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    HphI
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    HphI 5 000 units
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    New England Biolabs hphi
    HphI
    HphI 5 000 units
    https://www.bioz.com/result/hphi/product/New England Biolabs
    Average 94 stars, based on 48 article reviews
    Price from $9.99 to $1999.99
    hphi - by Bioz Stars, 2020-09
    94/100 stars

    Images

    1) Product Images from "Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch MigraseCentral Role of the Holliday Junction Helicase RuvAB in vlsE Recombination and Infectivity of Borrelia burgdorferi"

    Article Title: Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch MigraseCentral Role of the Holliday Junction Helicase RuvAB in vlsE Recombination and Infectivity of Borrelia burgdorferi

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000680

    Restriction fragment length polymorphism assay for switching at vlsE . A portion of the vlsE expression site containing the variable regions was amplified using primers B248 and B249 to give a product of 776 bp. PCR reactions were performed on B. burgdorferi grown from ear biopsies taken at day 21 and the products were digested with HphI and run on a 1.2% agarose gel in TAE buffer at 75V for 1.5 hours and stained with ethidium bromide (see Materials and Methods ). Wild-type B. burgdorferi B31-5A4 recovered following infection of a C3H/HeN mouse was used as a template in lanes 1 and 2. An unswitched template (not exposed to mouse infection) is shown in lanes 3 and 4. PCR products from rep2 and mutS2 /1 DNA templates are found in lanes 5 6, and 7 8 respectively. M denotes a 100bp molecular weight marker.
    Figure Legend Snippet: Restriction fragment length polymorphism assay for switching at vlsE . A portion of the vlsE expression site containing the variable regions was amplified using primers B248 and B249 to give a product of 776 bp. PCR reactions were performed on B. burgdorferi grown from ear biopsies taken at day 21 and the products were digested with HphI and run on a 1.2% agarose gel in TAE buffer at 75V for 1.5 hours and stained with ethidium bromide (see Materials and Methods ). Wild-type B. burgdorferi B31-5A4 recovered following infection of a C3H/HeN mouse was used as a template in lanes 1 and 2. An unswitched template (not exposed to mouse infection) is shown in lanes 3 and 4. PCR products from rep2 and mutS2 /1 DNA templates are found in lanes 5 6, and 7 8 respectively. M denotes a 100bp molecular weight marker.

    Techniques Used: Polymorphism Assay, Expressing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Infection, Molecular Weight, Marker

    2) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.924951

    A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    3) Product Images from "ACVR1 Gene Mutation in Sporadic Korean Patients with Fibrodysplasia Ossificans Progressiva"

    Article Title: ACVR1 Gene Mutation in Sporadic Korean Patients with Fibrodysplasia Ossificans Progressiva

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2009.24.3.433

    Mutation analysis of the ACVR1 in FOP patients with definite clinical manifestations. ( A ) Direct sequencing of the PCR products of the ACVR1 showed the presence of the c.617G > A mutation. R=adenine or guanine, ( B ) Restriction endonuclease digestion of the PCR product (350 bp). The G allele (control) was digested by Cac8I to produce three bands, whereas the A allele appeared as two bands. Because FOP patients were heterozygous for this mutation, the 139 bp and 114 bp bands were also presented. The PCR product not digested with HphI corresponds to the G allele (control) in contrast to digested products corresponding to the A allele (FOP).
    Figure Legend Snippet: Mutation analysis of the ACVR1 in FOP patients with definite clinical manifestations. ( A ) Direct sequencing of the PCR products of the ACVR1 showed the presence of the c.617G > A mutation. R=adenine or guanine, ( B ) Restriction endonuclease digestion of the PCR product (350 bp). The G allele (control) was digested by Cac8I to produce three bands, whereas the A allele appeared as two bands. Because FOP patients were heterozygous for this mutation, the 139 bp and 114 bp bands were also presented. The PCR product not digested with HphI corresponds to the G allele (control) in contrast to digested products corresponding to the A allele (FOP).

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction

    4) Product Images from "Genome-scale investigation of phenotypically distinct but nearly clonal Trichoderma strains"

    Article Title: Genome-scale investigation of phenotypically distinct but nearly clonal Trichoderma strains

    Journal: PeerJ

    doi: 10.7717/peerj.2023

    Electrophoretic separation of Hph I digested PCR fragments. Lanes B, D and F represent the undigested 660 bp PCR fragments from LU132, LU140 and IMI204060, respectively. Lanes A, C and E represent the HphI digested PCR fragments for LU132, LU140 and IMI204060, respectively. The arrow indicates the 350 bp restriction fragment that was only found in LU132. Size standard was 1 Kb Plus DNA Ladder™ (Invitrogen) in lane G.
    Figure Legend Snippet: Electrophoretic separation of Hph I digested PCR fragments. Lanes B, D and F represent the undigested 660 bp PCR fragments from LU132, LU140 and IMI204060, respectively. Lanes A, C and E represent the HphI digested PCR fragments for LU132, LU140 and IMI204060, respectively. The arrow indicates the 350 bp restriction fragment that was only found in LU132. Size standard was 1 Kb Plus DNA Ladder™ (Invitrogen) in lane G.

    Techniques Used: Polymerase Chain Reaction

    5) Product Images from "Excess SeqA prolongs sequestration of oriC and delays nucleoid segregation and cell division"

    Article Title: Excess SeqA prolongs sequestration of oriC and delays nucleoid segregation and cell division

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdg020

    Fig. 1. Overexpression of SeqA protein extends the period of hemimethylation at oriC . ( A ) Schematic representation of the temperature shifts at the indicated times during the experiment. ( B ) Exponentially growing cultures (30°C) of MG1655 dnaC2 cells harbouring pFH2102 (control) or pMAK7 (excess SeqA) were synchronized with respect to initiation for 1 h at 38°C before shifting the temperature to 30°C. IPTG (0.5 mM) was added to the cultures three mass doublings prior to synchronized initiation. Chromosomal DNA was extracted at the indicated times after returning the cultures to 30°C. The DNA was digested with Xho I and Hph I, electrophoresed and hybridized to an oriC probe. The positions of cut and uncut fragments are indicated by arrows. The rightmost lane contains DNA isolated from a dam – strain which reveals the position of the cut fragment. ( C ) Quantification of the amount of hemimethylated oriC in (B). Squares represent dnaC 2/pFH2102 and triangles dnaC 2/pMAK7. The percentage of hemimethylated oriC is twice the percentage of the cut fragment in (B) since 50% of the hemimethylated fragments are digested. Time indicates minutes from shift to 30°C after 1 h synchronization at 38°C. ( D ) Western analyses of samples taken from the cultures at 0 and 20 min after returning the cultures to 30°C. A 1 µg aliquot of total protein of each sample was loaded.
    Figure Legend Snippet: Fig. 1. Overexpression of SeqA protein extends the period of hemimethylation at oriC . ( A ) Schematic representation of the temperature shifts at the indicated times during the experiment. ( B ) Exponentially growing cultures (30°C) of MG1655 dnaC2 cells harbouring pFH2102 (control) or pMAK7 (excess SeqA) were synchronized with respect to initiation for 1 h at 38°C before shifting the temperature to 30°C. IPTG (0.5 mM) was added to the cultures three mass doublings prior to synchronized initiation. Chromosomal DNA was extracted at the indicated times after returning the cultures to 30°C. The DNA was digested with Xho I and Hph I, electrophoresed and hybridized to an oriC probe. The positions of cut and uncut fragments are indicated by arrows. The rightmost lane contains DNA isolated from a dam – strain which reveals the position of the cut fragment. ( C ) Quantification of the amount of hemimethylated oriC in (B). Squares represent dnaC 2/pFH2102 and triangles dnaC 2/pMAK7. The percentage of hemimethylated oriC is twice the percentage of the cut fragment in (B) since 50% of the hemimethylated fragments are digested. Time indicates minutes from shift to 30°C after 1 h synchronization at 38°C. ( D ) Western analyses of samples taken from the cultures at 0 and 20 min after returning the cultures to 30°C. A 1 µg aliquot of total protein of each sample was loaded.

    Techniques Used: Over Expression, Isolation, Western Blot

    6) Product Images from "Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity"

    Article Title: Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-831

    DNA restriction analyses. A) i) Phi93 and Phi145 (DAM negative) cut with Dpn1 (cuts methylated GATC) and DpnII (cuts unmethylated GATC). ii) Phi145, Phi93 (MTase positive) and Phi15 (MTase negative) with HphI. B) i) Plasmid pPiM.93DAM cut with Dpn1 (cuts methylated GATC) and DpnII (cuts unmethylated GATC) under induced (I) and un-induced (NI) conditions. ii) Plasmid pPTPi (P), pPiM.145I cut with HphI under induced (I) and un-induced (NI) conditions. iii) Plasmid pPTPi (P), pPiM.145II cut with HphI under induced (I) and un-induced (NI) conditions.
    Figure Legend Snippet: DNA restriction analyses. A) i) Phi93 and Phi145 (DAM negative) cut with Dpn1 (cuts methylated GATC) and DpnII (cuts unmethylated GATC). ii) Phi145, Phi93 (MTase positive) and Phi15 (MTase negative) with HphI. B) i) Plasmid pPiM.93DAM cut with Dpn1 (cuts methylated GATC) and DpnII (cuts unmethylated GATC) under induced (I) and un-induced (NI) conditions. ii) Plasmid pPTPi (P), pPiM.145I cut with HphI under induced (I) and un-induced (NI) conditions. iii) Plasmid pPTPi (P), pPiM.145II cut with HphI under induced (I) and un-induced (NI) conditions.

    Techniques Used: Methylation, Plasmid Preparation

    7) Product Images from "90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †"

    Article Title: 90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00294-08

    Differentiation of intestinal Cryptosporidium spp. by RFLP analysis of hsp90 PCR gene products using StyI and HphI. Lanes 1 to 4, C. parvum ; lanes 5 to 9 and 28, C. hominis ; lanes 11 to 14, C. canis ; lanes 15 and 29, 100-bp molecular markers; lanes 16
    Figure Legend Snippet: Differentiation of intestinal Cryptosporidium spp. by RFLP analysis of hsp90 PCR gene products using StyI and HphI. Lanes 1 to 4, C. parvum ; lanes 5 to 9 and 28, C. hominis ; lanes 11 to 14, C. canis ; lanes 15 and 29, 100-bp molecular markers; lanes 16

    Techniques Used: Polymerase Chain Reaction

    8) Product Images from "Identification and functional characterization of a novel splicing mutation in RP gene PRPF31"

    Article Title: Identification and functional characterization of a novel splicing mutation in RP gene PRPF31

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2007.12.156

    Identification of a novel splicing mutation in the PRPF31 gene. (A) A single nucleotide change (G > T) at the position +1 of intron 1 was identified in the Chinese family. Left, mutation; right, normal. (B) Mutation IVS1+1G > T co-segregates with patients in the family. M, molecular weight marker; N, normal individual; P, affected individual (patient). C, asymptomatic mutation carrier. The IVS1+1G > T mutation generates an Hph I restriction site. All patients and carriers are heterozygous for the wild type allele, identified by a 177-bp fragment, and for the mutated allele characterized by two fragments of 222-bp and 45-bp (not detectable in this gel). (C) Representative results from RT-PCR are shown for the proband (P) and one normal family member. (D) DNA sequence analysis of wild type and the truncated transcripts from the proband and normal family member: isoform 1 (606 bp); isoform 2 (360 bp); isoform 3 (318 bp). (E) Schematic representation of wild type and three alternative splicing isoforms.
    Figure Legend Snippet: Identification of a novel splicing mutation in the PRPF31 gene. (A) A single nucleotide change (G > T) at the position +1 of intron 1 was identified in the Chinese family. Left, mutation; right, normal. (B) Mutation IVS1+1G > T co-segregates with patients in the family. M, molecular weight marker; N, normal individual; P, affected individual (patient). C, asymptomatic mutation carrier. The IVS1+1G > T mutation generates an Hph I restriction site. All patients and carriers are heterozygous for the wild type allele, identified by a 177-bp fragment, and for the mutated allele characterized by two fragments of 222-bp and 45-bp (not detectable in this gel). (C) Representative results from RT-PCR are shown for the proband (P) and one normal family member. (D) DNA sequence analysis of wild type and the truncated transcripts from the proband and normal family member: isoform 1 (606 bp); isoform 2 (360 bp); isoform 3 (318 bp). (E) Schematic representation of wild type and three alternative splicing isoforms.

    Techniques Used: Mutagenesis, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction, Sequencing

    9) Product Images from "Inactivation of Individual SeqA Binding Sites of the E. coli Origin Reveals Robustness of Replication Initiation Synchrony"

    Article Title: Inactivation of Individual SeqA Binding Sites of the E. coli Origin Reveals Robustness of Replication Initiation Synchrony

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0166722

    Sequence of oriC showing its major protein binding sites. (A) Sequence of oriC that includes the minimal region (coordinates 1–246) required for origin function. The coordinate 1 correspondence to 3923744 of gb|U00096.3|. The region includes several GATC sites (shown in red) which are methylated by the Dam methylase enzyme. There are three 13-mer repeats of AT rich sequences where the origin initially unwinds. The remainder of the origin has mainly 9-mer DnaA binding sites as well as sites for binding IHF and FIS proteins. DnaA sites have either high (R1, R2 and R4) or low (τ1, R5, τ2, I1, I2, C3, C2, I3 and C1) affinity for DnaA. The numbers #1–9 mark the GATC sites studied here. A TaqI site (TCGA) overlapping each of the nine GATC sites was created by converting their two upstream bases to TC ( NNGA TC to TCGA TC). (B) A linear map of oriC features described above. The map also shows location of sites for restriction enzymes MboII and HphI that naturally occurs in oriC .
    Figure Legend Snippet: Sequence of oriC showing its major protein binding sites. (A) Sequence of oriC that includes the minimal region (coordinates 1–246) required for origin function. The coordinate 1 correspondence to 3923744 of gb|U00096.3|. The region includes several GATC sites (shown in red) which are methylated by the Dam methylase enzyme. There are three 13-mer repeats of AT rich sequences where the origin initially unwinds. The remainder of the origin has mainly 9-mer DnaA binding sites as well as sites for binding IHF and FIS proteins. DnaA sites have either high (R1, R2 and R4) or low (τ1, R5, τ2, I1, I2, C3, C2, I3 and C1) affinity for DnaA. The numbers #1–9 mark the GATC sites studied here. A TaqI site (TCGA) overlapping each of the nine GATC sites was created by converting their two upstream bases to TC ( NNGA TC to TCGA TC). (B) A linear map of oriC features described above. The map also shows location of sites for restriction enzymes MboII and HphI that naturally occurs in oriC .

    Techniques Used: Sequencing, Protein Binding, Methylation, Binding Assay, Immunohistofluorescence

    Related Articles

    Polymerase Chain Reaction:

    Article Title: ACVR1 Gene Mutation in Sporadic Korean Patients with Fibrodysplasia Ossificans Progressiva
    Article Snippet: .. Results were further verified by restriction endonuclease digestion of PCR products using Cac8I (New England Biolabs, Beverly, MA, U.S.A.) and HphI (New England Biolabs). .. RESULTS DNA sequence analysis demonstrated the invariable presence of a heterozygous point mutation of c.617G > A in all ten patients with obvious clinical manifestations ( ).

    Article Title: Identification and functional characterization of a novel splicing mutation in RP gene PRPF31
    Article Snippet: .. The 222 bp PCR product was digested with 1 unit of Hph I (NEB, USA) at 37°C overnight, and the resulting products were separated on a 2.0% agarose gel. .. Total RNA was extracted from peripheral whole blood samples from individuals III-13, III-15, IV-5, IV-13, IV-15, V-2, V-3, V-6, V-12, V-16 and V-24 (see ) using the Trizol reagent (Invitrogen, USA).

    Article Title: Genome-scale investigation of phenotypically distinct but nearly clonal Trichoderma strains
    Article Snippet: .. All PCR products were digested to completion with Hph I (New England Biolabs). shows the banding patterns for LU132, LU140 and IMI206040. .. The SNP1-containing region could also be amplified from five T. atroviride strains from Europe and Asia and three T. atroviride strains from New Zealand but the digest banding pattern resembled that of LU140 and IMI206040.

    Article Title: Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch MigraseCentral Role of the Holliday Junction Helicase RuvAB in vlsE Recombination and Infectivity of Borrelia burgdorferi
    Article Snippet: .. Approximately 200ng of PCR product was digested with 2 units of HphI (New England BioLabs) for 1.5 hours. .. Reaction products were analyzed on a 1.2% agarose gel run at 75 V for 1.5 hours in Tris-acetate buffer, stained with 0.5 µg/ml ethidium bromide and images acquired using a FluorChem 8900 imaging system.

    Article Title: 90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †
    Article Snippet: .. Ten microliters of the secondary PCR products of the Hsp90 gene was digested with StyI, HphI, or BbsI (New England BioLabs, Ipswich, MA) under the conditions suggested by the manufacturer, and the products were visualized by electrophoresis through a 2% agarose gel. ..

    Electrophoresis:

    Article Title: 90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †
    Article Snippet: .. Ten microliters of the secondary PCR products of the Hsp90 gene was digested with StyI, HphI, or BbsI (New England BioLabs, Ipswich, MA) under the conditions suggested by the manufacturer, and the products were visualized by electrophoresis through a 2% agarose gel. ..

    Agarose Gel Electrophoresis:

    Article Title: Identification and functional characterization of a novel splicing mutation in RP gene PRPF31
    Article Snippet: .. The 222 bp PCR product was digested with 1 unit of Hph I (NEB, USA) at 37°C overnight, and the resulting products were separated on a 2.0% agarose gel. .. Total RNA was extracted from peripheral whole blood samples from individuals III-13, III-15, IV-5, IV-13, IV-15, V-2, V-3, V-6, V-12, V-16 and V-24 (see ) using the Trizol reagent (Invitrogen, USA).

    Article Title: 90-Kilodalton Heat Shock Protein, Hsp90, as a Target for Genotyping Cryptosporidium spp. Known To Infect Humans ▿ spp. Known To Infect Humans ▿ †
    Article Snippet: .. Ten microliters of the secondary PCR products of the Hsp90 gene was digested with StyI, HphI, or BbsI (New England BioLabs, Ipswich, MA) under the conditions suggested by the manufacturer, and the products were visualized by electrophoresis through a 2% agarose gel. ..

    Article Title: Telomere damage induces internal loops that generate telomeric circles
    Article Snippet: .. The DNA was then digested overnight with 50 units each of RsaI, AluI, MboI, HinfI, MspI, HphI, MnlI (NEB) and then separated on a 0.7% low-melting agarose gel (SeaPlaque Agarose, Lonza, 50100), without ethidium bromide. .. Fragments migrating above the 5 kb band of the marker were extracted using the Silica Bead DNA gel extraction kit (Thermo Fisher Scientific, K0513) following the manufacturer’s instructions, except that once the DNA was bound, the beads were not resuspended to avoid mechanical shearing of the DNA.

    Plasmid Preparation:

    Article Title: Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity
    Article Snippet: .. Restriction endonuclease digests were performed on phage DNA, plasmid DNA and bacterial genomic DNA using DpnI and DpnII (Roche, United States), or HphI (NEB, United States), all according to the manufacturer’s instructions. .. Nucleotide sequence accession numbers All the sequences generated have been submitted to GenBank database with the following accession numbers: Phi15 [GenBank: KM091442], Phi93 [GenBank: KM091443] and Phi145 [GenBank: KM091444].

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    New England Biolabs hphi
    Restriction fragment length polymorphism assay for switching at vlsE . A portion of the vlsE expression site containing the variable regions was amplified using primers B248 and B249 to give a product of 776 bp. <t>PCR</t> reactions were performed on B. burgdorferi grown from ear biopsies taken at day 21 and the products were digested with <t>HphI</t> and run on a 1.2% agarose gel in TAE buffer at 75V for 1.5 hours and stained with ethidium bromide (see Materials and Methods ). Wild-type B. burgdorferi B31-5A4 recovered following infection of a C3H/HeN mouse was used as a template in lanes 1 and 2. An unswitched template (not exposed to mouse infection) is shown in lanes 3 and 4. PCR products from rep2 and mutS2 /1 DNA templates are found in lanes 5 6, and 7 8 respectively. M denotes a 100bp molecular weight marker.
    Hphi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hphi/product/New England Biolabs
    Average 94 stars, based on 25 article reviews
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    Restriction fragment length polymorphism assay for switching at vlsE . A portion of the vlsE expression site containing the variable regions was amplified using primers B248 and B249 to give a product of 776 bp. PCR reactions were performed on B. burgdorferi grown from ear biopsies taken at day 21 and the products were digested with HphI and run on a 1.2% agarose gel in TAE buffer at 75V for 1.5 hours and stained with ethidium bromide (see Materials and Methods ). Wild-type B. burgdorferi B31-5A4 recovered following infection of a C3H/HeN mouse was used as a template in lanes 1 and 2. An unswitched template (not exposed to mouse infection) is shown in lanes 3 and 4. PCR products from rep2 and mutS2 /1 DNA templates are found in lanes 5 6, and 7 8 respectively. M denotes a 100bp molecular weight marker.

    Journal: PLoS Pathogens

    Article Title: Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch MigraseCentral Role of the Holliday Junction Helicase RuvAB in vlsE Recombination and Infectivity of Borrelia burgdorferi

    doi: 10.1371/journal.ppat.1000680

    Figure Lengend Snippet: Restriction fragment length polymorphism assay for switching at vlsE . A portion of the vlsE expression site containing the variable regions was amplified using primers B248 and B249 to give a product of 776 bp. PCR reactions were performed on B. burgdorferi grown from ear biopsies taken at day 21 and the products were digested with HphI and run on a 1.2% agarose gel in TAE buffer at 75V for 1.5 hours and stained with ethidium bromide (see Materials and Methods ). Wild-type B. burgdorferi B31-5A4 recovered following infection of a C3H/HeN mouse was used as a template in lanes 1 and 2. An unswitched template (not exposed to mouse infection) is shown in lanes 3 and 4. PCR products from rep2 and mutS2 /1 DNA templates are found in lanes 5 6, and 7 8 respectively. M denotes a 100bp molecular weight marker.

    Article Snippet: Approximately 200ng of PCR product was digested with 2 units of HphI (New England BioLabs) for 1.5 hours.

    Techniques: Polymorphism Assay, Expressing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Infection, Molecular Weight, Marker

    A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Journal: bioRxiv

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    doi: 10.1101/2020.01.29.924951

    Figure Lengend Snippet: A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Article Snippet: The DNA was then digested overnight with 50 units each of RsaI, AluI, MboI, HinfI, MspI, HphI, MnlI (NEB) and then separated on a 0.7% low-melting agarose gel (SeaPlaque Agarose, Lonza, 50100), without ethidium bromide.

    Techniques: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    Mutation analysis of the ACVR1 in FOP patients with definite clinical manifestations. ( A ) Direct sequencing of the PCR products of the ACVR1 showed the presence of the c.617G > A mutation. R=adenine or guanine, ( B ) Restriction endonuclease digestion of the PCR product (350 bp). The G allele (control) was digested by Cac8I to produce three bands, whereas the A allele appeared as two bands. Because FOP patients were heterozygous for this mutation, the 139 bp and 114 bp bands were also presented. The PCR product not digested with HphI corresponds to the G allele (control) in contrast to digested products corresponding to the A allele (FOP).

    Journal: Journal of Korean Medical Science

    Article Title: ACVR1 Gene Mutation in Sporadic Korean Patients with Fibrodysplasia Ossificans Progressiva

    doi: 10.3346/jkms.2009.24.3.433

    Figure Lengend Snippet: Mutation analysis of the ACVR1 in FOP patients with definite clinical manifestations. ( A ) Direct sequencing of the PCR products of the ACVR1 showed the presence of the c.617G > A mutation. R=adenine or guanine, ( B ) Restriction endonuclease digestion of the PCR product (350 bp). The G allele (control) was digested by Cac8I to produce three bands, whereas the A allele appeared as two bands. Because FOP patients were heterozygous for this mutation, the 139 bp and 114 bp bands were also presented. The PCR product not digested with HphI corresponds to the G allele (control) in contrast to digested products corresponding to the A allele (FOP).

    Article Snippet: Results were further verified by restriction endonuclease digestion of PCR products using Cac8I (New England Biolabs, Beverly, MA, U.S.A.) and HphI (New England Biolabs).

    Techniques: Mutagenesis, Sequencing, Polymerase Chain Reaction

    Electrophoretic separation of Hph I digested PCR fragments. Lanes B, D and F represent the undigested 660 bp PCR fragments from LU132, LU140 and IMI204060, respectively. Lanes A, C and E represent the HphI digested PCR fragments for LU132, LU140 and IMI204060, respectively. The arrow indicates the 350 bp restriction fragment that was only found in LU132. Size standard was 1 Kb Plus DNA Ladder™ (Invitrogen) in lane G.

    Journal: PeerJ

    Article Title: Genome-scale investigation of phenotypically distinct but nearly clonal Trichoderma strains

    doi: 10.7717/peerj.2023

    Figure Lengend Snippet: Electrophoretic separation of Hph I digested PCR fragments. Lanes B, D and F represent the undigested 660 bp PCR fragments from LU132, LU140 and IMI204060, respectively. Lanes A, C and E represent the HphI digested PCR fragments for LU132, LU140 and IMI204060, respectively. The arrow indicates the 350 bp restriction fragment that was only found in LU132. Size standard was 1 Kb Plus DNA Ladder™ (Invitrogen) in lane G.

    Article Snippet: All PCR products were digested to completion with Hph I (New England Biolabs). shows the banding patterns for LU132, LU140 and IMI206040.

    Techniques: Polymerase Chain Reaction