hpai  (New England Biolabs)


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    Name:
    HpaI
    Description:
    HpaI 2 500 units
    Catalog Number:
    R0105L
    Price:
    261
    Category:
    Restriction Enzymes
    Size:
    2 500 units
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    New England Biolabs hpai
    HpaI
    HpaI 2 500 units
    https://www.bioz.com/result/hpai/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hpai - by Bioz Stars, 2021-04
    95/100 stars

    Images

    1) Product Images from "Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases"

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.43.8.4002-4009.2005

    RFLP-Southern blot analysis of HpaI- or EcoRV-digested DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were
    Figure Legend Snippet: RFLP-Southern blot analysis of HpaI- or EcoRV-digested DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were

    Techniques Used: Southern Blot, Isolation

    2) Product Images from "Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways"

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1252

    Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P
    Figure Legend Snippet: Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P

    Techniques Used: Homologous Recombination, Functional Assay, Plasmid Preparation, Expressing, FACS, Transfection, Southern Blot, Titration, In Vivo, Labeling, In Vitro, Purification, Radioactivity

    3) Product Images from "Recombination regulator PRDM9 influences the instability of its own coding sequence in humans"

    Article Title: Recombination regulator PRDM9 influences the instability of its own coding sequence in humans

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1220813110

    De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products
    Figure Legend Snippet: De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products

    Techniques Used: Mutagenesis, Amplification

    4) Product Images from "Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways"

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1252

    Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P
    Figure Legend Snippet: Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P

    Techniques Used: Homologous Recombination, Functional Assay, Plasmid Preparation, Expressing, FACS, Transfection, Southern Blot, Titration, In Vivo, Labeling, In Vitro, Purification, Radioactivity

    5) Product Images from "A new mouse model to study restoration of interleukin-6 (IL-6) expression in a Cre-dependent manner: microglial IL-6 regulation of experimental autoimmune encephalomyelitis"

    Article Title: A new mouse model to study restoration of interleukin-6 (IL-6) expression in a Cre-dependent manner: microglial IL-6 regulation of experimental autoimmune encephalomyelitis

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-020-01969-0

    Generation of a conditional reversible KO mouse for IL-6. a Outline of the targeting strategy . Left: 5614 bp HpaI-PacI (pink; upstream) and a 5241 bp XhoI-NotI (orange; downstream) flanking Il6 DIOex2 fragments. Right: the final product that consists of a PacI-SalI fragment with inverted exon 2 flanked by double lox sites, and three fragments containing Pgk -DT a and HSV-TK genes for negative selection and frt -flanked SvNeo gene for positive selection. b Southern blot for Il6 gene of the positive ES clones using the probe ( left ) revealed a ~11 kb band on the wild type allele and a ~8 kb band on the targeted allele, as expected ( right ); those clones with two bands were selected. c Left: The Il6 gene-modified sequence from IL6-DIO-KO mouse was sequenced by the Sanger method. The resulting sequence had 1361 bp and exon 1 (yellow), exon 2 (blue), lox2272 cassettes (green), loxP cassettes (purple), and FRT (red) were genetic structures observed in that sequence. Middle : PCR products for Il6 gene of WT, IL6-DIO-Het, and IL6-DIO-KO mice yielded the expected bands. Right : IL-6 protein in serum was undetectable by ELISA in IL6-DIO-KO mice after LPS administration. All results are represented as mean ± SEM; ★ p ≤ 0.05 vs. LPS-C57BL/6 mice
    Figure Legend Snippet: Generation of a conditional reversible KO mouse for IL-6. a Outline of the targeting strategy . Left: 5614 bp HpaI-PacI (pink; upstream) and a 5241 bp XhoI-NotI (orange; downstream) flanking Il6 DIOex2 fragments. Right: the final product that consists of a PacI-SalI fragment with inverted exon 2 flanked by double lox sites, and three fragments containing Pgk -DT a and HSV-TK genes for negative selection and frt -flanked SvNeo gene for positive selection. b Southern blot for Il6 gene of the positive ES clones using the probe ( left ) revealed a ~11 kb band on the wild type allele and a ~8 kb band on the targeted allele, as expected ( right ); those clones with two bands were selected. c Left: The Il6 gene-modified sequence from IL6-DIO-KO mouse was sequenced by the Sanger method. The resulting sequence had 1361 bp and exon 1 (yellow), exon 2 (blue), lox2272 cassettes (green), loxP cassettes (purple), and FRT (red) were genetic structures observed in that sequence. Middle : PCR products for Il6 gene of WT, IL6-DIO-Het, and IL6-DIO-KO mice yielded the expected bands. Right : IL-6 protein in serum was undetectable by ELISA in IL6-DIO-KO mice after LPS administration. All results are represented as mean ± SEM; ★ p ≤ 0.05 vs. LPS-C57BL/6 mice

    Techniques Used: Selection, Southern Blot, Clone Assay, Modification, Sequencing, Polymerase Chain Reaction, Mouse Assay, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Characterization of Two New CTX-M-25-Group Extended-Spectrum ?-Lactamase Variants Identified in Escherichia coli Isolates from Israel"

    Article Title: Characterization of Two New CTX-M-25-Group Extended-Spectrum ?-Lactamase Variants Identified in Escherichia coli Isolates from Israel

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046329

    HpaI plasmid profiles of bla CTX-M-94 -, bla CMY-2 - and bla CTX-M-100 -harbouring plasmids. A 0.7% agarose gel was loaded with HpaI restricted plasmid DNA purified from three recombinant E. coli TOP10 strains containing the (A) bla CTX-M-94 -, (B) bla CMY-2 - and (C) bla CTX-M-100 -harbouring plasmids, respectively, and stained with GelRed. Relative mobility calculations, based on known band sizes of the 1 Kb DNA Extension Ladder (M), estimated total plasmid sizes to be (A) 93 kb, (B) 98 kb and (C) 130 kb.
    Figure Legend Snippet: HpaI plasmid profiles of bla CTX-M-94 -, bla CMY-2 - and bla CTX-M-100 -harbouring plasmids. A 0.7% agarose gel was loaded with HpaI restricted plasmid DNA purified from three recombinant E. coli TOP10 strains containing the (A) bla CTX-M-94 -, (B) bla CMY-2 - and (C) bla CTX-M-100 -harbouring plasmids, respectively, and stained with GelRed. Relative mobility calculations, based on known band sizes of the 1 Kb DNA Extension Ladder (M), estimated total plasmid sizes to be (A) 93 kb, (B) 98 kb and (C) 130 kb.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Purification, Recombinant, Staining

    Related Articles

    Ethanol Precipitation:

    Article Title: Recombination regulator PRDM9 influences the instability of its own coding sequence in humans
    Article Snippet: .. Aliquots of 25 μg DNA were digested with 240 U HpaI plus 240 U PvuII-HF (New England BioLabs) in 500 μL NEB4 buffer at 37 °C for 2.5 h. Digested DNA was recovered by ethanol precipitation, dissolved in 5 mM Tris-HCl, pH 7.5, and 15 μg DNA was loaded into a 2.5-cm-wide slot in a 40-cm-long 0.9% (wt/vol) SeaKem HGT (Lonza) agarose gel in 0.5 × tris-borate EDTA buffer containing 0.5 μg/mL ethidium bromide. .. Adjacent size markers were 4 μg λ DNA × HindIII plus 2 μg ϕX174 DNA × HaeIII, together with 2 μg of ladder ML ( ) consisting of PCR products matched in size and GC content to HpaI–PvuII fragments encoding PRDM9 ZnF arrays with 11–17 repeats.

    Agarose Gel Electrophoresis:

    Article Title: Recombination regulator PRDM9 influences the instability of its own coding sequence in humans
    Article Snippet: .. Aliquots of 25 μg DNA were digested with 240 U HpaI plus 240 U PvuII-HF (New England BioLabs) in 500 μL NEB4 buffer at 37 °C for 2.5 h. Digested DNA was recovered by ethanol precipitation, dissolved in 5 mM Tris-HCl, pH 7.5, and 15 μg DNA was loaded into a 2.5-cm-wide slot in a 40-cm-long 0.9% (wt/vol) SeaKem HGT (Lonza) agarose gel in 0.5 × tris-borate EDTA buffer containing 0.5 μg/mL ethidium bromide. .. Adjacent size markers were 4 μg λ DNA × HindIII plus 2 μg ϕX174 DNA × HaeIII, together with 2 μg of ladder ML ( ) consisting of PCR products matched in size and GC content to HpaI–PvuII fragments encoding PRDM9 ZnF arrays with 11–17 repeats.

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: .. Samples were digested using HpaI and NcoI (New England Biolabs) for 4 h, run on a 2% native agarose gel and then transferred to Genescreen membrane for 5 h in 0.4 N NaOH. .. The DNA was then crosslinked to the membrane by UV irradiation.

    Article Title: A Transactivator Function of Cottontail Rabbit Papillomavirus E2 Is Essential for Tumor Induction in Rabbits
    Article Snippet: Cells were harvested 48 h after transfection, and low-molecular-weight DNA was purified as described previously ( ). .. DNA aliquots of equal volumes were incubated with the restriction enzymes Dpn I (5 U) and Hpa I (15 U) (both from New England Biolabs) and 10 μg of RNAse A at 37°C for 5 h. The DNAs were separated on a 0.8% agarose gel. .. DNA was transferred onto a nylon membrane (GeneScreen Plus; NEN Life Science Products) by capillary transfer with 0.4 M NaOH as a transfer buffer for 5 h. Eighty nanograms of linearized CRPV-pGL3-NCR served as a hybridization probe; the probe was labeled with 50 μCi of [α-32 P]dCTP (specific activity, 6,000 Ci/mmol; Amersham Pharmacia) and Ready-To-Go DNA labeling beads (without dCTP) (Amersham Pharmacia) and was purified by NucTrapProbe (Stratagene).

    Article Title: An Improved RSP Method to Detect HpaI Polymorphism in the Apolipoprotein C-1 Gene Promoter
    Article Snippet: Thermal cycling was done in a Perkin-Elmer GeneAmp PCR System 9600 Thermal Cycler with an initial 2 min denaturation at 94°C followed by 35 cycles of denaturing at 94°C for 30 sec., annealing at 60°C for 30 sec., extending at 72°C for 1 min, and a final extension of 5 min at 72°C. .. Genotyping One third of the PCR product was digested with 5 U Hpa I (Cat. No. 105S, New England Biolabs, Inc. Beverly, MA) for one and half hours in a 37°C water bath, followed by separation on a 2% agarose gel electrophoresis for 1 to 1.5 hours at constant voltage of 110 V. The gels were stained by 0.5 ug/ml of ethidium bromide. .. The image was captured on the thermal paper using the Eagle Eye II Still Video System (Stratagene, La Jolla, CA).

    Incubation:

    Article Title: A Transactivator Function of Cottontail Rabbit Papillomavirus E2 Is Essential for Tumor Induction in Rabbits
    Article Snippet: Cells were harvested 48 h after transfection, and low-molecular-weight DNA was purified as described previously ( ). .. DNA aliquots of equal volumes were incubated with the restriction enzymes Dpn I (5 U) and Hpa I (15 U) (both from New England Biolabs) and 10 μg of RNAse A at 37°C for 5 h. The DNAs were separated on a 0.8% agarose gel. .. DNA was transferred onto a nylon membrane (GeneScreen Plus; NEN Life Science Products) by capillary transfer with 0.4 M NaOH as a transfer buffer for 5 h. Eighty nanograms of linearized CRPV-pGL3-NCR served as a hybridization probe; the probe was labeled with 50 μCi of [α-32 P]dCTP (specific activity, 6,000 Ci/mmol; Amersham Pharmacia) and Ready-To-Go DNA labeling beads (without dCTP) (Amersham Pharmacia) and was purified by NucTrapProbe (Stratagene).

    Generated:

    Article Title: IRES-mediated translation of foot-and-mouth disease virus (FMDV) in cultured cells derived from FMDV-susceptible and -insusceptible animals
    Article Snippet: .. Reporter genes were excised from pRF/FMDV-IRES using the restriction endonucleases Eco RV (Toyobo) and Hpa I (NEB). pCAGGS/FMDV-IRES was generated by inserting a reporter gene into pCAGGS/MSC(F), which was then treated with SmaI (Takara) and rAPid Alkaline Phosphatase (Roche) using Mighty Mix (Takara). .. DNA fragments were purified with the Big Dye XTerminator Purification kit, followed by sequencing via capillary electrophoresis on the ABI PRISM310 genetic analyzer.

    Article Title: Features of Infections Due to Klebsiella pneumoniae Carbapenemase-Producing Escherichia coli: Emergence of Sequence Type 131
    Article Snippet: The plasmids were extracted from these transconjugants or transformants using the standard alkaline lysis method. .. Fingerprints of the plasmids were generated by digesting them with Eco RI or Hpa I (New England Biolabs). .. A total of 13 patients with infection due to KPC-producing E. coli was identified from the study period.

    Polymerase Chain Reaction:

    Article Title: An Improved RSP Method to Detect HpaI Polymorphism in the Apolipoprotein C-1 Gene Promoter
    Article Snippet: Thermal cycling was done in a Perkin-Elmer GeneAmp PCR System 9600 Thermal Cycler with an initial 2 min denaturation at 94°C followed by 35 cycles of denaturing at 94°C for 30 sec., annealing at 60°C for 30 sec., extending at 72°C for 1 min, and a final extension of 5 min at 72°C. .. Genotyping One third of the PCR product was digested with 5 U Hpa I (Cat. No. 105S, New England Biolabs, Inc. Beverly, MA) for one and half hours in a 37°C water bath, followed by separation on a 2% agarose gel electrophoresis for 1 to 1.5 hours at constant voltage of 110 V. The gels were stained by 0.5 ug/ml of ethidium bromide. .. The image was captured on the thermal paper using the Eagle Eye II Still Video System (Stratagene, La Jolla, CA).

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: DNA was visualized using SYBR safe DNA gel stain (Invitrogen). .. To generate the substrate used for the thermal stability experiments pECFP-289-NtCFP constructs with appropriate RSS mutations were digested with HpaI and NdeI (New England Biolabs) and a 1.6 Kb fragment was gel purified and then used for PCR amplification using NtCFP_upPCR 5′-CGCGCCGAGGTGAAGTTCGAGG-3′ and NtCFP_downPCR 5′-TGCCCCAGGATGTTGCCGTCCTCC-3′ to generate a 477 bp PCR product. .. PCR amplification was performed as follows: 94°C, 2 min; 94°C, 15 s; 65°C, 30 s; 72°C, 30 s for 25 cycles; and 72°C, 7 min. Student's T-test assuming equal variance was used to calculate statistical significance.

    Staining:

    Article Title: An Improved RSP Method to Detect HpaI Polymorphism in the Apolipoprotein C-1 Gene Promoter
    Article Snippet: Thermal cycling was done in a Perkin-Elmer GeneAmp PCR System 9600 Thermal Cycler with an initial 2 min denaturation at 94°C followed by 35 cycles of denaturing at 94°C for 30 sec., annealing at 60°C for 30 sec., extending at 72°C for 1 min, and a final extension of 5 min at 72°C. .. Genotyping One third of the PCR product was digested with 5 U Hpa I (Cat. No. 105S, New England Biolabs, Inc. Beverly, MA) for one and half hours in a 37°C water bath, followed by separation on a 2% agarose gel electrophoresis for 1 to 1.5 hours at constant voltage of 110 V. The gels were stained by 0.5 ug/ml of ethidium bromide. .. The image was captured on the thermal paper using the Eagle Eye II Still Video System (Stratagene, La Jolla, CA).

    Construct:

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: DNA was visualized using SYBR safe DNA gel stain (Invitrogen). .. To generate the substrate used for the thermal stability experiments pECFP-289-NtCFP constructs with appropriate RSS mutations were digested with HpaI and NdeI (New England Biolabs) and a 1.6 Kb fragment was gel purified and then used for PCR amplification using NtCFP_upPCR 5′-CGCGCCGAGGTGAAGTTCGAGG-3′ and NtCFP_downPCR 5′-TGCCCCAGGATGTTGCCGTCCTCC-3′ to generate a 477 bp PCR product. .. PCR amplification was performed as follows: 94°C, 2 min; 94°C, 15 s; 65°C, 30 s; 72°C, 30 s for 25 cycles; and 72°C, 7 min. Student's T-test assuming equal variance was used to calculate statistical significance.

    Purification:

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: DNA was visualized using SYBR safe DNA gel stain (Invitrogen). .. To generate the substrate used for the thermal stability experiments pECFP-289-NtCFP constructs with appropriate RSS mutations were digested with HpaI and NdeI (New England Biolabs) and a 1.6 Kb fragment was gel purified and then used for PCR amplification using NtCFP_upPCR 5′-CGCGCCGAGGTGAAGTTCGAGG-3′ and NtCFP_downPCR 5′-TGCCCCAGGATGTTGCCGTCCTCC-3′ to generate a 477 bp PCR product. .. PCR amplification was performed as follows: 94°C, 2 min; 94°C, 15 s; 65°C, 30 s; 72°C, 30 s for 25 cycles; and 72°C, 7 min. Student's T-test assuming equal variance was used to calculate statistical significance.

    Amplification:

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways
    Article Snippet: DNA was visualized using SYBR safe DNA gel stain (Invitrogen). .. To generate the substrate used for the thermal stability experiments pECFP-289-NtCFP constructs with appropriate RSS mutations were digested with HpaI and NdeI (New England Biolabs) and a 1.6 Kb fragment was gel purified and then used for PCR amplification using NtCFP_upPCR 5′-CGCGCCGAGGTGAAGTTCGAGG-3′ and NtCFP_downPCR 5′-TGCCCCAGGATGTTGCCGTCCTCC-3′ to generate a 477 bp PCR product. .. PCR amplification was performed as follows: 94°C, 2 min; 94°C, 15 s; 65°C, 30 s; 72°C, 30 s for 25 cycles; and 72°C, 7 min. Student's T-test assuming equal variance was used to calculate statistical significance.

    Isolation:

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases
    Article Snippet: A 318-bp digoxigenin (DIG)-labeled cpb2 -specific DNA probe was prepared by using a two-step PCR amplification method as previously described ( ). .. Isolated C. perfringens DNA samples, prepared as previously described ( , ), were digested with EcoRV or HpaI (New England Biolabs), separated by electrophoresis on 1% agarose gels, and Southern transferred. .. The blots were hybridized with the DIG-labeled cpb2 probe, which was then detected using a DIG chemiluminescence detection system utilizing CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)tricyclo[3,3.1.13.7]decane}-4-yl)phenyl phosphate] ready-to-use substrate (Roche) as described earlier ( ).

    Electrophoresis:

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases
    Article Snippet: A 318-bp digoxigenin (DIG)-labeled cpb2 -specific DNA probe was prepared by using a two-step PCR amplification method as previously described ( ). .. Isolated C. perfringens DNA samples, prepared as previously described ( , ), were digested with EcoRV or HpaI (New England Biolabs), separated by electrophoresis on 1% agarose gels, and Southern transferred. .. The blots were hybridized with the DIG-labeled cpb2 probe, which was then detected using a DIG chemiluminescence detection system utilizing CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)tricyclo[3,3.1.13.7]decane}-4-yl)phenyl phosphate] ready-to-use substrate (Roche) as described earlier ( ).

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    New England Biolabs hpai
    RFLP-Southern blot analysis of <t>HpaI-</t> or <t>EcoRV-digested</t> DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were
    Hpai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RFLP-Southern blot analysis of HpaI- or EcoRV-digested DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were

    Journal: Journal of Clinical Microbiology

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases

    doi: 10.1128/JCM.43.8.4002-4009.2005

    Figure Lengend Snippet: RFLP-Southern blot analysis of HpaI- or EcoRV-digested DNA from horse GI disease isolates. Total DNA isolated from each of the specified C. perfringens strains was digested with HpaI (A) or EcoRV (B) and then Southern transferred. The Southern blots were

    Article Snippet: Isolated C. perfringens DNA samples, prepared as previously described ( , ), were digested with EcoRV or HpaI (New England Biolabs), separated by electrophoresis on 1% agarose gels, and Southern transferred.

    Techniques: Southern Blot, Isolation

    Gel patterns of Hpa I polymorphism. Lane 1 shows the homozygous H1/H1 genotype, lane 2 is heterozygous H1/H2 and lane 3 is homozygous H2/H2. Lane 4 is a 72- to 1,353-bp DNA ladder.

    Journal: BMC Medical Genetics

    Article Title: An Improved RSP Method to Detect HpaI Polymorphism in the Apolipoprotein C-1 Gene Promoter

    doi: 10.1186/1471-2350-3-13

    Figure Lengend Snippet: Gel patterns of Hpa I polymorphism. Lane 1 shows the homozygous H1/H1 genotype, lane 2 is heterozygous H1/H2 and lane 3 is homozygous H2/H2. Lane 4 is a 72- to 1,353-bp DNA ladder.

    Article Snippet: Genotyping One third of the PCR product was digested with 5 U Hpa I (Cat. No. 105S, New England Biolabs, Inc. Beverly, MA) for one and half hours in a 37°C water bath, followed by separation on a 2% agarose gel electrophoresis for 1 to 1.5 hours at constant voltage of 110 V. The gels were stained by 0.5 ug/ml of ethidium bromide.

    Techniques:

    Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P

    Journal: Nucleic Acids Research

    Article Title: Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways

    doi: 10.1093/nar/gkp1252

    Figure Lengend Snippet: Certain non-consensus RSS affect handling of SEs after cleavage. ( A ) Homologous recombination assay. Repair of a non-functional CFP gene by homologous recombination after RAG cleavage of plasmid 289-ntCFP leads to the expression of the CFP gene resulting in the detection of blue fluorescent cells, measurable by FACS analysis. ( B ) Quantification of FACS data ( n = 6). Background, as measured by CFP expression in cells transfected with a catalytically inactive RAG1 allele, DDE, was subtracted from each sample. Con denotes consensus RSS. ( C ) Southern blot showing products of RAG cleavage of 289-ntCFP by cores RAG1 and RAG2. Hirt preps were digested with HpaI and NcoI to generate a 1626 bp fragment. Cleavage by the V(D)J recombinase generates a pair of SEs that are 816 bp (12RSS) and 558 bp (23RSS) and can be visualized using an internally radiolabeled probe. First four lanes show a titration of Hirt DNA from a transfection with a consensus pair of RSS. Percent of DNA loaded is found above each lane. Lanes 4–10 show levels of SEs in vivo at various non-consensus RSSs, labeled above each lane. ( D ) Quantification of in vitro cleavage by purified RAG proteins, measured as the combined amount of radioactivity from cleaved products divided by the total amount of radioactivity. Examples of gels are shown in Figure 1 C in the proteinase K treated lane. * denotes P

    Article Snippet: Samples were digested using HpaI and NcoI (New England Biolabs) for 4 h, run on a 2% native agarose gel and then transferred to Genescreen membrane for 5 h in 0.4 N NaOH.

    Techniques: Homologous Recombination, Functional Assay, Plasmid Preparation, Expressing, FACS, Transfection, Southern Blot, Titration, In Vivo, Labeling, In Vitro, Purification, Radioactivity

    De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Recombination regulator PRDM9 influences the instability of its own coding sequence in humans

    doi: 10.1073/pnas.1220813110

    Figure Lengend Snippet: De novo mutation at PRDM9 . ( A ) Restriction sites used for size-enriching ZnF repeat array mutants, plus primers (blue arrows) for single molecule amplification of the array (boxes). Using HpaI and PvuII ensured that any residual partial digest products

    Article Snippet: Aliquots of 25 μg DNA were digested with 240 U HpaI plus 240 U PvuII-HF (New England BioLabs) in 500 μL NEB4 buffer at 37 °C for 2.5 h. Digested DNA was recovered by ethanol precipitation, dissolved in 5 mM Tris-HCl, pH 7.5, and 15 μg DNA was loaded into a 2.5-cm-wide slot in a 40-cm-long 0.9% (wt/vol) SeaKem HGT (Lonza) agarose gel in 0.5 × tris-borate EDTA buffer containing 0.5 μg/mL ethidium bromide.

    Techniques: Mutagenesis, Amplification