long-range polymerase chain reaction pcr Search Results


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  • 88
    TaKaRa long range polymerase chain reaction pcr
    An SVA retrotransposal insertion induces abnormal splicing in <t>FCMD</t> a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of <t>RT-PCR</t> product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin . Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts ( d ) and model mice ( e ). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH ( LDLRAP1 , left), NLSDM ( PNPLA2 , middle), and human ( AB627340 , right).
    Long Range Polymerase Chain Reaction Pcr, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 23635 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Illumina Inc long range polymerase chain reaction pcr
    Laboratory procedure used to obtain MHC II <t>DRB</t> short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: <t>PCR</t> primers
    Long Range Polymerase Chain Reaction Pcr, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    long range polymerase chain reaction pcr - by Bioz Stars, 2020-08
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    85
    Illumina Inc sequencing long range polymerase chain reaction pcr
    Laboratory procedure used to obtain MHC II <t>DRB</t> short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: <t>PCR</t> primers
    Sequencing Long Range Polymerase Chain Reaction Pcr, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 4 article reviews
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    92
    Eurofins special long range polymerase chain reaction
    Laboratory procedure used to obtain MHC II <t>DRB</t> short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: <t>PCR</t> primers
    Special Long Range Polymerase Chain Reaction, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche long range pcr
    Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective <t>HLA</t> gene haplotype phasing. The size selection also determines an actual molar concentration for bridge <t>PCR</t> to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
    Long Range Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 990 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc long range polymerase chain reaction trusight hla v2 sequencing panel
    Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective <t>HLA</t> gene haplotype phasing. The size selection also determines an actual molar concentration for bridge <t>PCR</t> to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
    Long Range Polymerase Chain Reaction Trusight Hla V2 Sequencing Panel, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen longrange polymerase chain reaction pcr kit
    <t>RT-PCR</t> analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the <t>MHO_710–770</t> gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).
    Longrange Polymerase Chain Reaction Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    An SVA retrotransposal insertion induces abnormal splicing in FCMD a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of RT-PCR product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin . Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts ( d ) and model mice ( e ). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH ( LDLRAP1 , left), NLSDM ( PNPLA2 , middle), and human ( AB627340 , right).

    Journal: Nature

    Article Title: Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy

    doi: 10.1038/nature10456

    Figure Lengend Snippet: An SVA retrotransposal insertion induces abnormal splicing in FCMD a, Expression analysis of various regions of fukutin mRNA in lymphoblasts. Gray bar, the ratio of RT-PCR product in FCMD patients relative to the normal control; Numbers on the X axis, nucleotide positions of both forward and reverse primers in fukutin . Error bars, s.e.m. b, Long range PCR using primers flanking the expression-decreasing area (nucleotide position 1061 to 5941) detected a 3-kb PCR product in FCMD lymphoblast cDNA (open arrow) and 8-kb product in FCMD genomic DNA (closed arrow). In the normal control, cDNA and genomic DNA both showed 5-kb PCR products. The 8-kb band was weak probably because VNTR region of SVA is GC-rich (82%). c, Schematic representation of genomic DNA and cDNA in FCMD. Black and white arrows, forward and reverse sequencing primers. The intronic sequence in FCMD is indicated in lower case. The authentic stop codon is colored in red, and the new stop codon is colored in blue. d, e, Northern blot analysis of fukutin in human lymphoblasts ( d ) and model mice ( e ). F, FCMD; N, nomal control. The wild-type mouse fukutin mRNA was detected at a size of 6.1 kb. Both skeletal muscle (left) and brain (right) showed smaller, abnormal bands (open arrows) in Hp/Hp mice. Wt, wild type; Hn, Hn/Hn mice; Hp, Hp/Hp mice. f, Schematic representation of genomic DNA and cDNA in ARH ( LDLRAP1 , left), NLSDM ( PNPLA2 , middle), and human ( AB627340 , right).

    Article Snippet: To detect abnormally-spliced RT-PCR products from FCMD, ARH, and NLSDM patients, and from human brain AB627340 cDNA, long range PCR was performed using LA Taq with LA Taq Buffer II (Takara), adding dimethyl sulfoxide and 7-deaza-dGTP (Roche).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Northern Blot, Mouse Assay

    AON cocktail rescues normal fukutin mRNA a, RT-PCR diagram of three primers designed to assess normal fukutin mRNA recovery (upper). Black closed arrow, a common forward primer located on fukutin coding region; black open arrow, a reverse primer to detect the abnormal RT-PCR product (161 bp); gray closed arrow, the other reverse primer to detect the restored normal RT-PCR product (129 bp). The effect on Hp/Hp ES cells treated with each single or a cocktail of AONs (lower). F, FCMD; N, normal sample. b, Rescue from abnormal splicing in VMO-treated in Hp/Hp mice and Hp/− mice. Local injection of AED cocktail into TA (n=3). Dys, a negative control. c, Rescue from abnormal splicing in VMO-treated human FCMD lymphoblasts (left, n=2) and myotubes (right, n=2). The Y axis shows the percent recovery of normal mRNA (* p

    Journal: Nature

    Article Title: Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy

    doi: 10.1038/nature10456

    Figure Lengend Snippet: AON cocktail rescues normal fukutin mRNA a, RT-PCR diagram of three primers designed to assess normal fukutin mRNA recovery (upper). Black closed arrow, a common forward primer located on fukutin coding region; black open arrow, a reverse primer to detect the abnormal RT-PCR product (161 bp); gray closed arrow, the other reverse primer to detect the restored normal RT-PCR product (129 bp). The effect on Hp/Hp ES cells treated with each single or a cocktail of AONs (lower). F, FCMD; N, normal sample. b, Rescue from abnormal splicing in VMO-treated in Hp/Hp mice and Hp/− mice. Local injection of AED cocktail into TA (n=3). Dys, a negative control. c, Rescue from abnormal splicing in VMO-treated human FCMD lymphoblasts (left, n=2) and myotubes (right, n=2). The Y axis shows the percent recovery of normal mRNA (* p

    Article Snippet: To detect abnormally-spliced RT-PCR products from FCMD, ARH, and NLSDM patients, and from human brain AB627340 cDNA, long range PCR was performed using LA Taq with LA Taq Buffer II (Takara), adding dimethyl sulfoxide and 7-deaza-dGTP (Roche).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Injection, Negative Control

    Estimated CTX prophage region structure of V. cholerae strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and PCR of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain M25, the representative of group ET‐8. Profiles of CTX prophage region‐specific RFLP and PCR of strain M25 are unique and it was categorized as an independent group, ET‐8. The best estimated model for CTX prophage region of strain M25 is “TLC–RS1–CTX‐1–RS1–RTX” on chromosome I and there are no CTX prophage‐associated genes on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C7, the representative of group ET‐6. Profiles of CTX prophage region‐specific RFLP and PCR of strain C7 are unique and it was categorized as an independent group, ET‐6. The best estimated model for CTX prophage region of strain C7 is “TLC– + CTX‐1– # CTX‐1– + CTX‐1– + CTX‐1–RTX” on chromosome I; no CTX prophage‐associated genes are present on chromosome II. + CTX‐1, CTX‐1 harboring SNPs in rstA (G301A), rstB (T84C), and in ctxA (G622A); # CTX‐1, CTX‐1 harboring SNPs in rstB (T84C) and in ctxA (G622A).

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain C7, the representative of group ET‐6. Profiles of CTX prophage region‐specific RFLP and PCR of strain C7 are unique and it was categorized as an independent group, ET‐6. The best estimated model for CTX prophage region of strain C7 is “TLC– + CTX‐1– # CTX‐1– + CTX‐1– + CTX‐1–RTX” on chromosome I; no CTX prophage‐associated genes are present on chromosome II. + CTX‐1, CTX‐1 harboring SNPs in rstA (G301A), rstB (T84C), and in ctxA (G622A); # CTX‐1, CTX‐1 harboring SNPs in rstB (T84C) and in ctxA (G622A).

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain P2, the representative of group ET‐5. Four V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐5. The strain P2 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain P2, the representative of group ET‐5. Four V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐5. The strain P2 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain P16, the representative of group ET‐7. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐7. The strain P16 was chosen as a representative and sequenced. The best estimated model for CTX prophage region of strain P16 is “TLC–CTX‐1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and “VCET1_GI–VCET1_GI” on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain P16, the representative of group ET‐7. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR, and were categorized into a group designated as ET‐7. The strain P16 was chosen as a representative and sequenced. The best estimated model for CTX prophage region of strain P16 is “TLC–CTX‐1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and “VCET1_GI–VCET1_GI” on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C1, the representative of group ET‐3. Profiles of CTX prophage region‐specific RFLP and PCR of strain C1 are unique and it was categorized as an independent group, ET‐3. The best estimated model for CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain C1, the representative of group ET‐3. Profiles of CTX prophage region‐specific RFLP and PCR of strain C1 are unique and it was categorized as an independent group, ET‐3. The best estimated model for CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain J6, the representative of group ET‐2. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR and were categorized into a group designated as ET‐2. The strain J6 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain J6, the representative of group ET‐2. Two V. cholerae wave 1 strains revealed similar profiles of CTX prophage region‐specific RFLP and PCR and were categorized into a group designated as ET‐2. The strain J6 was chosen as a representative and sequenced. The best estimated model of CTX prophage region is “TLC–CTX‐1–*RS1–CTX‐1–RTX” for chromosome I; no CTX prophage‐associated genes are present on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Estimated CTX prophage region structure of V. cholerae strain C2, the representative of group ET‐4. Profiles of CTX prophage region‐specific RFLP and PCR of strain C2 are unique and it was categorized as an independent group, ET‐4. The best estimated model for CTX prophage region of strain C2 is “TLC–CTX‐1–RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and a single “VCET1_GI” on chromosome II.

    Journal: Microbiology and Immunology

    Article Title: Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia

    doi: 10.1111/1348-0421.12648

    Figure Lengend Snippet: Estimated CTX prophage region structure of V. cholerae strain C2, the representative of group ET‐4. Profiles of CTX prophage region‐specific RFLP and PCR of strain C2 are unique and it was categorized as an independent group, ET‐4. The best estimated model for CTX prophage region of strain C2 is “TLC–CTX‐1–RS1–CTX‐1–VCET1_GI–VCET1_GI–RTX” on chromosome I and a single “VCET1_GI” on chromosome II.

    Article Snippet: The CTX prophage region, which spans from the 3′‐end portion of the TLC gene cluster corresponding to a primer TLC3F sequence to the 5 ′‐end portion of the RTX gene cluster corresponding to a primer RTX5R sequence (Table ) , of each representative V. cholerae strain was amplified from the bacterial genome by long range PCR using Prime STAR Max DNA polymerase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction

    Activation of NFκB-response element in the 2 nd intron of MnSOD gene by BTG2. (A) MnSOD promoter analysis; Cells (0.5×10 5 /12 wells) were cotransfected with BTG2 cDNA and promoter DNA of MnSOD gene and then subjected to luciferase analysis. Transfection of BTG2 up to 100 ng failed to activate promoter of MnSOD. Sp1 cDNA was employed as a positive control. Lower panel shows protein expressions of the transfected DNAs and loading control. (B) Cells were cotransfected with BTG2 cDNA and the κB-RE before luciferase assay. Expression of MnSOD was significantly increased with transfection of BTG2, indicating the activation of κB-RE by BTG2. Immunoblot analysis shows protein expression of BTG2-HA and loading control. (C) To further investigate the regulation of NFκB activation and MnSOD induction by BTG2 expression, IκBα degradation was examined by immunoblot analysis. Note the degradation of IκBα and MnSOD expression in the BTG2-dependent manner. (D) Transduction of HeLa cells with Ad-BTG2 was performed and then treated with 10 μM of MG132 for 1 h before RT-PCR analysis. MG132 abolished induction of MnSOD expression by BTG2, suggesting the upregulation of MnSOD expression by BTG2 via proteasomal degradation of IκBα. (E) Cell lysates with the LacZ or the BTG2 expressers were subjected to ChIP analysis with anti-p65 antibody, and then the interaction was verified by PCR reaction with the primers written in the Additional file 5 . To verify our analysis, HeLa cells were treated with or without 100 ng of 12- O -tetradecanoylphorbol-13-acetate (TPA) for 2 h and then applied to ChIP assay as a positive and negative control. Unstimulated IgG was employed to exclude the nonspecific interaction. Inputs indicate total amount of κB-RE present in the samples. Note the interactions of p65 with κB-RE only in the BTG2 expressers and the TPA treated positive cells. (F) Immunoblot analyses showing the activation of IKKα/β in the BTG2 overexpressers without any changes in the expression of other anti-oxidant enzymes.

    Journal: Cell Communication and Signaling : CCS

    Article Title: B-cell translocation gene 2 mediates crosstalk between PI3K/Akt1 and NF?B pathways which enhances transcription of MnSOD by accelerating I?B? degradation in normal and cancer cells

    doi: 10.1186/1478-811X-11-69

    Figure Lengend Snippet: Activation of NFκB-response element in the 2 nd intron of MnSOD gene by BTG2. (A) MnSOD promoter analysis; Cells (0.5×10 5 /12 wells) were cotransfected with BTG2 cDNA and promoter DNA of MnSOD gene and then subjected to luciferase analysis. Transfection of BTG2 up to 100 ng failed to activate promoter of MnSOD. Sp1 cDNA was employed as a positive control. Lower panel shows protein expressions of the transfected DNAs and loading control. (B) Cells were cotransfected with BTG2 cDNA and the κB-RE before luciferase assay. Expression of MnSOD was significantly increased with transfection of BTG2, indicating the activation of κB-RE by BTG2. Immunoblot analysis shows protein expression of BTG2-HA and loading control. (C) To further investigate the regulation of NFκB activation and MnSOD induction by BTG2 expression, IκBα degradation was examined by immunoblot analysis. Note the degradation of IκBα and MnSOD expression in the BTG2-dependent manner. (D) Transduction of HeLa cells with Ad-BTG2 was performed and then treated with 10 μM of MG132 for 1 h before RT-PCR analysis. MG132 abolished induction of MnSOD expression by BTG2, suggesting the upregulation of MnSOD expression by BTG2 via proteasomal degradation of IκBα. (E) Cell lysates with the LacZ or the BTG2 expressers were subjected to ChIP analysis with anti-p65 antibody, and then the interaction was verified by PCR reaction with the primers written in the Additional file 5 . To verify our analysis, HeLa cells were treated with or without 100 ng of 12- O -tetradecanoylphorbol-13-acetate (TPA) for 2 h and then applied to ChIP assay as a positive and negative control. Unstimulated IgG was employed to exclude the nonspecific interaction. Inputs indicate total amount of κB-RE present in the samples. Note the interactions of p65 with κB-RE only in the BTG2 expressers and the TPA treated positive cells. (F) Immunoblot analyses showing the activation of IKKα/β in the BTG2 overexpressers without any changes in the expression of other anti-oxidant enzymes.

    Article Snippet: RT-PCR Total cellular RNAs (1.0 μg) isolated with RNAiso Plus were used for cDNA preparation and then amplified by PCR kit (Takara Inc., Japan); First strand cDNA was synthesized using oligo-dT by reverse transcription reaction in 10 μl of reaction volume.

