kpn1  (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 kpn1
    KpnI HF
    KpnI HF 20 000 units
    https://www.bioz.com/result/kpn1/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kpn1 - by Bioz Stars, 2021-03
    94/100 stars

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    Related Articles

    Plasmid Preparation:

    Article Title: A split transcriptional repressor that links protein solubility to an orthogonal genetic circuit
    Article Snippet: Samples yielding normalized fluorescence at least 5σ lower than that of the sample transformed with pTAC were selected, purified vectors were sequenced, and the sTetR variants were named after the last residue of the N-terminal TetR fragment followed by the letters designating the coiled-coil fused to the termini at the split site, where EK signifies IAAL-E3 and IAAL-K3 and SZ17/18 signifies SYNZIP17 and SYNZIP18. .. The TetR gene and pGEX vector backbone were PCR amplified from pcDNA6/TR (Invitrogen) and pGEX2TK-IFP1.4, respectively, and these were assembled to create a plasmid that expresses TetR using tac promoter (pTAC-TetR) via Gibson Assembly using the Gibson Assembly® Master Mix (New England Biolabs). pTAC-TetR was digested with BamHI-HF and KpnI-HF (New England Biolabs), treated with Mung Bean Nuclease (New England Biolabs) to create blunt ends, and self-ligated to generate pTAC, the control vector lacking the TetR gene. .. To generate pair of vectors for independent expression of the TetR fragments encoding each SYNZIP variant, the individual fragments were PCR amplified from the vector identified in our library screen, the ORFs encoding the first TetR fragments fused to SYNZIP17 was cloned into pQE80 (QIAGEN), and the ORFs encoding SYNZIP18 fused to the second TetR fragments were cloned into pTara.

    Article Title: Hypoxia-inducible lipid droplet-associated protein inhibits adipose triglyceride lipase
    Article Snippet: The vector was isolated using Qiagen plasmid maxi kit (Qiagen) according to the manufacturer’s instructions. .. The EGFP fragment was then excised from the pEGFP-N2 parent vector by enzyme digestion with Kpn I-HF and Not I-HF. .. The vector was gel-purified with QIAquick gel extraction kit (Qiagen) and the fragments of mCherry, sYFP2, or mTurquoise2 ( ) were ligated using T4 DNA ligase (Thermo Fisher Scientific) into the pEGFP-N2 vector, which was digested with Kpn I and Not I.

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: The amplified DNA fragment was inserted into pQK663 derived from pYA3332 [ ] between the Nco I and BamH I digestion sites to generate pQK163, which was then transformed into the S. Typhimurium Δasd Δcrp strain for a maltose fermentation assay. .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Article Title: Influence of promoter/enhancer region haplotypes on MGMT transcriptional regulation: a potential biomarker for human sensitivity to alkylating agents
    Article Snippet: The MGMT P/E region inserts were then PCR amplified with these two new primers from the identified positive pGEM-T-MGMT clones followed the PCR protocol described above. .. Both the amplicons and pGL4.11 vector were double digested by HindIII-HF and KpnI-HF (NEB) and then gel purified by QIAquick Gel Extraction Kit (Qiagen). .. The purified MGMT P/E DNA fragments were inserted into pGL4.11 vector and transformed into the competent E. coli JM109 cells.

    Polymerase Chain Reaction:

    Article Title: A split transcriptional repressor that links protein solubility to an orthogonal genetic circuit
    Article Snippet: Samples yielding normalized fluorescence at least 5σ lower than that of the sample transformed with pTAC were selected, purified vectors were sequenced, and the sTetR variants were named after the last residue of the N-terminal TetR fragment followed by the letters designating the coiled-coil fused to the termini at the split site, where EK signifies IAAL-E3 and IAAL-K3 and SZ17/18 signifies SYNZIP17 and SYNZIP18. .. The TetR gene and pGEX vector backbone were PCR amplified from pcDNA6/TR (Invitrogen) and pGEX2TK-IFP1.4, respectively, and these were assembled to create a plasmid that expresses TetR using tac promoter (pTAC-TetR) via Gibson Assembly using the Gibson Assembly® Master Mix (New England Biolabs). pTAC-TetR was digested with BamHI-HF and KpnI-HF (New England Biolabs), treated with Mung Bean Nuclease (New England Biolabs) to create blunt ends, and self-ligated to generate pTAC, the control vector lacking the TetR gene. .. To generate pair of vectors for independent expression of the TetR fragments encoding each SYNZIP variant, the individual fragments were PCR amplified from the vector identified in our library screen, the ORFs encoding the first TetR fragments fused to SYNZIP17 was cloned into pQE80 (QIAGEN), and the ORFs encoding SYNZIP18 fused to the second TetR fragments were cloned into pTara.

    Article Title: The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders
    Article Snippet: Genotyping To identify the ter allele by PCR, primers terFor2 5′-GTCTGGTCTTAAGTGCTTGG-′3 and terRev2 5′-TCACTGCTTCACCACAGAAC-3′ amplified a 560 bp sequence in a total volume of 25 µl containing 12.5 µl HotStarTaq Master Mix Kit (Qiagen, Hilden, Germany), 10 pmol of each primer and 1 µl template DNA (15 min at 95°C; 35 cycles: 95°C for 30 s, 54°C for 30 s, 72°C for 30 s; 72°C for 1 min). .. The PCR products were digested by adding 0.5 µl Kpn I-HF, 3 µl NEB4 buffer and 0.3 µl BSA (100×) (NEB, Ipswich, MA). .. Cleavage fragments from PCR products of wild type Dnd1 (180 bp and 380 bp) and unchanged 560 bp ter allele products were separated by electrophoresis in a 1.5% agarose gel in 1× TBE buffer.

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: The amplified DNA fragment was inserted into pQK663 derived from pYA3332 [ ] between the Nco I and BamH I digestion sites to generate pQK163, which was then transformed into the S. Typhimurium Δasd Δcrp strain for a maltose fermentation assay. .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    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.

    Amplification:

    Article Title: A split transcriptional repressor that links protein solubility to an orthogonal genetic circuit
    Article Snippet: Samples yielding normalized fluorescence at least 5σ lower than that of the sample transformed with pTAC were selected, purified vectors were sequenced, and the sTetR variants were named after the last residue of the N-terminal TetR fragment followed by the letters designating the coiled-coil fused to the termini at the split site, where EK signifies IAAL-E3 and IAAL-K3 and SZ17/18 signifies SYNZIP17 and SYNZIP18. .. The TetR gene and pGEX vector backbone were PCR amplified from pcDNA6/TR (Invitrogen) and pGEX2TK-IFP1.4, respectively, and these were assembled to create a plasmid that expresses TetR using tac promoter (pTAC-TetR) via Gibson Assembly using the Gibson Assembly® Master Mix (New England Biolabs). pTAC-TetR was digested with BamHI-HF and KpnI-HF (New England Biolabs), treated with Mung Bean Nuclease (New England Biolabs) to create blunt ends, and self-ligated to generate pTAC, the control vector lacking the TetR gene. .. To generate pair of vectors for independent expression of the TetR fragments encoding each SYNZIP variant, the individual fragments were PCR amplified from the vector identified in our library screen, the ORFs encoding the first TetR fragments fused to SYNZIP17 was cloned into pQE80 (QIAGEN), and the ORFs encoding SYNZIP18 fused to the second TetR fragments were cloned into pTara.

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: The amplified DNA fragment was inserted into pQK663 derived from pYA3332 [ ] between the Nco I and BamH I digestion sites to generate pQK163, which was then transformed into the S. Typhimurium Δasd Δcrp strain for a maltose fermentation assay. .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Expressing:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: The amplified DNA fragment was inserted into pQK663 derived from pYA3332 [ ] between the Nco I and BamH I digestion sites to generate pQK163, which was then transformed into the S. Typhimurium Δasd Δcrp strain for a maltose fermentation assay. .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Sequencing:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: The amplified DNA fragment was inserted into pQK663 derived from pYA3332 [ ] between the Nco I and BamH I digestion sites to generate pQK163, which was then transformed into the S. Typhimurium Δasd Δcrp strain for a maltose fermentation assay. .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Positron Emission Tomography:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: The amplified DNA fragment was inserted into pQK663 derived from pYA3332 [ ] between the Nco I and BamH I digestion sites to generate pQK163, which was then transformed into the S. Typhimurium Δasd Δcrp strain for a maltose fermentation assay. .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Purification:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: The amplified DNA fragment was inserted into pQK663 derived from pYA3332 [ ] between the Nco I and BamH I digestion sites to generate pQK163, which was then transformed into the S. Typhimurium Δasd Δcrp strain for a maltose fermentation assay. .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Article Title: Influence of promoter/enhancer region haplotypes on MGMT transcriptional regulation: a potential biomarker for human sensitivity to alkylating agents
    Article Snippet: The MGMT P/E region inserts were then PCR amplified with these two new primers from the identified positive pGEM-T-MGMT clones followed the PCR protocol described above. .. Both the amplicons and pGL4.11 vector were double digested by HindIII-HF and KpnI-HF (NEB) and then gel purified by QIAquick Gel Extraction Kit (Qiagen). .. The purified MGMT P/E DNA fragments were inserted into pGL4.11 vector and transformed into the competent E. coli JM109 cells.

    Subcloning:

    Article Title: High-performance chemical- and light-inducible recombinases in mammalian cells and mice
    Article Snippet: For insertion into a N-terminal CID or LID sub-cloning vector, recombinase terminating fragments were first PCR amplified out of a full recombinase sequence template to contain a 5′ sequence to code for an Kpn I-HF restriction site and a 3′ sequence to code for an SV40 nuclear localization signal, stop codon, and an Eco RI-HF restriction site. .. A similar process was followed as before with the C-terminal CID and LID sub-cloning vectors; however, Kpn I-HF and Eco RI-HF restriction digests (New England Biolabs) were carried out rather than with Mlu I and Bsp EI. .. To swap between CID and LID domains, sequenced fragments were gel isolated from sequenced vectors using Mlu I/Bsp EI or Kpn I-HF/Eco RI-HF and inserted into corresponding CID or LID sub-cloning vectors.

    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.

    Gel Extraction:

    Article Title: Influence of promoter/enhancer region haplotypes on MGMT transcriptional regulation: a potential biomarker for human sensitivity to alkylating agents
    Article Snippet: The MGMT P/E region inserts were then PCR amplified with these two new primers from the identified positive pGEM-T-MGMT clones followed the PCR protocol described above. .. Both the amplicons and pGL4.11 vector were double digested by HindIII-HF and KpnI-HF (NEB) and then gel purified by QIAquick Gel Extraction Kit (Qiagen). .. The purified MGMT P/E DNA fragments were inserted into pGL4.11 vector and transformed into the competent E. coli JM109 cells.

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    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, 2021-03
    98/100 stars
      Buy from Supplier

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    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

    Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a Kpn I restriction site used for genotyping of the ter allele performing PCR amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.

    Journal: PLoS ONE

    Article Title: The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

    doi: 10.1371/journal.pone.0038001

    Figure Lengend Snippet: Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a Kpn I restriction site used for genotyping of the ter allele performing PCR amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.

    Article Snippet: The PCR products were digested by adding 0.5 µl Kpn I-HF, 3 µl NEB4 buffer and 0.3 µl BSA (100×) (NEB, Ipswich, MA).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Negative Control