation:

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: The plasmid was isolated from E. coli using a Plasmid Giga Kit (Qiagen). .. CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight.

    Avidin-Biotin Assay:

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight. .. The PCR product of CEN3biotin was incubated in PBS with avidin protein (Sigma) on ice for 30 min, resulting in CEN3biotin-avidin .

    Labeling:

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight. .. For CEN3 CDEIII-end biotin-avidin labeling, two PCR primers were synthesized (Sigma) with a 5’-biotin modification on the reverse primer.

    Purification:

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: .. Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter). .. Approximately, 1 µg of the digested plasmid was then used for in vitro transcription (MEGAscript T7 Transcription Kit, ThermoFisher Scientific).

    Article Title: Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting
    Article Snippet: .. The minigenes were linearized with EcoRV-HF (NEB), purified using a PCR cleanup protocol with EconoSpin columns (Epoch Life Sciences), and in vitro transcribed using a T7 High Yield RNA Synthesis Kit (NEB E2040S) according to the manufacturer’s instructions, with subsequent deoxyribonuclease I treatment. .. Following RNA purification using the PureLink RNA Mini Kit, RNA was quantified by NanoDrop and using a Fragment Analyzer (Advanced Analytical DNF-489 Standard Sensitivity RNA Analysis Kit), mixed at varied concentrations, aliquoted, and stored at −80°C.

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: .. CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight. .. For CEN3 CDEIII-end biotin-avidin labeling, two PCR primers were synthesized (Sigma) with a 5’-biotin modification on the reverse primer.

    Polymerase Chain Reaction:

    Article Title: A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae) 1
    Article Snippet: Clone the PCR fusion product (3.5 μL) into pDONR P5-P2 in a BP reaction using Invitrogen MultiSite Gateway Pro according to the manufacturer’s instructions (Life Technologies). .. For the CESA5 entry clones, 2.5 units each of EcoRI-HF and EcoRV-HF were used in 20-μL reactions according to the manufacturer’s instructions (New England Biolabs).

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: DNA purification and concentration of restriction digests and PCR products were performed with DNA Clean & Concentrator TM-5 (Zymo). .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs).

    Article Title: Dynamics and processes of copy number instability in human ?-globin genes
    Article Snippet: For each blood and sperm sample, 230 μg genomic DNA were digested with EcoRV-HF (New England BioLabs) plus NdeI (Fermentas) according to the manufacturers’ recommendations, then electrophoresed in a 0.8% SeaKem HGT agarose gel (Cambrex Bio Science Rockland), as described previously ( ). .. Progenitor molecules were monitored in each fraction by long PCR with the γ-globin primers G18.4F (CGT ACT TTA GGC TTG TAA TGT G) and G23.2R (GTT AGA GAG AAG GGC GCA G) to amplify a 4.76-kb region spanning the A γ-globin gene.

    Article Title: Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase
    Article Snippet: To construct the pGRA-ROP17-3xHA plasmid, pGRA1plus -HPT-3xHA plasmid ( ) was first digested using EcoRV-HF and NcoI (New England Biolabs) for 4 h at 37°C to remove the GRA1 promoter. .. The empty vector backbone was amplified by PCR using Herculase II polymerase (Agilent) and primers 5′-CACATTTGTGTCACCCCAAATGAGAATTCGATATCAAGCTTGATCAGCAC-3′ and 5′-GAGGCGGCTTTATTACAGAAGGAGCCATGGTACCCGTACGACGTCCCG-3′ with each having 23- and 24-bp overhangs to ROP17 , respectively.

    Article Title: Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting
    Article Snippet: .. The minigenes were linearized with EcoRV-HF (NEB), purified using a PCR cleanup protocol with EconoSpin columns (Epoch Life Sciences), and in vitro transcribed using a T7 High Yield RNA Synthesis Kit (NEB E2040S) according to the manufacturer’s instructions, with subsequent deoxyribonuclease I treatment. .. Following RNA purification using the PureLink RNA Mini Kit, RNA was quantified by NanoDrop and using a Fragment Analyzer (Advanced Analytical DNF-489 Standard Sensitivity RNA Analysis Kit), mixed at varied concentrations, aliquoted, and stored at −80°C.

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight. .. For CEN3 CDEIII-end biotin-avidin labeling, two PCR primers were synthesized (Sigma) with a 5’-biotin modification on the reverse primer.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials
    Article Snippet: Prior to rheology measurements, 1624 ng sPX (final concentration of 0.24 × 10−6 m in 175 μL final sample volume; R = 0.01) were incubated with 5 μL of EcoRV-HF (20 000 units per milliliter, New England Biolabs Inc., USA) and 1.75 μL of 10x CutSmart buffer in a final volume of 20 μL for 1 h at 37 °C. .. Corresponding PAGE analysis showed a digestion rate of at least 80% after 1 h of incubation (Figure S2, ).

    Activated Clotting Time Assay:

    Article Title: Dynamics and processes of copy number instability in human ?-globin genes
    Article Snippet: For each blood and sperm sample, 230 μg genomic DNA were digested with EcoRV-HF (New England BioLabs) plus NdeI (Fermentas) according to the manufacturers’ recommendations, then electrophoresed in a 0.8% SeaKem HGT agarose gel (Cambrex Bio Science Rockland), as described previously ( ). .. Progenitor molecules were monitored in each fraction by long PCR with the γ-globin primers G18.4F (CGT ACT TTA GGC TTG TAA TGT G) and G23.2R (GTT AGA GAG AAG GGC GCA G) to amplify a 4.76-kb region spanning the A γ-globin gene.

    Plasmid Preparation:

    Article Title: A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae) 1
    Article Snippet: Isolate plasmid DNA from four overnight cultures inoculated with single colonies using a commercial kit according the manufacturer’s instructions (e.g., QIAprep Spin Miniprep Kit, QIAGEN, Venlo, The Netherlands). .. For the CESA5 entry clones, 2.5 units each of EcoRI-HF and EcoRV-HF were used in 20-μL reactions according to the manufacturer’s instructions (New England Biolabs).

    Article Title: A Requirement for Global Transcription Factor Lrp in Licensing Replication of Vibrio cholerae Chromosome 2
    Article Snippet: Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, CA, United States), digested with EcoRV-HF (NEB, MA, United States), and ligated overnight at 16°C with T4 DNA ligase (NEB). .. Plasmid DNA was extracted from individual colonies using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced using the primers supplied in the EZ-Tn5TM transposome kit to identify the locations of Tn insertion.

    Article Title: Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells
    Article Snippet: .. The luciferase reporter gene Luc2 (Photinus pyralis ), encoded within the vector pGL4.20 (Luc2 /Puro) (Promega® , Ipswich, USA), was digested with the restriction enzymes, EcoRV-HF® and BamHI-HF® (New England Biolabs® , San Diego, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro® -mcs (InvivoGen® , San Diego, USA) to generate the mammalian bioluminescence plasmid pVITRO2-Luc2 (Supplementary ). .. Nucleofection Early passage MSCs (1 × 106 cells) were nucleofected with 5 μ g pVITRO2-Luc2 and 2 μ g pmax-GFP® , according to the manufacturer's instructions (Lonza™, Basel, Switzerland), using the Nucleofector™ 2b device (Lonza™).

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: .. Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter). .. Approximately, 1 µg of the digested plasmid was then used for in vitro transcription (MEGAscript T7 Transcription Kit, ThermoFisher Scientific).

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: Paragraph title: 2.2. General methods for vector and plasmid assembly ... Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs).

    Article Title: Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase
    Article Snippet: .. To construct the pGRA-ROP17-3xHA plasmid, pGRA1plus -HPT-3xHA plasmid ( ) was first digested using EcoRV-HF and NcoI (New England Biolabs) for 4 h at 37°C to remove the GRA1 promoter. .. Product was incubated with Antarctic phosphatase (New England Biolabs) and gel extracted.

    Article Title: Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting
    Article Snippet: All synthetic clones were ordered as minigenes in the pIDTBlue vector (IDT), which incorporates a T7 promoter to allow for in vitro transcription. .. The minigenes were linearized with EcoRV-HF (NEB), purified using a PCR cleanup protocol with EconoSpin columns (Epoch Life Sciences), and in vitro transcribed using a T7 High Yield RNA Synthesis Kit (NEB E2040S) according to the manufacturer’s instructions, with subsequent deoxyribonuclease I treatment.

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: The plasmid was isolated from E. coli using a Plasmid Giga Kit (Qiagen). .. CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight.

    Functional Assay:

    Article Title: Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials
    Article Snippet: To investigate the necessity of two actin binding domains on one crosslinker for the stiffening of reconstituted actin networks, bulk rheology measurements were performed on actin with functional, undigested, as well as EcoRV-HF-digested sPX (incubated for 1 h at 37 °C). .. Prior to rheology measurements, 1624 ng sPX (final concentration of 0.24 × 10−6 m in 175 μL final sample volume; R = 0.01) were incubated with 5 μL of EcoRV-HF (20 000 units per milliliter, New England Biolabs Inc., USA) and 1.75 μL of 10x CutSmart buffer in a final volume of 20 μL for 1 h at 37 °C.

    Selection:

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs). ..

    Agarose Gel Electrophoresis:

    Article Title: Dynamics and processes of copy number instability in human ?-globin genes
    Article Snippet: .. For each blood and sperm sample, 230 μg genomic DNA were digested with EcoRV-HF (New England BioLabs) plus NdeI (Fermentas) according to the manufacturers’ recommendations, then electrophoresed in a 0.8% SeaKem HGT agarose gel (Cambrex Bio Science Rockland), as described previously ( ). ..

    In Vitro:

    Article Title: The delivery of mRNA to colon inflammatory lesions by lipid-nano-particles containing environmentally-sensitive lipid-like materials with oleic acid scaffolds
    Article Snippet: Paragraph title: In vitro transcription (IVT) of mRNA ... The pDNA was linearized using EcoRV-HF (New England Biolab).

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: Each spike-in sequence contained a T7 promoter for in vitro transcription. .. Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter).

    Article Title: Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting
    Article Snippet: .. The minigenes were linearized with EcoRV-HF (NEB), purified using a PCR cleanup protocol with EconoSpin columns (Epoch Life Sciences), and in vitro transcribed using a T7 High Yield RNA Synthesis Kit (NEB E2040S) according to the manufacturer’s instructions, with subsequent deoxyribonuclease I treatment. .. Following RNA purification using the PureLink RNA Mini Kit, RNA was quantified by NanoDrop and using a Fragment Analyzer (Advanced Analytical DNF-489 Standard Sensitivity RNA Analysis Kit), mixed at varied concentrations, aliquoted, and stored at −80°C.

    Spectrophotometry:

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: Nucleic acid concentrations were measured with a UV-Vis spectrophotometer NanoDrop 2000c. .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs).

    Concentration Assay:

    Article Title: Identification of Guide-Intrinsic Determinants of Cas9 Specificity
    Article Snippet: For cutting reactions run in the presence of human gDNA, gDNA was present at a final concentration of 750 ng/μL. .. Briefly, 10 μL 10× CutSmart (NEB), 5 μL EcoRV-HF (NEB), and 75 μL NF water were added to a 10 μL sample.

    Article Title: Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials
    Article Snippet: .. Prior to rheology measurements, 1624 ng sPX (final concentration of 0.24 × 10−6 m in 175 μL final sample volume; R = 0.01) were incubated with 5 μL of EcoRV-HF (20 000 units per milliliter, New England Biolabs Inc., USA) and 1.75 μL of 10x CutSmart buffer in a final volume of 20 μL for 1 h at 37 °C. .. Corresponding PAGE analysis showed a digestion rate of at least 80% after 1 h of incubation (Figure S2, ).

    Article Title: Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells
    Article Snippet: Approximately 1.5 µg of each plasmid was digested with 10 U of EcoRV-HF (New England BioLabs) and purified with DNA-binding magnetic beads (SPRI-select, Beckman Coulter). .. The final concentration was then determined with the TapeStation (Agilent Technologies).

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: DNA purification and concentration of restriction digests and PCR products were performed with DNA Clean & Concentrator TM-5 (Zymo). .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs).

    Fractionation:

    Article Title: Dynamics and processes of copy number instability in human ?-globin genes
    Article Snippet: Paragraph title: DNA Fractionation to Recover γ-Globin Gene Rearrangements. ... For each blood and sperm sample, 230 μg genomic DNA were digested with EcoRV-HF (New England BioLabs) plus NdeI (Fermentas) according to the manufacturers’ recommendations, then electrophoresed in a 0.8% SeaKem HGT agarose gel (Cambrex Bio Science Rockland), as described previously ( ).

    DNA Purification:

    Article Title: Development of a cyanobacterial heterologous polyketide production platform
    Article Snippet: DNA purification and concentration of restriction digests and PCR products were performed with DNA Clean & Concentrator TM-5 (Zymo). .. Briefly, DNA devices including S. elongatus recombination or replicon sequences, E. coli origin of replication, antibiotic selection markers and expression cassettes were released from donor plasmids by restriction digests using Zral or EcoRV-HF (New England BioLabs).

    Recombinant:

    Article Title: Architecture of the CBF3-Centromere Complex of the Budding Yeast Kinetochore
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant CBF3−CEN3 complex ... CEN3 was purified with resource Q anion exchange chromatography (GE Healthcare) after digestion with EcoRV-HF (NEB) at 37 °C overnight.

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    New England Biolabs ecorv hf
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