    Techniques: Activation Assay, Luciferase, Transfection, Positive Control, Expressing, Transduction, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control

    Expression of the OsPSK gene. ( A ) The steady state levels of OsPSK transcripts in rice Oc culture cells. Total RNAs (20 μg) extracted from Oc cells cultured for 3 (lane 1), 7 (lane 2), 10 (lane 3), or 14 days (lane 4) were separated by electrophoresis and hybridized with labeled cDNA ( OsPSK ) at 60°C. The blot was reprobed with the labeled rice actin cDNA ( OsRAc1 ) to indicate equal loading of RNA. ( B ) OsPSK transcripts in transgenic Oc cells. Total RNAs (20 μg) were extracted from S2 sense (lane 1), A2 antisense (lane 2), or C1 control (lane 3) Oc cells cultured for 3 days and used to perform RNA blot analysis as described in A . ( C ) Expression of the OsPSK gene in rice seedlings. Total RNAs (1.5 μg) isolated from the first leaves (lane 1), second leaves (lane 2), shoot apexes (lane 3), crown roots (lane 4), and seminal roots (lane 5) of rice seedlings 8 days after germination were individually used to perform RT-PCR and visualized by hybridization as described above. A 0.5-kb band was predictably proliferated and hybridized with the labeled OsPSK cDNA at 65°C.

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

    Article Title: Oryza sativa PSK gene encodes a precursor of phytosulfokine-?, a sulfated peptide growth factor found in plants

    doi:

    Figure Lengend Snippet: Expression of the OsPSK gene. ( A ) The steady state levels of OsPSK transcripts in rice Oc culture cells. Total RNAs (20 μg) extracted from Oc cells cultured for 3 (lane 1), 7 (lane 2), 10 (lane 3), or 14 days (lane 4) were separated by electrophoresis and hybridized with labeled cDNA ( OsPSK ) at 60°C. The blot was reprobed with the labeled rice actin cDNA ( OsRAc1 ) to indicate equal loading of RNA. ( B ) OsPSK transcripts in transgenic Oc cells. Total RNAs (20 μg) were extracted from S2 sense (lane 1), A2 antisense (lane 2), or C1 control (lane 3) Oc cells cultured for 3 days and used to perform RNA blot analysis as described in A . ( C ) Expression of the OsPSK gene in rice seedlings. Total RNAs (1.5 μg) isolated from the first leaves (lane 1), second leaves (lane 2), shoot apexes (lane 3), crown roots (lane 4), and seminal roots (lane 5) of rice seedlings 8 days after germination were individually used to perform RT-PCR and visualized by hybridization as described above. A 0.5-kb band was predictably proliferated and hybridized with the labeled OsPSK cDNA at 65°C.

    Article Snippet: A 22-mer primer (5′-CATCTTGGGAGTAGATATAATC-3′) was synthesized and used to obtain mutated cDNA with an LA PCR In Vitro Mutagenesis Kit (Takara).

    Techniques: Expressing, Cell Culture, Electrophoresis, Labeling, Transgenic Assay, Northern blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Hybridization

    Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p

    Journal: Veterinary Research

    Article Title: The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

    doi: 10.1186/s13567-018-0589-8

    Figure Lengend Snippet: Identification of the mutant strain RA1062. A PCR amplification. M: Takara DL2000 marker; lanes 1–2: R. anatipestifer 16S rRNA was amplified from the WT strain CH3 (lane 1), the mutant strain RA1062 (lane 2), showing a 744-bp fragment of 16S rRNA; lanes 4–5: a 678-bp fragment of M949_RS01035 was amplified from the WT strain CH3 (lane 4), but not the mutant strain RA1062 (lane 5); lanes 7–8: the 644-bp fragment of erm gene was not amplified from the WT strain CH3 (lane 7), but amplified from the mutant strain RA1062 (lane 8); lanes 3, 6 and 9: the avian pathogenic E. coli strain (APEC, CVCC1547), as negative controls. B Southern blot analysis of the transposon Tn4351 insertion. Lane 1, 10 μg of pEP 4351 digested with Xba I (positive control); Lane 2, 10 μg of chromosomal DNA from mutant strain RA1062 digested with Xba I; Lane 3, 10 μg of chromosomal DNA from the WT strain CH3 digested with Xba I (negative control). The digested sample was resolved on a 0.7% agarose gel and Southern blot analysis was performed using a TnDIG-labeled probe. C Schematic chart of Tn4351 insertion in RA1062 chromosome at 318 bp of the gene, which is 678 nucleotides in length. D qPCR analysis. The expression of the mRNAs were expressed as fold change and calculated using the comparative C T (2 −∆∆CT ) method. Data were normalized to the housekeeping gene ldh and expressed as fold changes. The expression of M949_RS01035 in the mutant strain RA1062 was disrupted. However, no change was shown for its upstream M949_RS10475 gene and downstream M949_RS01030 gene. Error bars represent standard deviations from three replicates (*** p

    Article Snippet: Primer pairs specific for Tn4351 (primers TN-1 and IS4351-F) were used to amplify the sequences adjacent to the insertion site using the LA PCR kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Marker, Southern Blot, Positive Control, Negative Control, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction, Expressing

    Laboratory procedure used to obtain MHC II DRB short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: PCR primers

    Journal: Heredity

    Article Title: A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra)

    doi: 10.1038/s41437-018-0070-5

    Figure Lengend Snippet: Laboratory procedure used to obtain MHC II DRB short and long amplicons, and assembly pipelines. The short amplicon was sequenced by standard Sanger sequencing (step 1), while the long amplicon was sequenced with Illumina MiSeq and nanopore MinION after gel extraction (step 2). The gene structure is based on Bos taurus (Ensembl: ENSBTAG00000013919) and Ovis aries (GenBank AM884914) structures. Boxes: coding regions, lines: introns, horizontal arrows: PCR primers

    Article Snippet: More in detail, we designed primers on DRB exons 1 and 3 and set-up a long-range PCR coupled with Illumina NGS sequencing to a very high coverage.

    Techniques: Amplification, Sequencing, Gel Extraction, Polymerase Chain Reaction

    Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

    Journal: BMC Genomics

    Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing

    doi: 10.1186/1471-2164-14-355

    Figure Lengend Snippet: Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

    Article Snippet: The six highly polymorphic HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1 ) were amplified by long-range PCR and the PCR amplicons covering full sequences of the genes were subjected to the MiSeq sequencer via the transposase-based library preparation.

    Techniques: Selection, Agarose Gel Electrophoresis, Sample Prep, Concentration Assay, Bridge PCR, Amplification

    RT-PCR analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the MHO_710–770 gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).

    Journal: PLoS ONE

    Article Title: Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection

    doi: 10.1371/journal.pone.0181383

    Figure Lengend Snippet: RT-PCR analysis of the postulated ABC transporter genes 730–760. The positions of the different amplicons are shown below the scheme of the MHO_710–770 gene region. The primers used ( Table 1 ) and the lengths of the amplicons (A-G) are indicated. PCR products (A–G) for genomic DNA (g), mRNA (m) and cDNA (c) were separated on a 0.6% agarose gel and stained with ethidium bromide. M, Gene Ruler 1 kb DNA ladder (Fermentas).

    Article Snippet: Overlapping regions of the MHO genes 720–770 were amplified using the Long Range PCR Kit (Qiagen, Hilden, Germany) by standard PCR conditions (initial cycle of 3 min at 93°C; 35 cycles of 15 s at 93°C, 30 s at 50°C, 10 min at 68°C).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining