tcsc5d amplification  (New England Biolabs)


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    New England Biolabs tcsc5d amplification
    HpaI
    HpaI 2 500 units
    https://www.bioz.com/result/tcsc5d amplification/product/New England Biolabs
    Average 85 stars, based on 394 article reviews
    Price from $9.99 to $1999.99
    tcsc5d amplification - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product"

    Article Title: A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001777

    Key polymorphic sites in the T. cruzi TcSC5D gene. A: diploid genotypes built from identified highly informative sites (see main text), showing a section of one chromatogram around the tetra-allelic SNP at position 657. B: Summary of observed nucleotide changes between DTUs for these six sites. In the case of the comparisons with the TcII DTU, this is the minimum expected number of changes (marked with * in the figure), because of the diversity observed at position 618 in strains from this DTU.
    Figure Legend Snippet: Key polymorphic sites in the T. cruzi TcSC5D gene. A: diploid genotypes built from identified highly informative sites (see main text), showing a section of one chromatogram around the tetra-allelic SNP at position 657. B: Summary of observed nucleotide changes between DTUs for these six sites. In the case of the comparisons with the TcII DTU, this is the minimum expected number of changes (marked with * in the figure), because of the diversity observed at position 618 in strains from this DTU.

    Techniques Used:

    The TcSC5D locus as a lineage discriminant marker. A: schematic view of the TcSC5D amplicon, with all identified SNPs, including key discriminant positions (marked in blue), and polymorphic HpaI/SphI restriction enzyme sites, showing their presence/absence in each lineage. B: Restriction fragment length polymorphism analysis of the TcSC5D amplification product. Fragments of the SphI/HpaI double digestion were resolved in a 2% TBE-agarose gel. Lanes in the gel correspond to: molecular size markers (lane 1), and DNA from T. cruzi strains (lanes 2–21). These are: Sylvio X10 (lane 3), Dm28c (4), and CAI72 (5) for DTU TcI; MAS1 cl1 (6), TU18 cl93 (7), and IVV cl4 (8) for DTU TcII; M6241 cl6 (9), M5631 cl5 (10), and X109/2 (11) for DUT TcIII; CanIII cl1(12), Dog Theis (13) and 92122102R (14) for DTU TcIV; Sc43 (15), MN cl2 (16) and Teh53 (17) for DTU TcV; CL-Brener (18), P63 cl1 (19), and Tulahuen cl2 (20) for DTU TcVI; and TCC1994 and TCC1122 for DTU Tcbat. The corresponding locus from T. cruzi marinkellei was analyzed in lanes 22/23 (stocks B3 and B7).
    Figure Legend Snippet: The TcSC5D locus as a lineage discriminant marker. A: schematic view of the TcSC5D amplicon, with all identified SNPs, including key discriminant positions (marked in blue), and polymorphic HpaI/SphI restriction enzyme sites, showing their presence/absence in each lineage. B: Restriction fragment length polymorphism analysis of the TcSC5D amplification product. Fragments of the SphI/HpaI double digestion were resolved in a 2% TBE-agarose gel. Lanes in the gel correspond to: molecular size markers (lane 1), and DNA from T. cruzi strains (lanes 2–21). These are: Sylvio X10 (lane 3), Dm28c (4), and CAI72 (5) for DTU TcI; MAS1 cl1 (6), TU18 cl93 (7), and IVV cl4 (8) for DTU TcII; M6241 cl6 (9), M5631 cl5 (10), and X109/2 (11) for DUT TcIII; CanIII cl1(12), Dog Theis (13) and 92122102R (14) for DTU TcIV; Sc43 (15), MN cl2 (16) and Teh53 (17) for DTU TcV; CL-Brener (18), P63 cl1 (19), and Tulahuen cl2 (20) for DTU TcVI; and TCC1994 and TCC1122 for DTU Tcbat. The corresponding locus from T. cruzi marinkellei was analyzed in lanes 22/23 (stocks B3 and B7).

    Techniques Used: Marker, Amplification, Agarose Gel Electrophoresis

    Proposed typing strategy based on the TcSC5D locus. A : the highly streamlined TcSC5D-PCR-RFLP assay can discriminate all non-hybrid lineages. If discrimination of DTUs TcV from TcVI is necessary, sequencing of the TcSC5D amplification locus is required. B : alternatively, if the method of choice is the PCR-RFLP, then a second locus can be assayed to resolve these DTUs. This additional locus can be the TcMK gene as shown in the figure (this work, see main text), or the gp72 gene (polymorphic Taq I site [41] ).
    Figure Legend Snippet: Proposed typing strategy based on the TcSC5D locus. A : the highly streamlined TcSC5D-PCR-RFLP assay can discriminate all non-hybrid lineages. If discrimination of DTUs TcV from TcVI is necessary, sequencing of the TcSC5D amplification locus is required. B : alternatively, if the method of choice is the PCR-RFLP, then a second locus can be assayed to resolve these DTUs. This additional locus can be the TcMK gene as shown in the figure (this work, see main text), or the gp72 gene (polymorphic Taq I site [41] ).

    Techniques Used: Polymerase Chain Reaction, RFLP Assay, Sequencing, Amplification

    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 "Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry"

    Article Title: Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006766

    Phospholipid scramblase associates with glycoprotein L to restore membrane architecture. (A) CaSki cells were mock-infected or synchronously infected with HSV-2(G), HSV-1(KOS), complemented or non-complemented gL-2 deletion virus (ΔgL-2 +/- and ΔgL-2 -/- , respectively), non-complemented gH-2 deletion (ΔgH-2 -/- ), or non-complemented gL-1 (ΔgL-1 -/- ) or gH-1 deleted viruses (ΔgH-1 -/- ) (MOI equivalent to ~ 1 pfu/cell). Fifteen minutes after the temperature shift, the cells were fixed and stained with the indicated murine HSV-serotype common mAbs and a rabbit polyclonal antibody against PLSCR1 and then probed with species-specific proximity ligation secondary antibodies. Results are representative of at least 3 independent experiments. (B). Western blots of dextran gradient-purified ΔgL or ΔgH virus isolated 24 hours after infection of 79VB4 (ΔgL-2 +/- ), F6 (ΔgH-2 +/- ) or Vero (ΔgL-2 −/− and ΔgH-2 -/- ) cells. Protein expression was assessed for viral glycoproteins H and L with serotype-specific mAbs. (C). CaSki cells were synchronously infected with HSV-2(G) (5 pfu/cell) (+) or mock-infected (-) and cell lysates were prepared 15 min post-temperature shift, immunoprecipitated (IP) with serotype common mouse anti-gL, mouse ant-gH or rabbit anti-PLSCR1 and equivalent volumes of supernatant, pellet or whole cell lysates analyzed by preparing Western blots (WB) and probing with rabbit anti-PLSCR1, mouse anti-gL, mouse anti-gH or mouse anti-αvβ3. Blots are representative of results obtained in 2 independent experiments. (D). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) and at the indicated times post-infection, cells were fixed and stained with antibodies to phosphatidylserine (red) or Akt (green); nuclei were stained blue. Mock-infected cells were included as a negative control. Representative extended focus images from two experiments are shown; scale bar 10μm. (E). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) for 30 minutes and then the inoculum removed, cells washed with a low pH citrate buffer and then incubated for 1, 2 or 4 h. R5421 (or DMSO) was added to the medium at the time of infection (t = 0 minutes) or immediately following citrate treatment (t = 30 minutes). Cells were stained as in Panel D. Representative images from two experiments are shown; bar = 10μm.
    Figure Legend Snippet: Phospholipid scramblase associates with glycoprotein L to restore membrane architecture. (A) CaSki cells were mock-infected or synchronously infected with HSV-2(G), HSV-1(KOS), complemented or non-complemented gL-2 deletion virus (ΔgL-2 +/- and ΔgL-2 -/- , respectively), non-complemented gH-2 deletion (ΔgH-2 -/- ), or non-complemented gL-1 (ΔgL-1 -/- ) or gH-1 deleted viruses (ΔgH-1 -/- ) (MOI equivalent to ~ 1 pfu/cell). Fifteen minutes after the temperature shift, the cells were fixed and stained with the indicated murine HSV-serotype common mAbs and a rabbit polyclonal antibody against PLSCR1 and then probed with species-specific proximity ligation secondary antibodies. Results are representative of at least 3 independent experiments. (B). Western blots of dextran gradient-purified ΔgL or ΔgH virus isolated 24 hours after infection of 79VB4 (ΔgL-2 +/- ), F6 (ΔgH-2 +/- ) or Vero (ΔgL-2 −/− and ΔgH-2 -/- ) cells. Protein expression was assessed for viral glycoproteins H and L with serotype-specific mAbs. (C). CaSki cells were synchronously infected with HSV-2(G) (5 pfu/cell) (+) or mock-infected (-) and cell lysates were prepared 15 min post-temperature shift, immunoprecipitated (IP) with serotype common mouse anti-gL, mouse ant-gH or rabbit anti-PLSCR1 and equivalent volumes of supernatant, pellet or whole cell lysates analyzed by preparing Western blots (WB) and probing with rabbit anti-PLSCR1, mouse anti-gL, mouse anti-gH or mouse anti-αvβ3. Blots are representative of results obtained in 2 independent experiments. (D). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) and at the indicated times post-infection, cells were fixed and stained with antibodies to phosphatidylserine (red) or Akt (green); nuclei were stained blue. Mock-infected cells were included as a negative control. Representative extended focus images from two experiments are shown; scale bar 10μm. (E). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) for 30 minutes and then the inoculum removed, cells washed with a low pH citrate buffer and then incubated for 1, 2 or 4 h. R5421 (or DMSO) was added to the medium at the time of infection (t = 0 minutes) or immediately following citrate treatment (t = 30 minutes). Cells were stained as in Panel D. Representative images from two experiments are shown; bar = 10μm.

    Techniques Used: Infection, Staining, Ligation, Western Blot, Purification, Isolation, Expressing, Immunoprecipitation, Negative Control, Incubation

    4) Product Images from "Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry"

    Article Title: Herpes simplex viruses activate phospholipid scramblase to redistribute phosphatidylserines and Akt to the outer leaflet of the plasma membrane and promote viral entry

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006766

    Phospholipid scramblase associates with glycoprotein L to restore membrane architecture. (A) CaSki cells were mock-infected or synchronously infected with HSV-2(G), HSV-1(KOS), complemented or non-complemented gL-2 deletion virus (ΔgL-2 +/- and ΔgL-2 -/- , respectively), non-complemented gH-2 deletion (ΔgH-2 -/- ), or non-complemented gL-1 (ΔgL-1 -/- ) or gH-1 deleted viruses (ΔgH-1 -/- ) (MOI equivalent to ~ 1 pfu/cell). Fifteen minutes after the temperature shift, the cells were fixed and stained with the indicated murine HSV-serotype common mAbs and a rabbit polyclonal antibody against PLSCR1 and then probed with species-specific proximity ligation secondary antibodies. Results are representative of at least 3 independent experiments. (B). Western blots of dextran gradient-purified ΔgL or ΔgH virus isolated 24 hours after infection of 79VB4 (ΔgL-2 +/- ), F6 (ΔgH-2 +/- ) or Vero (ΔgL-2 −/− and ΔgH-2 -/- ) cells. Protein expression was assessed for viral glycoproteins H and L with serotype-specific mAbs. (C). CaSki cells were synchronously infected with HSV-2(G) (5 pfu/cell) (+) or mock-infected (-) and cell lysates were prepared 15 min post-temperature shift, immunoprecipitated (IP) with serotype common mouse anti-gL, mouse ant-gH or rabbit anti-PLSCR1 and equivalent volumes of supernatant, pellet or whole cell lysates analyzed by preparing Western blots (WB) and probing with rabbit anti-PLSCR1, mouse anti-gL, mouse anti-gH or mouse anti-αvβ3. Blots are representative of results obtained in 2 independent experiments. (D). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) and at the indicated times post-infection, cells were fixed and stained with antibodies to phosphatidylserine (red) or Akt (green); nuclei were stained blue. Mock-infected cells were included as a negative control. Representative extended focus images from two experiments are shown; scale bar 10μm. (E). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) for 30 minutes and then the inoculum removed, cells washed with a low pH citrate buffer and then incubated for 1, 2 or 4 h. R5421 (or DMSO) was added to the medium at the time of infection (t = 0 minutes) or immediately following citrate treatment (t = 30 minutes). Cells were stained as in Panel D. Representative images from two experiments are shown; bar = 10μm.
    Figure Legend Snippet: Phospholipid scramblase associates with glycoprotein L to restore membrane architecture. (A) CaSki cells were mock-infected or synchronously infected with HSV-2(G), HSV-1(KOS), complemented or non-complemented gL-2 deletion virus (ΔgL-2 +/- and ΔgL-2 -/- , respectively), non-complemented gH-2 deletion (ΔgH-2 -/- ), or non-complemented gL-1 (ΔgL-1 -/- ) or gH-1 deleted viruses (ΔgH-1 -/- ) (MOI equivalent to ~ 1 pfu/cell). Fifteen minutes after the temperature shift, the cells were fixed and stained with the indicated murine HSV-serotype common mAbs and a rabbit polyclonal antibody against PLSCR1 and then probed with species-specific proximity ligation secondary antibodies. Results are representative of at least 3 independent experiments. (B). Western blots of dextran gradient-purified ΔgL or ΔgH virus isolated 24 hours after infection of 79VB4 (ΔgL-2 +/- ), F6 (ΔgH-2 +/- ) or Vero (ΔgL-2 −/− and ΔgH-2 -/- ) cells. Protein expression was assessed for viral glycoproteins H and L with serotype-specific mAbs. (C). CaSki cells were synchronously infected with HSV-2(G) (5 pfu/cell) (+) or mock-infected (-) and cell lysates were prepared 15 min post-temperature shift, immunoprecipitated (IP) with serotype common mouse anti-gL, mouse ant-gH or rabbit anti-PLSCR1 and equivalent volumes of supernatant, pellet or whole cell lysates analyzed by preparing Western blots (WB) and probing with rabbit anti-PLSCR1, mouse anti-gL, mouse anti-gH or mouse anti-αvβ3. Blots are representative of results obtained in 2 independent experiments. (D). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) and at the indicated times post-infection, cells were fixed and stained with antibodies to phosphatidylserine (red) or Akt (green); nuclei were stained blue. Mock-infected cells were included as a negative control. Representative extended focus images from two experiments are shown; scale bar 10μm. (E). CaSki cells were infected with HSV-2(G) (MOI 10 pfu/cell) for 30 minutes and then the inoculum removed, cells washed with a low pH citrate buffer and then incubated for 1, 2 or 4 h. R5421 (or DMSO) was added to the medium at the time of infection (t = 0 minutes) or immediately following citrate treatment (t = 30 minutes). Cells were stained as in Panel D. Representative images from two experiments are shown; bar = 10μm.

    Techniques Used: Infection, Staining, Ligation, Western Blot, Purification, Isolation, Expressing, Immunoprecipitation, Negative Control, Incubation

    5) 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

    6) 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

    7) Product Images from "An Improved RSP Method to Detect HpaI Polymorphism in the Apolipoprotein C-1 Gene Promoter"

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

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-3-13

    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.
    Figure Legend 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.

    Techniques Used:

    8) 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

    9) Product Images from "A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product"

    Article Title: A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001777

    Restriction fragment length polymorphism analysis of the TcMK amplification product. A: a multiple sequence alignment of the 5′-UTR just upstream of the translational start codon, showing the polymorphic Xho I site. A solid line box marks the Xho I site in TcV strains. B: Agarose gel electrophoresis showing a PCR-RFLP analysis of selected strains. Strains analyzed were: Sc43 cl9, MN cl2, LL014 and Teh53 for DTU TcV; and Tulahuen cl2, CL-Brener, Tul2 and P63 cl1 for DTU TcVI.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis of the TcMK amplification product. A: a multiple sequence alignment of the 5′-UTR just upstream of the translational start codon, showing the polymorphic Xho I site. A solid line box marks the Xho I site in TcV strains. B: Agarose gel electrophoresis showing a PCR-RFLP analysis of selected strains. Strains analyzed were: Sc43 cl9, MN cl2, LL014 and Teh53 for DTU TcV; and Tulahuen cl2, CL-Brener, Tul2 and P63 cl1 for DTU TcVI.

    Techniques Used: Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    10) Product Images from "IRES-mediated translation of foot-and-mouth disease virus (FMDV) in cultured cells derived from FMDV-susceptible and -insusceptible animals"

    Article Title: IRES-mediated translation of foot-and-mouth disease virus (FMDV) in cultured cells derived from FMDV-susceptible and -insusceptible animals

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-016-0694-8

    pRF/FMDV-IRES and pCAGGS/FMDV-IRES plasmid construction. Structure of the bicistronic luciferase reporter construct containing the FMDV-IRES element located between Renilla luciferase and firefly luciferase (pRF-FMDV-IRS). Reporter gene was excised from this plasmid construct using the restriction enzymes Eco RV and Hpa I, and was o the pCAGGS/MCS(F) vector treated with Sma I and rAPid Alkaline Phosphatase using Mighty Mix
    Figure Legend Snippet: pRF/FMDV-IRES and pCAGGS/FMDV-IRES plasmid construction. Structure of the bicistronic luciferase reporter construct containing the FMDV-IRES element located between Renilla luciferase and firefly luciferase (pRF-FMDV-IRS). Reporter gene was excised from this plasmid construct using the restriction enzymes Eco RV and Hpa I, and was o the pCAGGS/MCS(F) vector treated with Sma I and rAPid Alkaline Phosphatase using Mighty Mix

    Techniques Used: Plasmid Preparation, Luciferase, Construct

    11) 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

    12) Product Images from "A Transactivator Function of Cottontail Rabbit Papillomavirus E2 Is Essential for Tumor Induction in Rabbits"

    Article Title: A Transactivator Function of Cottontail Rabbit Papillomavirus E2 Is Essential for Tumor Induction in Rabbits

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.22.11209-11215.2002

    Autoradiograph of a transient DNA replication assay. SCC-13 cells were transfected with plasmid CRPV-pGL3-NCR either alone (−) or together with an expression vector for CRPV E1 and an expression vector for CRPV wt E2 or the R37K, R37A, E39Q, E39A, I73L, or I73A mutant E2 protein. Low-molecular-weight DNA was extracted, digested with Dpn I and Hpa I, and analyzed by Southern blot hybridization. The position of Dpn I-resistant CRPV-pGL3-NCR DNA is indicated by an arrow. Percentages given refer to the amounts of replicating plasmids of the respective E2 mutants in relation to the wt E2 protein (100%).
    Figure Legend Snippet: Autoradiograph of a transient DNA replication assay. SCC-13 cells were transfected with plasmid CRPV-pGL3-NCR either alone (−) or together with an expression vector for CRPV E1 and an expression vector for CRPV wt E2 or the R37K, R37A, E39Q, E39A, I73L, or I73A mutant E2 protein. Low-molecular-weight DNA was extracted, digested with Dpn I and Hpa I, and analyzed by Southern blot hybridization. The position of Dpn I-resistant CRPV-pGL3-NCR DNA is indicated by an arrow. Percentages given refer to the amounts of replicating plasmids of the respective E2 mutants in relation to the wt E2 protein (100%).

    Techniques Used: Autoradiography, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Molecular Weight, Southern Blot, Hybridization

    13) Product Images from "Features of Infections Due to Klebsiella pneumoniae Carbapenemase-Producing Escherichia coli: Emergence of Sequence Type 131"

    Article Title: Features of Infections Due to Klebsiella pneumoniae Carbapenemase-Producing Escherichia coli: Emergence of Sequence Type 131

    Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

    doi: 10.1093/cid/cis387

    Plasmid profiles of Klebsiella pneumoniae carbapenemase–producing Escherichia coli– non– E. coli pairs from 5 cases using restriction enzyme Hpa I. Lanes 1 and 2, E. coli and K. pneumoniae from case 1; lanes 3, 4, and 5, E. coli ,
    Figure Legend Snippet: Plasmid profiles of Klebsiella pneumoniae carbapenemase–producing Escherichia coli– non– E. coli pairs from 5 cases using restriction enzyme Hpa I. Lanes 1 and 2, E. coli and K. pneumoniae from case 1; lanes 3, 4, and 5, E. coli ,

    Techniques Used: Plasmid Preparation

    Related Articles

    Amplification:

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

    Agarose Gel Electrophoresis:

    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: An Improved RSP Method to Detect HpaI Polymorphism in the Apolipoprotein C-1 Gene Promoter
    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. .. The image was captured on the thermal paper using the Eagle Eye II Still Video System (Stratagene, La Jolla, CA).

    Article Title: A Transactivator Function of Cottontail Rabbit Papillomavirus E2 Is Essential for Tumor Induction in Rabbits
    Article Snippet: .. 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: 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.

    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.

    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.

    Construct:

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

    Electrophoresis:

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

    Incubation:

    Article Title: A Transactivator Function of Cottontail Rabbit Papillomavirus E2 Is Essential for Tumor Induction in Rabbits
    Article Snippet: .. 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).

    Isolation:

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

    Polymerase Chain Reaction:

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

    Plasmid Preparation:

    Article Title: Characterization of Two New CTX-M-25-Group Extended-Spectrum ?-Lactamase Variants Identified in Escherichia coli Isolates from Israel
    Article Snippet: .. Plasmid profiles of the transformants carrying unique plasmids were determined by restriction digestion of their plasmid DNA with HpaI (New England Biolabs, Ipswich, MA, USA). .. Restriction products were separated along with a 1 Kb DNA Extension Ladder (Invitrogen) on a 0.7% agarose gel for 10 h at 2.5 V/cm with 0.5x Tris-borate-EDTA buffer.

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    New England Biolabs hpai
    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 <t>HpaI</t> and <t>NcoI</t> 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
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    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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0046329

    Figure Lengend 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.

    Article Snippet: Plasmid profiles of the transformants carrying unique plasmids were determined by restriction digestion of their plasmid DNA with HpaI (New England Biolabs, Ipswich, MA, USA).

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

    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

    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: