hindiii  (TaKaRa)

 
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  • 99
    Name:
    Hind III
    Description:
    A | AGCT TT TCGA | A
    Catalog Number:
    1060b
    Price:
    None
    Size:
    50 000 Units
    Category:
    HindIII Restriction enzymes Cloning
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    Structured Review

    TaKaRa hindiii
    The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, <t>lambda/HindIII</t> fragments or a concatemer of lambda DNA was used as a size marker.
    A | AGCT TT TCGA | A
    https://www.bioz.com/result/hindiii/product/TaKaRa
    Average 99 stars, based on 24 article reviews
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    hindiii - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis"

    Article Title: Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-300

    The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.
    Figure Legend Snippet: The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.

    Techniques Used: Selection, Marker, Isolation, Labeling, Homologous Recombination, Plasmid Preparation, Southern Blot, Lambda DNA Preparation

    Cloning of BAC inserts into the BGM vector. ( A ) Genomic structures of mouse chromosome 7 and the BAC inserts used in this study. The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3 , black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene [ 20 ]. ( B , C ) The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in B and C ) and Southern blotting (right panels in B and C ). The representative BGM vectors with transferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector. Purified original BAC clones and genomic DNA from representative BGM clones were digested with EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences were overlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII fragments or a lambda DNA concatemer was used as a size marker.
    Figure Legend Snippet: Cloning of BAC inserts into the BGM vector. ( A ) Genomic structures of mouse chromosome 7 and the BAC inserts used in this study. The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3 , black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene [ 20 ]. ( B , C ) The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in B and C ) and Southern blotting (right panels in B and C ). The representative BGM vectors with transferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector. Purified original BAC clones and genomic DNA from representative BGM clones were digested with EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences were overlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII fragments or a lambda DNA concatemer was used as a size marker.

    Techniques Used: Clone Assay, BAC Assay, Plasmid Preparation, Southern Blot, Purification, Lambda DNA Preparation, Marker

    Targeted insertion of a reporter gene. ( A ) In the first step, IRES-tauEGFP was inserted into the targeted site 3 bp downstream of the MOR42-3 stop codon by transformation with a purified DNA fragment consisting of L and R arms (1.0 kb each) homologous to MOR42-3 and the c I- spc selection cassette. In the second step, the selection cassette was removed by transformation with a fragment lacking the selection marker. ( B ) The targeted insertion of the reporter gene was confirmed by Southern blot analysis using an R homology region as a probe. The genomic DNA of each BGM clone was digested with BamHI and resolved by CHEF, and the changes in the sizes of the BamHI fragments were as expected, indicating the successful targeted insertion of IRES-tauEGFP . The sensitivity to antibiotics is shown. R, resistant; S, sensitive. The BamHI sites are represented by “B”. Lane M, lambda/HindIII fragments were used as a size marker.
    Figure Legend Snippet: Targeted insertion of a reporter gene. ( A ) In the first step, IRES-tauEGFP was inserted into the targeted site 3 bp downstream of the MOR42-3 stop codon by transformation with a purified DNA fragment consisting of L and R arms (1.0 kb each) homologous to MOR42-3 and the c I- spc selection cassette. In the second step, the selection cassette was removed by transformation with a fragment lacking the selection marker. ( B ) The targeted insertion of the reporter gene was confirmed by Southern blot analysis using an R homology region as a probe. The genomic DNA of each BGM clone was digested with BamHI and resolved by CHEF, and the changes in the sizes of the BamHI fragments were as expected, indicating the successful targeted insertion of IRES-tauEGFP . The sensitivity to antibiotics is shown. R, resistant; S, sensitive. The BamHI sites are represented by “B”. Lane M, lambda/HindIII fragments were used as a size marker.

    Techniques Used: Transformation Assay, Purification, Selection, Marker, Southern Blot

    2) Product Images from "Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus ▿ O Antigen Gene Locus ▿ †"

    Article Title: Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus ▿ O Antigen Gene Locus ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02946-09

    Dendrogram of Proteus O antigen gene clusters generated by using the BioNumerics software program following restriction digestion by HindIII and EcoRI.
    Figure Legend Snippet: Dendrogram of Proteus O antigen gene clusters generated by using the BioNumerics software program following restriction digestion by HindIII and EcoRI.

    Techniques Used: Generated, Software

    PCR-RFLP analyses of putative O antigen gene clusters following restriction with HindIII and EcoRI. Lane M, DNA marker; lane 1, O69 (G2667); lane 2, O71 (G2669); lane 3, O72a (G2670); lane 4, O73ac (G2673); lane 5, O74 (G2674); lane 6, O75 (G2675).
    Figure Legend Snippet: PCR-RFLP analyses of putative O antigen gene clusters following restriction with HindIII and EcoRI. Lane M, DNA marker; lane 1, O69 (G2667); lane 2, O71 (G2669); lane 3, O72a (G2670); lane 4, O73ac (G2673); lane 5, O74 (G2674); lane 6, O75 (G2675).

    Techniques Used: Polymerase Chain Reaction, Marker

    3) Product Images from "Structural and Functional Characterization of the Actin-1 Gene Promoter From the Antheraea pernyi (Lepidoptera: Saturniidae)"

    Article Title: Structural and Functional Characterization of the Actin-1 Gene Promoter From the Antheraea pernyi (Lepidoptera: Saturniidae)

    Journal: Journal of Insect Science

    doi: 10.1093/jisesa/ieu035

    Reverse transcription PCR analysis of GFP expression after a series of deletions of A1 core promoter region in Sf9 cells. (A) A series of actin A1 promoter fragments with 5′-end deletions were amplified using PCR method. The amplified products were digested with HindIII and BamHI and inserted into pGFP-N2 to generate expression vectors F1, F2, F3, and F4. The coordinates of the relative deletion fragments are indicated. The transcription start sites (ATG) are shown by arrows. (B) Ratio (GFP/kanamycin) of mRNA expression level in Sf9 cells determined by reverse transcription PCR and normalized to kanamycin. Averages of three independent experiments ± SD are shown. (C) Melting curve of qPCR of GFP and kanamycin resistance gene.
    Figure Legend Snippet: Reverse transcription PCR analysis of GFP expression after a series of deletions of A1 core promoter region in Sf9 cells. (A) A series of actin A1 promoter fragments with 5′-end deletions were amplified using PCR method. The amplified products were digested with HindIII and BamHI and inserted into pGFP-N2 to generate expression vectors F1, F2, F3, and F4. The coordinates of the relative deletion fragments are indicated. The transcription start sites (ATG) are shown by arrows. (B) Ratio (GFP/kanamycin) of mRNA expression level in Sf9 cells determined by reverse transcription PCR and normalized to kanamycin. Averages of three independent experiments ± SD are shown. (C) Melting curve of qPCR of GFP and kanamycin resistance gene.

    Techniques Used: Polymerase Chain Reaction, Expressing, Amplification, Real-time Polymerase Chain Reaction

    4) Product Images from "Construction of the POT1 promoter report gene vector, and the effect and underlying mechanism of the POT1 promoter in regulating telomerase and telomere length"

    Article Title: Construction of the POT1 promoter report gene vector, and the effect and underlying mechanism of the POT1 promoter in regulating telomerase and telomere length

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7127

    Identification of the recombinant plasmid POT1-promoter-2. (A) Electrophoresis of BglII and HindIII digested products. M, molecular weight marker (200–5,000 bp); lane 1 and 2, enzyme digestion products (5,057 and 461 bp). (B) Electrophoresis of the PCR products. M, molecular weight marker (100–600 bp); lane 1–4, the PCR products (461 bp) of the POT1-promoter-2 plasmid using primers for POT1-promoter-2. (C) Sequencing results of POT1-promoter-2. POT1, protection of telomere 1; PCR, polymerase chain reaction; bp, base pairs.
    Figure Legend Snippet: Identification of the recombinant plasmid POT1-promoter-2. (A) Electrophoresis of BglII and HindIII digested products. M, molecular weight marker (200–5,000 bp); lane 1 and 2, enzyme digestion products (5,057 and 461 bp). (B) Electrophoresis of the PCR products. M, molecular weight marker (100–600 bp); lane 1–4, the PCR products (461 bp) of the POT1-promoter-2 plasmid using primers for POT1-promoter-2. (C) Sequencing results of POT1-promoter-2. POT1, protection of telomere 1; PCR, polymerase chain reaction; bp, base pairs.

    Techniques Used: Recombinant, Plasmid Preparation, Electrophoresis, Molecular Weight, Marker, Polymerase Chain Reaction, Sequencing

    Identification of the recombinant plasmid POT1-promoter-1. (A) Electrophoresis of BglII and HindIII digested products. M, molecular weight marker (200–5,000 bp); lane 1–3, enzyme digestion products (5,057 and 207 bp). (B) Electrophoresis of the PCR products. M, molecular weight marker (100–600 bp); lane 1–4, the PCR products (207 bp) of the POT1-promoter-1 plasmid using primers for POT1-promoter-1. (C) Sequencing results of POT1-promoter-1. POT1, protection of telomere 1; PCR, polymerase chain reaction; bp, base pairs.
    Figure Legend Snippet: Identification of the recombinant plasmid POT1-promoter-1. (A) Electrophoresis of BglII and HindIII digested products. M, molecular weight marker (200–5,000 bp); lane 1–3, enzyme digestion products (5,057 and 207 bp). (B) Electrophoresis of the PCR products. M, molecular weight marker (100–600 bp); lane 1–4, the PCR products (207 bp) of the POT1-promoter-1 plasmid using primers for POT1-promoter-1. (C) Sequencing results of POT1-promoter-1. POT1, protection of telomere 1; PCR, polymerase chain reaction; bp, base pairs.

    Techniques Used: Recombinant, Plasmid Preparation, Electrophoresis, Molecular Weight, Marker, Polymerase Chain Reaction, Sequencing

    5) Product Images from "Characterization of Two Newly Identified Genes, vgaD and vatG, Conferring Resistance to Streptogramin A in Enterococcus faecium ▿"

    Article Title: Characterization of Two Newly Identified Genes, vgaD and vatG, Conferring Resistance to Streptogramin A in Enterococcus faecium ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00798-09

    Schematic diagram depicting a restriction map of a 5.6-kb HindIII fragment of E. faecium JS79 DNA containing two streptogramin A resistance genes and a transposase gene. Bold arrows reflect the orientations of vgaD and vatG and the transposase (IS); black
    Figure Legend Snippet: Schematic diagram depicting a restriction map of a 5.6-kb HindIII fragment of E. faecium JS79 DNA containing two streptogramin A resistance genes and a transposase gene. Bold arrows reflect the orientations of vgaD and vatG and the transposase (IS); black

    Techniques Used:

    Southern hybridization to identify vatG -containing E. faecium isolates from healthy humans, swine, poultry stool samples, and chicken meat. (A) Agarose gel electrophoresis of HindIII-digested plasmid DNA from E. faecium isolates. (B) Southern hybridization
    Figure Legend Snippet: Southern hybridization to identify vatG -containing E. faecium isolates from healthy humans, swine, poultry stool samples, and chicken meat. (A) Agarose gel electrophoresis of HindIII-digested plasmid DNA from E. faecium isolates. (B) Southern hybridization

    Techniques Used: Hybridization, Agarose Gel Electrophoresis, Plasmid Preparation

    6) Product Images from "Detection and Discrimination of Cryptosporidium parvum and C. hominis in Water Samples by Immunomagnetic Separation-PCR"

    Article Title: Detection and Discrimination of Cryptosporidium parvum and C. hominis in Water Samples by Immunomagnetic Separation-PCR

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.71.2.898-903.2005

    RFLP of the Cpgp40/15 PCR product generated from three C. parvum and C. hominis strains. The products digested with HindIII (A), MflI (B), and RsaI (C) are in lanes 1 to 3, 4 to 6, and 7 to 9, respectively. Lanes 1, 4, and 7, C. parvum Iowa strain; lanes 2, 5, and 8, C. parvum HNJ-1 strain; lanes 3, 6, and 9, C. hominis Ogose strain. Lane M shows a 100-bp DNA ladder. The lower band of the Ogose strain digested with RsaI (lane 9) consists of two fragments of 160 and 170 bp.
    Figure Legend Snippet: RFLP of the Cpgp40/15 PCR product generated from three C. parvum and C. hominis strains. The products digested with HindIII (A), MflI (B), and RsaI (C) are in lanes 1 to 3, 4 to 6, and 7 to 9, respectively. Lanes 1, 4, and 7, C. parvum Iowa strain; lanes 2, 5, and 8, C. parvum HNJ-1 strain; lanes 3, 6, and 9, C. hominis Ogose strain. Lane M shows a 100-bp DNA ladder. The lower band of the Ogose strain digested with RsaI (lane 9) consists of two fragments of 160 and 170 bp.

    Techniques Used: Polymerase Chain Reaction, Generated

    7) Product Images from "An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments"

    Article Title: An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1425-4

    Cloning of BAC1 into the iREX. (a) One-step cloning of BAC1 into the iREX. Nm S , neomycin sensitive; Nm R , neomycin resistant; Spc S , spectinomycin sensitive; Spc R , spectinomycin resistant; I, I-PpoI recognition sequence. (b) iREX/BAC1 was digested with I-PpoI followed by CHEF gel electrophoresis. The BAC1 insert is indicated as an open arrowhead, and the BGM vector is indicated as a closed arrowhead. A lambda DNA concatemer was used as a size marker in lane M. (c) Original BAC1 and genomic DNA of the iREX recombinant were digested with EcoRI or HindIII and hybridized with the original BAC1 clone as a probe. Band patterns identical to the original BAC1 clones were confirmed in the iREX recombinant, except for the bands derived from the BAC end sequences. The closed arrowhead indicates a BAC1-specific signal, and the open arrowheads indicate BGM-specific signals. In lane M, lambda/HindIII fragments were used as a size marker.
    Figure Legend Snippet: Cloning of BAC1 into the iREX. (a) One-step cloning of BAC1 into the iREX. Nm S , neomycin sensitive; Nm R , neomycin resistant; Spc S , spectinomycin sensitive; Spc R , spectinomycin resistant; I, I-PpoI recognition sequence. (b) iREX/BAC1 was digested with I-PpoI followed by CHEF gel electrophoresis. The BAC1 insert is indicated as an open arrowhead, and the BGM vector is indicated as a closed arrowhead. A lambda DNA concatemer was used as a size marker in lane M. (c) Original BAC1 and genomic DNA of the iREX recombinant were digested with EcoRI or HindIII and hybridized with the original BAC1 clone as a probe. Band patterns identical to the original BAC1 clones were confirmed in the iREX recombinant, except for the bands derived from the BAC end sequences. The closed arrowhead indicates a BAC1-specific signal, and the open arrowheads indicate BGM-specific signals. In lane M, lambda/HindIII fragments were used as a size marker.

    Techniques Used: Clone Assay, Sequencing, Nucleic Acid Electrophoresis, Plasmid Preparation, Lambda DNA Preparation, Marker, Recombinant, Derivative Assay, BAC Assay

    Evaluation of the stability of the cloned DNA using inversion. (a) Schematic diagram of the evaluation of the cloned DNA stability using inversion. Tet S , tetracycline sensitive; Tet R , tetracycline resistant. (b) Numbers of tetracycline-resistant colonies in the BEST310 system and the iREX system. Error bars, s.d. n = 3. (c) Many tetracycline-resistant recombinants were observed in the BEST310 system. In the iREX system, the same results were obtained in the presence of xylose because of the induced RecA. In contrast, few colonies were observed using the iREX in the absence of xylose. This result indicates that the cloned DNA insert of the iREX is stably maintained due to the strong repression of recA . ( d and e ) Southern blot analysis using a tet probe revealed changes in the sizes of the signals, indicating the inversion of the BAC1 insert. The genomic DNA of the represented clones was digested with BamHI. In lane M, lambda/HindIII fragments were used as a size marker.
    Figure Legend Snippet: Evaluation of the stability of the cloned DNA using inversion. (a) Schematic diagram of the evaluation of the cloned DNA stability using inversion. Tet S , tetracycline sensitive; Tet R , tetracycline resistant. (b) Numbers of tetracycline-resistant colonies in the BEST310 system and the iREX system. Error bars, s.d. n = 3. (c) Many tetracycline-resistant recombinants were observed in the BEST310 system. In the iREX system, the same results were obtained in the presence of xylose because of the induced RecA. In contrast, few colonies were observed using the iREX in the absence of xylose. This result indicates that the cloned DNA insert of the iREX is stably maintained due to the strong repression of recA . ( d and e ) Southern blot analysis using a tet probe revealed changes in the sizes of the signals, indicating the inversion of the BAC1 insert. The genomic DNA of the represented clones was digested with BamHI. In lane M, lambda/HindIII fragments were used as a size marker.

    Techniques Used: Clone Assay, Stable Transfection, Southern Blot, Marker

    Construction of the recA -inducible BGM vector system. (a) The BEST310 and iREX constructs that possess two antibiotic resistance gene cassettes for BAC cloning: Pr- neo , a lambda Pr promoter fused to the neomycin resistance gene ( neo ), and c I- spc , which contains c I encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene ( spc ). The closed and open arrows indicate the BAC cloning site, and the red and blue lines indicate the pBR322 sequence. (b) The inducible recA expression cassette, pX-recA, was inserted at the amyE locus of the BEST310 genome via homologous recombination. amyE is not essential for the viability of B. subtilis [ 15 ] . cat , chloramphenicol acetyltransferase; H, HindIII; X, XhoI. (c) After introducing the pX-recA, the endogenous recA was replaced with the tetracycline resistance gene ( tet ) via homologous recombination. X, XhoI. (d) Southern blot analysis using an amyE probe indicated the correct insertion of pX-recA. The genomic DNA of the represented clones was digested with HindIII. The open arrowhead indicates the intact amyE in BEST310. The closed arrowheads indicate 5’- amyE and 3’- amyE divided by the insertion of pX-recA. (e) Southern blot analysis using a recA probe indicated the correct insertion of pCTP. The genomic DNA of the represented clones was digested with XhoI. The open arrowheads indicate the endogenous recA . The closed arrowheads indicate the inducible recA derived from pX-recA.
    Figure Legend Snippet: Construction of the recA -inducible BGM vector system. (a) The BEST310 and iREX constructs that possess two antibiotic resistance gene cassettes for BAC cloning: Pr- neo , a lambda Pr promoter fused to the neomycin resistance gene ( neo ), and c I- spc , which contains c I encoding the CI repressor protein, which binds to the Pr promoter, fused to the spectinomycin resistance gene ( spc ). The closed and open arrows indicate the BAC cloning site, and the red and blue lines indicate the pBR322 sequence. (b) The inducible recA expression cassette, pX-recA, was inserted at the amyE locus of the BEST310 genome via homologous recombination. amyE is not essential for the viability of B. subtilis [ 15 ] . cat , chloramphenicol acetyltransferase; H, HindIII; X, XhoI. (c) After introducing the pX-recA, the endogenous recA was replaced with the tetracycline resistance gene ( tet ) via homologous recombination. X, XhoI. (d) Southern blot analysis using an amyE probe indicated the correct insertion of pX-recA. The genomic DNA of the represented clones was digested with HindIII. The open arrowhead indicates the intact amyE in BEST310. The closed arrowheads indicate 5’- amyE and 3’- amyE divided by the insertion of pX-recA. (e) Southern blot analysis using a recA probe indicated the correct insertion of pCTP. The genomic DNA of the represented clones was digested with XhoI. The open arrowheads indicate the endogenous recA . The closed arrowheads indicate the inducible recA derived from pX-recA.

    Techniques Used: Plasmid Preparation, Construct, BAC Assay, Clone Assay, Sequencing, Expressing, Homologous Recombination, Southern Blot, Derivative Assay

    8) Product Images from "A Gaijin-like miniature inverted repeat transposable element is mobilized in rice during cell differentiation"

    Article Title: A Gaijin-like miniature inverted repeat transposable element is mobilized in rice during cell differentiation

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-135

    TD analysis of mGing in various rice cultivars and wild rice accessions. The gDNA samples were digested with HindIII and ligated to the HindIII cassettes for TD analysis. A collection of fifteen O. rufipogon accessions, including three lines from Guangdong province of China (1, S7943; 2, S7944; 3, S6238), three accessions from Guangxi province of China (4, 7023; 5, GX4; 6, GX6), three accessions from Fujian province of China (7, 7045; 8, 7074; 9, 7075), three accessions s from Hubei province of China (10, Wuye No. 1; 11, Wuye No. 2; 12, Wuye No. 3) and three accessions from Jiangxi province of China (13, 7028; 14, 7029; 15, 550-1), three javanica cultivars (lane 16 to 18: Aus 114, Jaya and Dular), five japonica cultivars (lane 19 to 23: 19, Nipponbare; 20, Yueguang; 21, Liming; 22, Nongken 58; 23,Qiuguang) and five indica cultivars (lane 24 to 28: IR 36, Tetep, Milyang 46, IR 24 and IR 30, respectively) were randomly selected.
    Figure Legend Snippet: TD analysis of mGing in various rice cultivars and wild rice accessions. The gDNA samples were digested with HindIII and ligated to the HindIII cassettes for TD analysis. A collection of fifteen O. rufipogon accessions, including three lines from Guangdong province of China (1, S7943; 2, S7944; 3, S6238), three accessions from Guangxi province of China (4, 7023; 5, GX4; 6, GX6), three accessions from Fujian province of China (7, 7045; 8, 7074; 9, 7075), three accessions s from Hubei province of China (10, Wuye No. 1; 11, Wuye No. 2; 12, Wuye No. 3) and three accessions from Jiangxi province of China (13, 7028; 14, 7029; 15, 550-1), three javanica cultivars (lane 16 to 18: Aus 114, Jaya and Dular), five japonica cultivars (lane 19 to 23: 19, Nipponbare; 20, Yueguang; 21, Liming; 22, Nongken 58; 23,Qiuguang) and five indica cultivars (lane 24 to 28: IR 36, Tetep, Milyang 46, IR 24 and IR 30, respectively) were randomly selected.

    Techniques Used:

    9) Product Images from "Novel Human Adenovirus Causing Nosocomial Epidemic Keratoconjunctivitis "

    Article Title: Novel Human Adenovirus Causing Nosocomial Epidemic Keratoconjunctivitis

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01835-07

    Genome type of Kobe-H. Viral genomic DNA was digested with three restriction enzymes: BamHI, HindIII, and SmaI.
    Figure Legend Snippet: Genome type of Kobe-H. Viral genomic DNA was digested with three restriction enzymes: BamHI, HindIII, and SmaI.

    Techniques Used:

    10) Product Images from "Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids"

    Article Title: Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

    Journal: Environmental Microbiology

    doi: 10.1111/j.1462-2920.2007.01518.x

    Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).
    Figure Legend Snippet: Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).

    Techniques Used: Polymerase Chain Reaction, Amplification

    11) Product Images from "Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids"

    Article Title: Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids

    Journal: Environmental Microbiology

    doi: 10.1111/j.1462-2920.2007.01518.x

    Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).
    Figure Legend Snippet: Capillary electrophoreses of IPCR (odd-numbered lanes) and PAI-PCR (even-numbered lanes) products amplified from DNA extracted from horse vermiform appendixes. The electrophoreses followed by the analysis of the electrophoregrams were automatically performed by using an Agilent 2100 Bioanalyser with a DNA 7500 LabChip® kit (TaKaRa-Bio). The results are shown as a gel-like image. L is the ladder obtained with DNA fragment size markers (bp). The green and pink peaks are the lowest 50 bp and highest 10 380 bp markers respectively. The substrates used for IPCR or PAI-PCR were BamHI-digested DNA (lanes 1 and 2), EcoRI-digested DNA (lanes 3 and 4), HindIII-digested DNA (lanes 5 and 6), PstI-digested DNA (lanes 7 and 8) and XbaI-digested DNA (lanes 9 and 10).

    Techniques Used: Polymerase Chain Reaction, Amplification

    12) Product Images from "A Novel Plasmid-Encoded Serotype Conversion Mechanism through Addition of Phosphoethanolamine to the O-Antigen of Shigella flexneri"

    Article Title: A Novel Plasmid-Encoded Serotype Conversion Mechanism through Addition of Phosphoethanolamine to the O-Antigen of Shigella flexneri

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046095

    Genomic structure and the presence of plasmid pSFxv_2 in serotype Xv and X strains. A , genomic structure of plasmid pSFxv_2. ORFs were shown as thick arrows, and encoding regions were marked according to the genome annotation (accession No. CP001385). The specific lpt-O gene was marked in red color, and primers positions were indicated using thin arrows. B , plasmid profiles of serotype Xv strains 2002017, 2008129, 2008131, 05AH022 and 05AH027 (lanes 1–5, respectively) and X strains 06HN400, 05BJ002, 03HL001, 03HL020 and 2003055 (lanes 6–10, respectively) strains. Plasmid DNA was separated by electrophoresis with a Chef Mapper system (Bio-Rad) on a 1% SeaKem Gold agarose gel and visualized by EB staining. Plasmids isolated from strain 2002017 (lane 1) were used as control. Lambda DNA cleaved with HindIII (TaKaRa, Japan) was used as molecular mass markers. C , Southern hybridization detection of the lpt-O gene in serotype Xv strains. PCR product amplified from strain 2002017 using primer pair lpt-O -2 was prepared as probe.
    Figure Legend Snippet: Genomic structure and the presence of plasmid pSFxv_2 in serotype Xv and X strains. A , genomic structure of plasmid pSFxv_2. ORFs were shown as thick arrows, and encoding regions were marked according to the genome annotation (accession No. CP001385). The specific lpt-O gene was marked in red color, and primers positions were indicated using thin arrows. B , plasmid profiles of serotype Xv strains 2002017, 2008129, 2008131, 05AH022 and 05AH027 (lanes 1–5, respectively) and X strains 06HN400, 05BJ002, 03HL001, 03HL020 and 2003055 (lanes 6–10, respectively) strains. Plasmid DNA was separated by electrophoresis with a Chef Mapper system (Bio-Rad) on a 1% SeaKem Gold agarose gel and visualized by EB staining. Plasmids isolated from strain 2002017 (lane 1) were used as control. Lambda DNA cleaved with HindIII (TaKaRa, Japan) was used as molecular mass markers. C , Southern hybridization detection of the lpt-O gene in serotype Xv strains. PCR product amplified from strain 2002017 using primer pair lpt-O -2 was prepared as probe.

    Techniques Used: Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Staining, Isolation, Lambda DNA Preparation, Hybridization, Polymerase Chain Reaction, Amplification

    Related Articles

    Clone Assay:

    Article Title: Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression
    Article Snippet: .. Filters were hybridized with the following probes: (i) 1400 bp fragment from human cyclin B1 cDNA obtained by Bam HI/Hind III digestion of a pCMX plasmid carrying the entire cyclin B1 cDNA cloned into Bam HI/Hind III sites, (ii) 1200 bp fragment from human cyclin A cDNA obtained by Bam HI/Hind III digestion of a pCMX plasmid carrying the entire cyclin B1 cDNA cloned into Bam HI/Hind III sites, (iii) 740 bp fragment from human MXI1 cDNA obtained by PCR on the pCR3 expression vector carrying MXI1 cDNA, (iv) β-actin (Clontech Laboratories Inc., CA, USA). .. The hybridization were performed at 42°C in a buffer containing 50% formamide, washed to a final stringency of 0.5×SSC, 0.1% SDS at 65°C and autoradiographed at −80°C.

    Article Title: Identification of CRMP4 as a Convergent Regulator of Axon Outgrowth Inhibition
    Article Snippet: .. To generate C4RIP-V5, the unique N-terminal domain of CRMP4b (residues 1–126) was introduced into the Bam HI and Eco RI sites of pcDNA 3.1V5-His. pHSVC4RIP was generated by subcloning C4RIP-V5 into the Hind III and Sal I sites of pHSVPrPUC. pHSVCRMP4bGFP was generated by cloning CRMP4b into the Hind III and Eco RI sites of pEGFP N2 (Clontech, Palo Alto, CA) and subsequently subcloning into the Hind III and Xba I sites of pHSVPrPUC. .. Chimeric CRMP4bNCRMP2-V5 was constructed by PCR by ligating residues 14–572 of CRMP2a into the Eco RI and Xho I sites of C4RIP-V5.

    Southern Blot:

    Article Title: Increased Production of α-Linolenic Acid in Soybean Seeds by Overexpression of Lesquerella FAD3-1
    Article Snippet: .. The PCR reactions were conducted using a thermal cycler (Takara, Japan) under the following conditions: 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 50–65°C for 30 s, and 72°C for 30–90 s, with a final extension at 72°C for 10 min. For Southern blot analysis, 10 µg genomic DNA from NT and transgenic leaf tissues was digested overnight with Hind III (Takara, Japan), fractionated on 0.8% agarose gels by electrophoresis, and transferred onto a Hybond N+ nylon membrane (Amersham Pharmacia, USA). .. Hybridization, washing, and detection were carried out with a digoxigenin (DIG)-labeled DNA probe and chemiluminescence system (Roche, Germany), according to the manufacturer's instructions.

    Subcloning:

    Article Title: Identification of CRMP4 as a Convergent Regulator of Axon Outgrowth Inhibition
    Article Snippet: .. To generate C4RIP-V5, the unique N-terminal domain of CRMP4b (residues 1–126) was introduced into the Bam HI and Eco RI sites of pcDNA 3.1V5-His. pHSVC4RIP was generated by subcloning C4RIP-V5 into the Hind III and Sal I sites of pHSVPrPUC. pHSVCRMP4bGFP was generated by cloning CRMP4b into the Hind III and Eco RI sites of pEGFP N2 (Clontech, Palo Alto, CA) and subsequently subcloning into the Hind III and Xba I sites of pHSVPrPUC. .. Chimeric CRMP4bNCRMP2-V5 was constructed by PCR by ligating residues 14–572 of CRMP2a into the Eco RI and Xho I sites of C4RIP-V5.

    Construct:

    Article Title: The Arabidopsis Dual-Affinity Nitrate Transporter Gene AtNRT1.1 (CHL1) Is Activated and Functions in Nascent Organ Development during Vegetative and Reproductive Growth
    Article Snippet: .. CHL1-GUS constructs were made with translational fusions of the 4.9-kb HindIII-XhoI fragment and the 6.1-kb HindIII-SpeI fragment into the HindIII-SalI and HindIII-XbaI sites of the pBI101.2-GUS vector, respectively (Clontech, Palo Alto, CA). .. Transgenic Arabidopsis plants were produced by vacuum infiltrating 4-week-old plants in Agrobacterium tumefaciens culture containing the appropriate construct ( ).

    Electrophoresis:

    Article Title: Increased Production of α-Linolenic Acid in Soybean Seeds by Overexpression of Lesquerella FAD3-1
    Article Snippet: .. The PCR reactions were conducted using a thermal cycler (Takara, Japan) under the following conditions: 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 50–65°C for 30 s, and 72°C for 30–90 s, with a final extension at 72°C for 10 min. For Southern blot analysis, 10 µg genomic DNA from NT and transgenic leaf tissues was digested overnight with Hind III (Takara, Japan), fractionated on 0.8% agarose gels by electrophoresis, and transferred onto a Hybond N+ nylon membrane (Amersham Pharmacia, USA). .. Hybridization, washing, and detection were carried out with a digoxigenin (DIG)-labeled DNA probe and chemiluminescence system (Roche, Germany), according to the manufacturer's instructions.

    Transgenic Assay:

    Article Title: Increased Production of α-Linolenic Acid in Soybean Seeds by Overexpression of Lesquerella FAD3-1
    Article Snippet: .. The PCR reactions were conducted using a thermal cycler (Takara, Japan) under the following conditions: 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 50–65°C for 30 s, and 72°C for 30–90 s, with a final extension at 72°C for 10 min. For Southern blot analysis, 10 µg genomic DNA from NT and transgenic leaf tissues was digested overnight with Hind III (Takara, Japan), fractionated on 0.8% agarose gels by electrophoresis, and transferred onto a Hybond N+ nylon membrane (Amersham Pharmacia, USA). .. Hybridization, washing, and detection were carried out with a digoxigenin (DIG)-labeled DNA probe and chemiluminescence system (Roche, Germany), according to the manufacturer's instructions.

    Generated:

    Article Title: Identification of CRMP4 as a Convergent Regulator of Axon Outgrowth Inhibition
    Article Snippet: .. To generate C4RIP-V5, the unique N-terminal domain of CRMP4b (residues 1–126) was introduced into the Bam HI and Eco RI sites of pcDNA 3.1V5-His. pHSVC4RIP was generated by subcloning C4RIP-V5 into the Hind III and Sal I sites of pHSVPrPUC. pHSVCRMP4bGFP was generated by cloning CRMP4b into the Hind III and Eco RI sites of pEGFP N2 (Clontech, Palo Alto, CA) and subsequently subcloning into the Hind III and Xba I sites of pHSVPrPUC. .. Chimeric CRMP4bNCRMP2-V5 was constructed by PCR by ligating residues 14–572 of CRMP2a into the Eco RI and Xho I sites of C4RIP-V5.

    Expressing:

    Article Title: Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression
    Article Snippet: .. Filters were hybridized with the following probes: (i) 1400 bp fragment from human cyclin B1 cDNA obtained by Bam HI/Hind III digestion of a pCMX plasmid carrying the entire cyclin B1 cDNA cloned into Bam HI/Hind III sites, (ii) 1200 bp fragment from human cyclin A cDNA obtained by Bam HI/Hind III digestion of a pCMX plasmid carrying the entire cyclin B1 cDNA cloned into Bam HI/Hind III sites, (iii) 740 bp fragment from human MXI1 cDNA obtained by PCR on the pCR3 expression vector carrying MXI1 cDNA, (iv) β-actin (Clontech Laboratories Inc., CA, USA). .. The hybridization were performed at 42°C in a buffer containing 50% formamide, washed to a final stringency of 0.5×SSC, 0.1% SDS at 65°C and autoradiographed at −80°C.

    Article Title: T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells
    Article Snippet: .. For mammalian expression of C-terminally GFP tagged proteins, TSAd and TSAd Δ239–274 were subcloned into the Hind III/Eco RI and Xho I/Eco RI sites of pEGFP-N3 (Clontech), respectively. .. The pEF-myc-Itk and pEF-Lck constructs were kindly provided by Professor Leslie Berg and Professor Tomas Mustelin, respectively.

    Polymerase Chain Reaction:

    Article Title: Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression
    Article Snippet: .. Filters were hybridized with the following probes: (i) 1400 bp fragment from human cyclin B1 cDNA obtained by Bam HI/Hind III digestion of a pCMX plasmid carrying the entire cyclin B1 cDNA cloned into Bam HI/Hind III sites, (ii) 1200 bp fragment from human cyclin A cDNA obtained by Bam HI/Hind III digestion of a pCMX plasmid carrying the entire cyclin B1 cDNA cloned into Bam HI/Hind III sites, (iii) 740 bp fragment from human MXI1 cDNA obtained by PCR on the pCR3 expression vector carrying MXI1 cDNA, (iv) β-actin (Clontech Laboratories Inc., CA, USA). .. The hybridization were performed at 42°C in a buffer containing 50% formamide, washed to a final stringency of 0.5×SSC, 0.1% SDS at 65°C and autoradiographed at −80°C.

    Article Title: Increased Production of α-Linolenic Acid in Soybean Seeds by Overexpression of Lesquerella FAD3-1
    Article Snippet: .. The PCR reactions were conducted using a thermal cycler (Takara, Japan) under the following conditions: 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 50–65°C for 30 s, and 72°C for 30–90 s, with a final extension at 72°C for 10 min. For Southern blot analysis, 10 µg genomic DNA from NT and transgenic leaf tissues was digested overnight with Hind III (Takara, Japan), fractionated on 0.8% agarose gels by electrophoresis, and transferred onto a Hybond N+ nylon membrane (Amersham Pharmacia, USA). .. Hybridization, washing, and detection were carried out with a digoxigenin (DIG)-labeled DNA probe and chemiluminescence system (Roche, Germany), according to the manufacturer's instructions.

