homology directed repair kpn i hf  (New England Biolabs)


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    Name:
    KpnI HF
    Description:
    KpnI HF 20 000 units
    Catalog Number:
    R3142L
    Price:
    269
    Category:
    Restriction Enzymes
    Size:
    20 000 units
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    Structured Review

    New England Biolabs homology directed repair kpn i hf
    KpnI HF
    KpnI HF 20 000 units
    https://www.bioz.com/result/homology directed repair kpn i hf/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    homology directed repair kpn i hf - by Bioz Stars, 2021-06
    98/100 stars

    Images

    1) Product Images from "Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair"

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36506-w

    CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p
    Figure Legend Snippet: CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Techniques Used: CRISPR, Introduce, Mutagenesis, Transfection

    Related Articles

    Plasmid Preparation:

    Article Title: Dysregulation of C-X-C motif ligand 10 during aging and association with cognitive performance
    Article Snippet: .. To methylate just the CXCL10 promoter within the plasmid (patch methylation), the pGL4-CXCL10 plasmid (10 μg) was first double digested with KpnI -HF and NheI -HF (20 U each; New England Biolabs) restriction enzymes, and the products were gel extracted and purified via the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). .. The extracted CXCL10 promoter insert (2 μg) was subject to the same aforementioned in vitro methylation treatment and purified with the NucleoSpin Gel and PCR Clean-up kit.

    Article Title: Characterization of Pseudomonas aeruginosa Growth on O-Acylcarnitines and Identification of a Short-Chain Acylcarnitine Hydrolase
    Article Snippet: The 213-bp product was ligated into the pCR-Zero Blunt vector using the Zero Blunt cloning kit (Invitrogen), with subsequent transformation and plasmid preparation as described above. .. The resulting plasmid was digested with KpnI-HF and BamHI-HF (NEB), and the 213-bp fragment was extracted from an agarose gel and ligated into the similarly digested pJAM8 to generate pJAM61. .. The pJAM61 plasmid was digested with KpnI-HF and HindIII-HF, and the ∼1.2-kbp fragment was ligated into the similarly cut plasmid pUC18-mini-Tn7T-Gm ( ) and transformed into chemically competent E. coli NEB5α.

    Methylation:

    Article Title: Dysregulation of C-X-C motif ligand 10 during aging and association with cognitive performance
    Article Snippet: .. To methylate just the CXCL10 promoter within the plasmid (patch methylation), the pGL4-CXCL10 plasmid (10 μg) was first double digested with KpnI -HF and NheI -HF (20 U each; New England Biolabs) restriction enzymes, and the products were gel extracted and purified via the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). .. The extracted CXCL10 promoter insert (2 μg) was subject to the same aforementioned in vitro methylation treatment and purified with the NucleoSpin Gel and PCR Clean-up kit.

    Purification:

    Article Title: Dysregulation of C-X-C motif ligand 10 during aging and association with cognitive performance
    Article Snippet: .. To methylate just the CXCL10 promoter within the plasmid (patch methylation), the pGL4-CXCL10 plasmid (10 μg) was first double digested with KpnI -HF and NheI -HF (20 U each; New England Biolabs) restriction enzymes, and the products were gel extracted and purified via the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). .. The extracted CXCL10 promoter insert (2 μg) was subject to the same aforementioned in vitro methylation treatment and purified with the NucleoSpin Gel and PCR Clean-up kit.

    Polymerase Chain Reaction:

    Article Title: Dysregulation of C-X-C motif ligand 10 during aging and association with cognitive performance
    Article Snippet: .. To methylate just the CXCL10 promoter within the plasmid (patch methylation), the pGL4-CXCL10 plasmid (10 μg) was first double digested with KpnI -HF and NheI -HF (20 U each; New England Biolabs) restriction enzymes, and the products were gel extracted and purified via the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). .. The extracted CXCL10 promoter insert (2 μg) was subject to the same aforementioned in vitro methylation treatment and purified with the NucleoSpin Gel and PCR Clean-up kit.

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: The Surveyor assay was performed following manufacturer’s instructions (Transgenomic, UK) and gene editing frequencies were calculated as described . .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Article Title: The eukaryotic replisome tolerates leading‐strand base damage by replicase switching
    Article Snippet: BbvCI (NEB) and DpnI (NEB) for 90 min at 37°C before heat inactivation at 80°C for 20 min. .. The region surrounding the lesion was PCR‐amplified (primer sequences in Appendix Table , Byp_FID_FWD and REV) and cloned into pUC19 using HindIII‐HF (NEB) and KpnI‐HF (NEB). .. Following transformation into NEB 5‐alpha Competent E. coli, individual clones were selected and grown overnight in a 96‐well plate before Sanger sequencing with the M13F primer (Source BioScience) using the Source BioScience Bugs2Bases service.

    Incubation:

    Article Title: Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding
    Article Snippet: Ligase was inactivated by heating to 65°C for 10 min, and efficient ligation was verified on an agarose gel. .. The reaction volume was increased to 200 μl with 1× Cut Smart buffer, 40 U of KpnI-HF (NEB) was added, and incubation was continued for an additional 1.5 h at 37°C. .. Twenty units of HindIII-HF then was added, and the mixture was incubated for an additional hour, subsequently cleaned up on MinElute columns (5 μg per column; Qiagen), and eluted in 20 μl H2O.

    Generated:

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: The Surveyor assay was performed following manufacturer’s instructions (Transgenomic, UK) and gene editing frequencies were calculated as described . .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Clone Assay:

    Article Title: The eukaryotic replisome tolerates leading‐strand base damage by replicase switching
    Article Snippet: BbvCI (NEB) and DpnI (NEB) for 90 min at 37°C before heat inactivation at 80°C for 20 min. .. The region surrounding the lesion was PCR‐amplified (primer sequences in Appendix Table , Byp_FID_FWD and REV) and cloned into pUC19 using HindIII‐HF (NEB) and KpnI‐HF (NEB). .. Following transformation into NEB 5‐alpha Competent E. coli, individual clones were selected and grown overnight in a 96‐well plate before Sanger sequencing with the M13F primer (Source BioScience) using the Source BioScience Bugs2Bases service.

    Agarose Gel Electrophoresis:

    Article Title: Characterization of Pseudomonas aeruginosa Growth on O-Acylcarnitines and Identification of a Short-Chain Acylcarnitine Hydrolase
    Article Snippet: The 213-bp product was ligated into the pCR-Zero Blunt vector using the Zero Blunt cloning kit (Invitrogen), with subsequent transformation and plasmid preparation as described above. .. The resulting plasmid was digested with KpnI-HF and BamHI-HF (NEB), and the 213-bp fragment was extracted from an agarose gel and ligated into the similarly digested pJAM8 to generate pJAM61. .. The pJAM61 plasmid was digested with KpnI-HF and HindIII-HF, and the ∼1.2-kbp fragment was ligated into the similarly cut plasmid pUC18-mini-Tn7T-Gm ( ) and transformed into chemically competent E. coli NEB5α.

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  • 98
    New England Biolabs homology directed repair kpn i hf
    CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) <t>ssODN</t> that remove the c.828TAAA insertion and introduce a <t>Kpn</t> I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p
    Homology Directed Repair Kpn I Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/homology directed repair kpn i hf/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    homology directed repair kpn i hf - by Bioz Stars, 2021-06
    98/100 stars
      Buy from Supplier

    Image Search Results


    CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Journal: Scientific Reports

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair

    doi: 10.1038/s41598-018-36506-w

    Figure Lengend Snippet: CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Article Snippet: In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers.

    Techniques: CRISPR, Introduce, Mutagenesis, Transfection