    Plasmid Preparation:

    Article Title: Mxi1 inhibits the proliferation of U87 glioma cells through down-regulation of cyclin B1 gene expression
    Article Snippet: .. Filters were hybridized with the following probes: (i) 1400 bp fragment from human cyclin B1 cDNA obtained by Bam HI/Hind III digestion of a pCMX plasmid carrying the entire cyclin B1 cDNA cloned into Bam HI/Hind III sites, (ii) 1200 bp fragment from human cyclin A cDNA obtained by Bam HI/Hind III digestion of a pCMX plasmid carrying the entire cyclin B1 cDNA cloned into Bam HI/Hind III sites, (iii) 740 bp fragment from human MXI1 cDNA obtained by PCR on the pCR3 expression vector carrying MXI1 cDNA, (iv) β-actin (Clontech Laboratories Inc., CA, USA). .. The hybridization were performed at 42°C in a buffer containing 50% formamide, washed to a final stringency of 0.5×SSC, 0.1% SDS at 65°C and autoradiographed at −80°C.

    Article Title: The Arabidopsis Dual-Affinity Nitrate Transporter Gene AtNRT1.1 (CHL1) Is Activated and Functions in Nascent Organ Development during Vegetative and Reproductive Growth
    Article Snippet: .. CHL1-GUS constructs were made with translational fusions of the 4.9-kb HindIII-XhoI fragment and the 6.1-kb HindIII-SpeI fragment into the HindIII-SalI and HindIII-XbaI sites of the pBI101.2-GUS vector, respectively (Clontech, Palo Alto, CA). .. Transgenic Arabidopsis plants were produced by vacuum infiltrating 4-week-old plants in Agrobacterium tumefaciens culture containing the appropriate construct ( ).

    Article Title: The Arabidopsis thaliana Dihydroxyacetone Phosphate Reductase Gene SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 Is Required for Glycerolipid Metabolism and for the Activation of Systemic Acquired Resistance W⃞
    Article Snippet: .. A 5-kb HindIII fragment then was further subcloned into the HindIII site of the binary vector pBI121 (Clonetech, San Fransisco, CA). .. The resultant plasmid pBI121-SFD1 was electroporated into the Agrobacterium tumefaciens strain GV3101.

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    TaKaRa 5 kb hindiii fragment
    Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a <t>5-kb</t> <t>HindIII</t> fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.
    5 Kb Hindiii Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.

    Journal: The Plant Cell

    Article Title: The Arabidopsis thaliana Dihydroxyacetone Phosphate Reductase Gene SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 Is Required for Glycerolipid Metabolism and for the Activation of Systemic Acquired Resistance W⃞

    doi: 10.1105/tpc.016907

    Figure Lengend Snippet: Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.

    Article Snippet: A 5-kb HindIII fragment then was further subcloned into the HindIII site of the binary vector pBI121 (Clonetech, San Fransisco, CA).

    Techniques: Clone Assay, Sequencing, Binding Assay, Transformation Assay, Staining, Transgenic Assay, Expressing, Plasmid Preparation

    Schemes of CHL1-GFP/GUS Constructs. (A) CHL1 gene structure. Closed boxes represent exons containing coding regions, open boxes represent 5′ and 3′ untranslated sequences, and numbers indicate nucleotides from the start of translation. (B) HaeII reporter construct with ∼4.9 kb of CHL1 promoter sequence and exon 1 (up to amino acid 34 of CHL1) fused in frame at the HaeII site to the coding sequence of GFP or GUS . NOS indicates the 3′ termination sequence of the nopaline synthase gene. (C) XhoI reporter construct with the 4.9-kb CHL1 promoter and intragenic sequences up to the middle of exon 4 (up to amino acid 269 of CHL1) fused in frame to the coding sequence of GFP or GUS .

    Journal: The Plant Cell

    Article Title: The Arabidopsis Dual-Affinity Nitrate Transporter Gene AtNRT1.1 (CHL1) Is Activated and Functions in Nascent Organ Development during Vegetative and Reproductive Growth

    doi: 10.11054/TPC.010126

    Figure Lengend Snippet: Schemes of CHL1-GFP/GUS Constructs. (A) CHL1 gene structure. Closed boxes represent exons containing coding regions, open boxes represent 5′ and 3′ untranslated sequences, and numbers indicate nucleotides from the start of translation. (B) HaeII reporter construct with ∼4.9 kb of CHL1 promoter sequence and exon 1 (up to amino acid 34 of CHL1) fused in frame at the HaeII site to the coding sequence of GFP or GUS . NOS indicates the 3′ termination sequence of the nopaline synthase gene. (C) XhoI reporter construct with the 4.9-kb CHL1 promoter and intragenic sequences up to the middle of exon 4 (up to amino acid 269 of CHL1) fused in frame to the coding sequence of GFP or GUS .

    Article Snippet: CHL1-GUS constructs were made with translational fusions of the 4.9-kb HindIII-XhoI fragment and the 6.1-kb HindIII-SpeI fragment into the HindIII-SalI and HindIII-XbaI sites of the pBI101.2-GUS vector, respectively (Clontech, Palo Alto, CA).

    Techniques: Construct, Sequencing

    The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.

    Journal: BMC Genomics

    Article Title: Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis

    doi: 10.1186/1471-2164-14-300

    Figure Lengend Snippet: The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. ( A ) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes c I- bsr , consisting of the c I and blasticidin S resistance genes (depicted as c B), and c I- spc (depicted as c S) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr -labeled Tg-110 clone (Tg-110CIBS) and added to the spc -labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. ( B ) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. ( C ) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.

    Article Snippet: The genomic DNA was digested with EcoRI, HindIII or BamHI (TaKaRa).

    Techniques: Selection, Marker, Isolation, Labeling, Homologous Recombination, Plasmid Preparation, Southern Blot, Lambda DNA Preparation

    Cloning of BAC inserts into the BGM vector. ( A ) Genomic structures of mouse chromosome 7 and the BAC inserts used in this study. The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3 , black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene [ 20 ]. ( B , C ) The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in B and C ) and Southern blotting (right panels in B and C ). The representative BGM vectors with transferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector. Purified original BAC clones and genomic DNA from representative BGM clones were digested with EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences were overlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII fragments or a lambda DNA concatemer was used as a size marker.

    Journal: BMC Genomics

    Article Title: Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis

    doi: 10.1186/1471-2164-14-300

    Figure Lengend Snippet: Cloning of BAC inserts into the BGM vector. ( A ) Genomic structures of mouse chromosome 7 and the BAC inserts used in this study. The BAC clones cover a portion of the class I OR locus on chromosome 7, share 82 kb overlapping region and are oriented in opposite directions in the BAC vector. A large triangle depicts MOR42-3 , black triangles indicate other OR genes, and the gray triangle represents the pseudo-OR gene [ 20 ]. ( B , C ) The cloning of BAC1 and BAC2 into the BGM vector was confirmed by I-PpoI/CHEF (left panels in B and C ) and Southern blotting (right panels in B and C ). The representative BGM vectors with transferred BAC1 and BAC2 inserts were digested with I-PpoI and resolved by CHEF. The open arrowheads indicate BAC insert bands, and the closed arrowheads indicate the BGM vector. Purified original BAC clones and genomic DNA from representative BGM clones were digested with EcoRI or HindIII and hybridized with the original BAC clones as probes. The BGM recombinants showed a band pattern identical to the original BAC clones (lane: BAC), except for the bands corresponding to the BAC end sequences from EcoRI-digested BAC1; the closed arrowhead indicates a BAC-specific signal, and the open arrowheads indicate BGM-specific signals from EcoRI-digested BAC1. In other digestion patterns, the bands of the BAC end sequences were overlapping and indistinguishable from insert signals predicted from restriction maps of BAC clones and the BGM vector. Numbers above lanes are the BGM clone numbers. In lane M, lambda/HindIII fragments or a lambda DNA concatemer was used as a size marker.

    Article Snippet: The genomic DNA was digested with EcoRI, HindIII or BamHI (TaKaRa).

    Techniques: Clone Assay, BAC Assay, Plasmid Preparation, Southern Blot, Purification, Lambda DNA Preparation, Marker

    Targeted insertion of a reporter gene. ( A ) In the first step, IRES-tauEGFP was inserted into the targeted site 3 bp downstream of the MOR42-3 stop codon by transformation with a purified DNA fragment consisting of L and R arms (1.0 kb each) homologous to MOR42-3 and the c I- spc selection cassette. In the second step, the selection cassette was removed by transformation with a fragment lacking the selection marker. ( B ) The targeted insertion of the reporter gene was confirmed by Southern blot analysis using an R homology region as a probe. The genomic DNA of each BGM clone was digested with BamHI and resolved by CHEF, and the changes in the sizes of the BamHI fragments were as expected, indicating the successful targeted insertion of IRES-tauEGFP . The sensitivity to antibiotics is shown. R, resistant; S, sensitive. The BamHI sites are represented by “B”. Lane M, lambda/HindIII fragments were used as a size marker.

    Journal: BMC Genomics

    Article Title: Bacillus subtilis genome vector-based complete manipulation and reconstruction of genomic DNA for mouse transgenesis

    doi: 10.1186/1471-2164-14-300

    Figure Lengend Snippet: Targeted insertion of a reporter gene. ( A ) In the first step, IRES-tauEGFP was inserted into the targeted site 3 bp downstream of the MOR42-3 stop codon by transformation with a purified DNA fragment consisting of L and R arms (1.0 kb each) homologous to MOR42-3 and the c I- spc selection cassette. In the second step, the selection cassette was removed by transformation with a fragment lacking the selection marker. ( B ) The targeted insertion of the reporter gene was confirmed by Southern blot analysis using an R homology region as a probe. The genomic DNA of each BGM clone was digested with BamHI and resolved by CHEF, and the changes in the sizes of the BamHI fragments were as expected, indicating the successful targeted insertion of IRES-tauEGFP . The sensitivity to antibiotics is shown. R, resistant; S, sensitive. The BamHI sites are represented by “B”. Lane M, lambda/HindIII fragments were used as a size marker.

    Article Snippet: The genomic DNA was digested with EcoRI, HindIII or BamHI (TaKaRa).

    Techniques: Transformation Assay, Purification, Selection, Marker, Southern Blot

    Dendrogram of Proteus O antigen gene clusters generated by using the BioNumerics software program following restriction digestion by HindIII and EcoRI.

    Journal: Applied and Environmental Microbiology

    Article Title: Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus ▿ O Antigen Gene Locus ▿ †

    doi: 10.1128/AEM.02946-09

    Figure Lengend Snippet: Dendrogram of Proteus O antigen gene clusters generated by using the BioNumerics software program following restriction digestion by HindIII and EcoRI.

    Article Snippet: Eight microliters of purified O antigen PCR product (the sizes are about 14,000 to 20,000 bp) was digested with 7.5 U of HindIII and 7.5 U of EcoRI (Takara).

    Techniques: Generated, Software

    PCR-RFLP analyses of putative O antigen gene clusters following restriction with HindIII and EcoRI. Lane M, DNA marker; lane 1, O69 (G2667); lane 2, O71 (G2669); lane 3, O72a (G2670); lane 4, O73ac (G2673); lane 5, O74 (G2674); lane 6, O75 (G2675).

    Journal: Applied and Environmental Microbiology

    Article Title: Molecular and Genetic Analyses of the Putative Proteus O Antigen Gene Locus ▿ O Antigen Gene Locus ▿ †

    doi: 10.1128/AEM.02946-09

    Figure Lengend Snippet: PCR-RFLP analyses of putative O antigen gene clusters following restriction with HindIII and EcoRI. Lane M, DNA marker; lane 1, O69 (G2667); lane 2, O71 (G2669); lane 3, O72a (G2670); lane 4, O73ac (G2673); lane 5, O74 (G2674); lane 6, O75 (G2675).

    Article Snippet: Eight microliters of purified O antigen PCR product (the sizes are about 14,000 to 20,000 bp) was digested with 7.5 U of HindIII and 7.5 U of EcoRI (Takara).

    Techniques: Polymerase Chain Reaction, Marker