topo ta cloning kit  (Thermo Fisher)


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    Name:
    TA Cloning Kit
    Description:
    The TA Cloning Kit with pCR 2 1 vector provides a quick one step cloning strategy for directly inserting a Taq amplified PCR product into a plasmid vector The TA Cloning Kit uses the pCR 2 1 cloning vector and ExpressLink T4 DNA Ligase to generate a ligation product in a fifteen minute room temperature ligation step Reactions typically yield 80 recombinants containing inserts Features of the TA Cloning Kit with pCR 2 1 vector • Fast convenient 15 minute room temperature ligation• Efficient blue white screening and 80 clones with correct insert• Flexible choice of kanamycin or ampicillin resistance for flexible antibiotic selection• Hassle free eliminates any enzymatic modifications of the PCR product• Streamlined does not require the use of PCR primers that contain restriction sitesThe pCR 2 1 vector provides • 3 T overhangs for direct ligation of Taq amplified PCR products• T7 promoter for in vitro RNA transcription and sequencing• A versatile polylinker with flanking EcoR I sites for easy excision of inserts• M13 forward and reverse primer sites for sequencingHow TA Cloning WorksTaq polymerase has a non template dependent activity that adds a single deoxyadenosine A to the 3 ends of PCR products The linearized vector supplied in this kit has single 3 deoxythymidine T residues This allows PCR inserts to ligate efficiently with the vector Kit Configurations The TA Cloning Kit is offered in a variety of configurations without competent cells K2020 20 and K2020 40 with One Shot INVF Chemically Competent E coli K2000 01 and K2000 40 with One Shot TOP10F Chemically Competent E coli K2030 01 and K2030 40 and with One Shot TOP10 Chemically Competent E coli K2040 01 and K2040 40 in 20 and 40 reaction kit sizes
    Catalog Number:
    k200001
    Price:
    None
    Applications:
    Cloning|PCR Cloning
    Category:
    DNA Vectors Clones Purified Nucleic Acids Libraries
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    Structured Review

    Thermo Fisher topo ta cloning kit
    Neighbor joining tree for N-terminal MiSp encoding sequences demonstrates variation within and among species. Variants are shown for L. hesperus ( Lh ) and <t>TOPO</t> clones from L. tredecimguttatus ( Lt ), L. geometricus ( Lg ), and S. grossa ( Sg ). Units are number of substitutions. Latrodectus clones are arbitrarily numbered, except that Lt M1-7 and Lg M1-5 resulted from amplification with primers designed from L. hesperus , while higher clone numbers resulted from amplification with species-specific primers. S. grossa TOPO clones resulted from two separate <t>PCR</t> reactions, which are indicated here as “A#” and “B#”. Completely sequenced clones are indicated by bolded names. See Table 1 and Additional file 1 : Table S4 for accession numbers. Boxes indicate distinct clusters identified in neighbor joining trees based on N-terminal sequences including adjacent repetitive sequences and in C-terminal and adjacent repetitive region encoding sequences (Additional file 1 : Figures S2 and S3)
    The TA Cloning Kit with pCR 2 1 vector provides a quick one step cloning strategy for directly inserting a Taq amplified PCR product into a plasmid vector The TA Cloning Kit uses the pCR 2 1 cloning vector and ExpressLink T4 DNA Ligase to generate a ligation product in a fifteen minute room temperature ligation step Reactions typically yield 80 recombinants containing inserts Features of the TA Cloning Kit with pCR 2 1 vector • Fast convenient 15 minute room temperature ligation• Efficient blue white screening and 80 clones with correct insert• Flexible choice of kanamycin or ampicillin resistance for flexible antibiotic selection• Hassle free eliminates any enzymatic modifications of the PCR product• Streamlined does not require the use of PCR primers that contain restriction sitesThe pCR 2 1 vector provides • 3 T overhangs for direct ligation of Taq amplified PCR products• T7 promoter for in vitro RNA transcription and sequencing• A versatile polylinker with flanking EcoR I sites for easy excision of inserts• M13 forward and reverse primer sites for sequencingHow TA Cloning WorksTaq polymerase has a non template dependent activity that adds a single deoxyadenosine A to the 3 ends of PCR products The linearized vector supplied in this kit has single 3 deoxythymidine T residues This allows PCR inserts to ligate efficiently with the vector Kit Configurations The TA Cloning Kit is offered in a variety of configurations without competent cells K2020 20 and K2020 40 with One Shot INVF Chemically Competent E coli K2000 01 and K2000 40 with One Shot TOP10F Chemically Competent E coli K2030 01 and K2030 40 and with One Shot TOP10 Chemically Competent E coli K2040 01 and K2040 40 in 20 and 40 reaction kit sizes
    https://www.bioz.com/result/topo ta cloning kit/product/Thermo Fisher
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    topo ta cloning kit - by Bioz Stars, 2020-11
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    Images

    1) Product Images from "Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders"

    Article Title: Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders

    Journal: BMC Evolutionary Biology

    doi: 10.1186/s12862-017-0927-x

    Neighbor joining tree for N-terminal MiSp encoding sequences demonstrates variation within and among species. Variants are shown for L. hesperus ( Lh ) and TOPO clones from L. tredecimguttatus ( Lt ), L. geometricus ( Lg ), and S. grossa ( Sg ). Units are number of substitutions. Latrodectus clones are arbitrarily numbered, except that Lt M1-7 and Lg M1-5 resulted from amplification with primers designed from L. hesperus , while higher clone numbers resulted from amplification with species-specific primers. S. grossa TOPO clones resulted from two separate PCR reactions, which are indicated here as “A#” and “B#”. Completely sequenced clones are indicated by bolded names. See Table 1 and Additional file 1 : Table S4 for accession numbers. Boxes indicate distinct clusters identified in neighbor joining trees based on N-terminal sequences including adjacent repetitive sequences and in C-terminal and adjacent repetitive region encoding sequences (Additional file 1 : Figures S2 and S3)
    Figure Legend Snippet: Neighbor joining tree for N-terminal MiSp encoding sequences demonstrates variation within and among species. Variants are shown for L. hesperus ( Lh ) and TOPO clones from L. tredecimguttatus ( Lt ), L. geometricus ( Lg ), and S. grossa ( Sg ). Units are number of substitutions. Latrodectus clones are arbitrarily numbered, except that Lt M1-7 and Lg M1-5 resulted from amplification with primers designed from L. hesperus , while higher clone numbers resulted from amplification with species-specific primers. S. grossa TOPO clones resulted from two separate PCR reactions, which are indicated here as “A#” and “B#”. Completely sequenced clones are indicated by bolded names. See Table 1 and Additional file 1 : Table S4 for accession numbers. Boxes indicate distinct clusters identified in neighbor joining trees based on N-terminal sequences including adjacent repetitive sequences and in C-terminal and adjacent repetitive region encoding sequences (Additional file 1 : Figures S2 and S3)

    Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction

    Arrangement of amino acid motifs in MiSp sequences of cobweb and orb-web weaving spiders. Latrodectus and S. grossa accession numbers are in Table 1 . Lh , L. hesperus ; Lt , L. tredecimguttatus ; Lg , L. geometricus ; Sg , S. grossa ; Pt , P. tepidariorum based on combining the incompletely assembled MiSp-encoding region from Scaffold 853 of the i5K genome with an ~2.3 kb TOPO-cloned PCR product, KX584004; Av , A. ventricosus, JX513956.1 [ 30 ]. Lengths of missing amino acid sequences in Pt MiSp_v1 are based on the length of gaps in Scaffold 853 of the i5K genome, assuming no introns are present within those gaps. The last gap was predicted to be 1782 bp long (594 aa if no intron) in Scaffold 853, but our TOPO clone added 2236 bp, including a 1175 bp intron, which was longer than the predicted gap. The repetitive encoding region at the 3’ end of our TOPO clone did not overlap with the encoding region present in the i5K genome prior to the last gap
    Figure Legend Snippet: Arrangement of amino acid motifs in MiSp sequences of cobweb and orb-web weaving spiders. Latrodectus and S. grossa accession numbers are in Table 1 . Lh , L. hesperus ; Lt , L. tredecimguttatus ; Lg , L. geometricus ; Sg , S. grossa ; Pt , P. tepidariorum based on combining the incompletely assembled MiSp-encoding region from Scaffold 853 of the i5K genome with an ~2.3 kb TOPO-cloned PCR product, KX584004; Av , A. ventricosus, JX513956.1 [ 30 ]. Lengths of missing amino acid sequences in Pt MiSp_v1 are based on the length of gaps in Scaffold 853 of the i5K genome, assuming no introns are present within those gaps. The last gap was predicted to be 1782 bp long (594 aa if no intron) in Scaffold 853, but our TOPO clone added 2236 bp, including a 1175 bp intron, which was longer than the predicted gap. The repetitive encoding region at the 3’ end of our TOPO clone did not overlap with the encoding region present in the i5K genome prior to the last gap

    Techniques Used: Clone Assay, Polymerase Chain Reaction

    2) Product Images from "Single-cell analysis of the fate of c-kit-positive bone marrow cells"

    Article Title: Single-cell analysis of the fate of c-kit-positive bone marrow cells

    Journal: NPJ Regenerative Medicine

    doi: 10.1038/s41536-017-0032-1

    c-kit-BMCs acquire distinct cardiac cell phenotypes in vivo. a Representative scatter plots illustrating the expression of c-kit, Thy1.2 and CD31 in cardiac cell populations isolated from c-kit-BMC-treated infarcted hearts. The percentage of positive cells is indicated. CTRL: isotype control; SSC: side scatter. b Transcripts for α-myosin heavy chain ( Myh6 ), c-kit, CD31, collagen type III α-1 ( Col3a1 ) and β-2 microglobulin ( B2M ) in isolated cardiomyocytes (Myo), c-kit-BMCs (c-kit), endothelial cells (ECs) and fibroblasts (Fbl). Myocardium (first lane, MC) was used as control. bp: base pairs. c Isolated cardiomyocytes expressing α-sarcomeric actin (α-SA, red), ECs expressing von Willebrand factor (vWF, yellow) and fibroblasts expressing procollagen (Pro-Col, green) are shown. Quantitative data are presented as mean ± SD. Scale bars: Left and central panels = 50 µm; Right panel = 20 µm. d PCR products run on agarose gel correspond to the sites of integration of the viral genome in the DNA of c-kit-BMCs and myocytes. These images correspond to representative examples of experiments conducted in 8 mice. The upper band shows the pCR4-TOPO TA vector. Molecular mass: 100 bp incremental ladders
    Figure Legend Snippet: c-kit-BMCs acquire distinct cardiac cell phenotypes in vivo. a Representative scatter plots illustrating the expression of c-kit, Thy1.2 and CD31 in cardiac cell populations isolated from c-kit-BMC-treated infarcted hearts. The percentage of positive cells is indicated. CTRL: isotype control; SSC: side scatter. b Transcripts for α-myosin heavy chain ( Myh6 ), c-kit, CD31, collagen type III α-1 ( Col3a1 ) and β-2 microglobulin ( B2M ) in isolated cardiomyocytes (Myo), c-kit-BMCs (c-kit), endothelial cells (ECs) and fibroblasts (Fbl). Myocardium (first lane, MC) was used as control. bp: base pairs. c Isolated cardiomyocytes expressing α-sarcomeric actin (α-SA, red), ECs expressing von Willebrand factor (vWF, yellow) and fibroblasts expressing procollagen (Pro-Col, green) are shown. Quantitative data are presented as mean ± SD. Scale bars: Left and central panels = 50 µm; Right panel = 20 µm. d PCR products run on agarose gel correspond to the sites of integration of the viral genome in the DNA of c-kit-BMCs and myocytes. These images correspond to representative examples of experiments conducted in 8 mice. The upper band shows the pCR4-TOPO TA vector. Molecular mass: 100 bp incremental ladders

    Techniques Used: In Vivo, Expressing, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Mouse Assay, Plasmid Preparation

    3) Product Images from "Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens, et al. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens"

    Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens, et al. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

    Journal: Xenotransplantation

    doi: 10.1111/xen.12524

    Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1
    Figure Legend Snippet: Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1

    Techniques Used: Sequencing, Polymerase Chain Reaction, Electrophoresis, Clone Assay, Transfection, Produced, CRISPR

    4) Product Images from "Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells"

    Article Title: Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601602

    Bisulphite sequencing of CIITA. Amplified PCR products were cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen) and plasmid DNA was purified. Sequencing reaction was performed using a Big-Dye terminator Kit (Applied Biosystems) and electrophoresed using an ABI3100 system (Applied Biosystems). CpG sites are shown above. Methylated alleles are shown as solid circles; unmethylated alleles as shown as open circles.
    Figure Legend Snippet: Bisulphite sequencing of CIITA. Amplified PCR products were cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen) and plasmid DNA was purified. Sequencing reaction was performed using a Big-Dye terminator Kit (Applied Biosystems) and electrophoresed using an ABI3100 system (Applied Biosystems). CpG sites are shown above. Methylated alleles are shown as solid circles; unmethylated alleles as shown as open circles.

    Techniques Used: Bisulfite Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, TA Cloning, Purification, Sequencing, Methylation

    5) Product Images from "CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose"

    Article Title: CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.12981

    Sequencing and Western blot analysis for the T 2 lines X‐KO #34‐4‐4 and F‐KO #6‐10‐6 and the F 2 line FX‐KO #20‐4. (a) Mutations in XylT 1 and 2 of X‐KO #34‐4‐4 as identified by Sanger sequencing of PCR amplicons ( XylT 1) and TOPO‐cloned PCR products ( XylT 2). The corresponding wild‐type sequence is shown above, the gRNA target sequences are indicated by coloured boxes, and the PAM sequences are shown in bold. (b) Mutations in FucT 1, 2, 3 and 4 of F‐KO #6‐10‐6. The PAM‐distal mismatch between the gRNA and Fuc T 3 and 4 is highlighted in yellow. (c) Mutations in FucT 1‐4 and XylT 1 and 2 of FX‐KO #20‐4. Five of six genes carry homozygous mutations, with the exception of FucT 3 which has biallelic mutations. (d) Western blot of FX‐KO #20‐4 in comparison to N. benthamiana wild type, F‐KO #6‐10‐6 and X‐KO #34‐4‐4. ~10 µg of total soluble protein were loaded for each sample. Anti‐fucose blot: 1st antibody rabbit‐anti‐α1,3‐fucose (1 : 10 000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed). Anti‐xylose blot: 1st antibody rabbit‐anti‐β1,2‐xylose (1 : 5000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed).
    Figure Legend Snippet: Sequencing and Western blot analysis for the T 2 lines X‐KO #34‐4‐4 and F‐KO #6‐10‐6 and the F 2 line FX‐KO #20‐4. (a) Mutations in XylT 1 and 2 of X‐KO #34‐4‐4 as identified by Sanger sequencing of PCR amplicons ( XylT 1) and TOPO‐cloned PCR products ( XylT 2). The corresponding wild‐type sequence is shown above, the gRNA target sequences are indicated by coloured boxes, and the PAM sequences are shown in bold. (b) Mutations in FucT 1, 2, 3 and 4 of F‐KO #6‐10‐6. The PAM‐distal mismatch between the gRNA and Fuc T 3 and 4 is highlighted in yellow. (c) Mutations in FucT 1‐4 and XylT 1 and 2 of FX‐KO #20‐4. Five of six genes carry homozygous mutations, with the exception of FucT 3 which has biallelic mutations. (d) Western blot of FX‐KO #20‐4 in comparison to N. benthamiana wild type, F‐KO #6‐10‐6 and X‐KO #34‐4‐4. ~10 µg of total soluble protein were loaded for each sample. Anti‐fucose blot: 1st antibody rabbit‐anti‐α1,3‐fucose (1 : 10 000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed). Anti‐xylose blot: 1st antibody rabbit‐anti‐β1,2‐xylose (1 : 5000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed).

    Techniques Used: Sequencing, Western Blot, Polymerase Chain Reaction, Clone Assay

    6) Product Images from "In vivo dissection of the chromosome condensation machinery"

    Article Title: In vivo dissection of the chromosome condensation machinery

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200109056

    Topo II and phospho-H3 are not essential for rDNA condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.
    Figure Legend Snippet: Topo II and phospho-H3 are not essential for rDNA condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.

    Techniques Used: Blocking Assay, Fluorescence In Situ Hybridization

    7) Product Images from "A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish"

    Article Title: A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.026765

    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged PCR products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a TOPO-cloning procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.
    Figure Legend Snippet: Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged PCR products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a TOPO-cloning procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.

    Techniques Used: Mutagenesis, Sequencing, Fluorescence In Situ Hybridization, Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    8) Product Images from "The Major Floral Promoter NtFT5 in Tobacco (Nicotiana tabacum) Is a Promising Target for Crop Improvement"

    Article Title: The Major Floral Promoter NtFT5 in Tobacco (Nicotiana tabacum) Is a Promising Target for Crop Improvement

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2019.01666

    CRISPR/Cas9 genome editing of NtFT5 in N. tabacum cv. SR1 and identification of induced mutations in T 0 plants. (A) Location of the NtFT5 -specific protospacer and the protospacer adjacent motif (PAM) on the antisense DNA strand in the 147–169 bp region of exon I. Exons are shown as boxes, introns as lines. The underlines indicate triplets corresponding to amino acids. (B) PCR-based screening of three independent transgenic T 0 lines (L1–L3) for mutated allelic variants of NtFT5 . NtFT5 (exon I) was amplified using the primer combination listed in Supplemental Table S1 , single amplicons were transferred to pCRII-TOPO and sequenced (n = 15 per line). Point mutations (mut), insertions and deletions are highlighted in red letters. (C) Sequence alignment of native NtFT5 with the protein variants encoded by the identified mutated alleles. Red letters indicate amino acid substitutions and red stops indicate premature termination. (D) Experimental workflow of NtFT5 knockout analysis under LD conditions. Flowering T 0 plant L2 was chosen for detailed characterization. In the T 1 generation (n = 107) all characterized individuals lacking the cas9 gene (n = 7) were non-flowering and showed a nullizygous Ntft5 – genotype. T 1 plant #78 was representatively selected for backcrossing, and flowering was induced by grafting onto wild-type ( NtFT5 + ) rootstock. After pollination using a wild-type donor, heterozygous Ntft5 – /NtFT5 + plants of the first backcross generation (BC 1 , n = 4) flowered and one of these (#2) was randomly self-fertilized. Detailed phenotyping of non-flowering nullizygous Ntft5 – plants and flowering NtFT5 + and Ntft5 – /NtFT5 + plants was finally carried out in the second backcross generation (BC 2 ).
    Figure Legend Snippet: CRISPR/Cas9 genome editing of NtFT5 in N. tabacum cv. SR1 and identification of induced mutations in T 0 plants. (A) Location of the NtFT5 -specific protospacer and the protospacer adjacent motif (PAM) on the antisense DNA strand in the 147–169 bp region of exon I. Exons are shown as boxes, introns as lines. The underlines indicate triplets corresponding to amino acids. (B) PCR-based screening of three independent transgenic T 0 lines (L1–L3) for mutated allelic variants of NtFT5 . NtFT5 (exon I) was amplified using the primer combination listed in Supplemental Table S1 , single amplicons were transferred to pCRII-TOPO and sequenced (n = 15 per line). Point mutations (mut), insertions and deletions are highlighted in red letters. (C) Sequence alignment of native NtFT5 with the protein variants encoded by the identified mutated alleles. Red letters indicate amino acid substitutions and red stops indicate premature termination. (D) Experimental workflow of NtFT5 knockout analysis under LD conditions. Flowering T 0 plant L2 was chosen for detailed characterization. In the T 1 generation (n = 107) all characterized individuals lacking the cas9 gene (n = 7) were non-flowering and showed a nullizygous Ntft5 – genotype. T 1 plant #78 was representatively selected for backcrossing, and flowering was induced by grafting onto wild-type ( NtFT5 + ) rootstock. After pollination using a wild-type donor, heterozygous Ntft5 – /NtFT5 + plants of the first backcross generation (BC 1 , n = 4) flowered and one of these (#2) was randomly self-fertilized. Detailed phenotyping of non-flowering nullizygous Ntft5 – plants and flowering NtFT5 + and Ntft5 – /NtFT5 + plants was finally carried out in the second backcross generation (BC 2 ).

    Techniques Used: CRISPR, Polymerase Chain Reaction, Transgenic Assay, Amplification, Sequencing, Knock-Out

    9) Product Images from "A Hypomorphic Cystathionine ß-Synthase Gene Contributes to Cavefish Eye Loss by Disrupting Optic Vasculature"

    Article Title: A Hypomorphic Cystathionine ß-Synthase Gene Contributes to Cavefish Eye Loss by Disrupting Optic Vasculature

    Journal: bioRxiv

    doi: 10.1101/805804

    cbsa gene regulation and structure. ( a ) F1 hybrid test. Left: Summary of procedure. Middle: Sequenced cbsa RT-PCR products from SF, PA-CF, and F1 hybrid heads showing SF and CF cbsa marker SNP (A and G nucleotides, respectively). Right: Marker region sequences of TOPO cloned RT-PCR products from F1 hybrid heads showing the CF allele marker in red. ( b, c ) Maps of the SF and PA-CF cbsa loci showing indels in CF populations (c, red letters) and predicted enhancer (E) in SF cbsa . Colored boxes: exons. White boxes: Flanking gemin8 and cryaa loci. ( d-f ) Expression of CMV-GFP constructs at 40 hpf. (d) cbsa enhancer E-CMV-GFP construct. (e) Control pCMV-GFP did not drive GFP expression in the head (arrow) in 54 injected SF embryos. (e) pcbsa-enhancer-CMV-GFP drove GFP expression in the head (arrow), including the eyes and brain, in 75 of 101 injected SF embryos.
    Figure Legend Snippet: cbsa gene regulation and structure. ( a ) F1 hybrid test. Left: Summary of procedure. Middle: Sequenced cbsa RT-PCR products from SF, PA-CF, and F1 hybrid heads showing SF and CF cbsa marker SNP (A and G nucleotides, respectively). Right: Marker region sequences of TOPO cloned RT-PCR products from F1 hybrid heads showing the CF allele marker in red. ( b, c ) Maps of the SF and PA-CF cbsa loci showing indels in CF populations (c, red letters) and predicted enhancer (E) in SF cbsa . Colored boxes: exons. White boxes: Flanking gemin8 and cryaa loci. ( d-f ) Expression of CMV-GFP constructs at 40 hpf. (d) cbsa enhancer E-CMV-GFP construct. (e) Control pCMV-GFP did not drive GFP expression in the head (arrow) in 54 injected SF embryos. (e) pcbsa-enhancer-CMV-GFP drove GFP expression in the head (arrow), including the eyes and brain, in 75 of 101 injected SF embryos.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker, Clone Assay, Expressing, Construct, Injection

    10) Product Images from "Calcium Influx and DREAM Protein Are Required For GnRH Gene Expression Pulse Activity"

    Article Title: Calcium Influx and DREAM Protein Are Required For GnRH Gene Expression Pulse Activity

    Journal: Molecular and cellular endocrinology

    doi: 10.1016/j.mce.2006.12.040

    RT-PCR analysis and nucleotide DNA sequence of the mouse DREAM gene cDNA from GT1-7 cells A, RT-PCR analysis of DREAM gene expression in GT1-7 cells. One microgram of total RNA was reversed transcribed and amplified with mouse specific primers for the DREAM gene cDNA yielding an expected product of 852 bp (DAM12), as described in Materials and methods are shown (lanes 2 and 3). The negative control (lane 4) contained water instead of cDNA template. One third of the PCR mixture was resolved on 1.0 % TAE-agarose gel stained with ethidium bromide. Two cDNA templates generated with total RNA isolated from GT1-7 cells were employed in the PCR amplifications (GT1-7 cDNA #1, lane 2 and GT1-7 cDNA #2, lane 3). A lambda Pst I digest (lane 1) and a 100 bp DNA ladder (lane 5) were used as molecular weight markers. B, Nucleotide DNA sequence resulting from six-independent cloned RT-PCR amplified fragments. The DAM12 fragments were cloned in pCR2.1 TOPO vector as outlined in Materials and methods . The deduced amino acid sequence is shown directly below the first base of each codon. Regions corresponding to primers DAM-F1 and DAM-R2 are underlined. The splice variant GAC (coding for an extra aspartic acid residue) is indicated in bold and enclosed in a box.
    Figure Legend Snippet: RT-PCR analysis and nucleotide DNA sequence of the mouse DREAM gene cDNA from GT1-7 cells A, RT-PCR analysis of DREAM gene expression in GT1-7 cells. One microgram of total RNA was reversed transcribed and amplified with mouse specific primers for the DREAM gene cDNA yielding an expected product of 852 bp (DAM12), as described in Materials and methods are shown (lanes 2 and 3). The negative control (lane 4) contained water instead of cDNA template. One third of the PCR mixture was resolved on 1.0 % TAE-agarose gel stained with ethidium bromide. Two cDNA templates generated with total RNA isolated from GT1-7 cells were employed in the PCR amplifications (GT1-7 cDNA #1, lane 2 and GT1-7 cDNA #2, lane 3). A lambda Pst I digest (lane 1) and a 100 bp DNA ladder (lane 5) were used as molecular weight markers. B, Nucleotide DNA sequence resulting from six-independent cloned RT-PCR amplified fragments. The DAM12 fragments were cloned in pCR2.1 TOPO vector as outlined in Materials and methods . The deduced amino acid sequence is shown directly below the first base of each codon. Regions corresponding to primers DAM-F1 and DAM-R2 are underlined. The splice variant GAC (coding for an extra aspartic acid residue) is indicated in bold and enclosed in a box.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, Expressing, Amplification, Negative Control, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Generated, Isolation, Molecular Weight, Clone Assay, Plasmid Preparation, Variant Assay

    11) Product Images from "Potent Antibody-Mediated Neutralization and Evolution of Antigenic Escape Variants of Simian Immunodeficiency Virus Strain SIVmac239 In Vivo ▿"

    Article Title: Potent Antibody-Mediated Neutralization and Evolution of Antigenic Escape Variants of Simian Immunodeficiency Virus Strain SIVmac239 In Vivo ▿

    Journal:

    doi: 10.1128/JVI.00871-08

    Amino acid sequence alignment of envelope clones isolated from Mm333 week 16 and week 42 plasma. The full-length env coding region was amplified and cloned from plasma viral RNA by RT-PCR, followed by TOPO-TA cloning. Ten individual envelope clones from
    Figure Legend Snippet: Amino acid sequence alignment of envelope clones isolated from Mm333 week 16 and week 42 plasma. The full-length env coding region was amplified and cloned from plasma viral RNA by RT-PCR, followed by TOPO-TA cloning. Ten individual envelope clones from

    Techniques Used: Sequencing, Clone Assay, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, TA Cloning

    12) Product Images from "MELK expression correlates with tumor mitotic activity but is not required for cancer growth"

    Article Title: MELK expression correlates with tumor mitotic activity but is not required for cancer growth

    Journal: eLife

    doi: 10.7554/eLife.32838

    Generation and characterization of MELK-KO clones in A375 and DLD1. ( A ) Sanger sequencing of either the PCR-amplified cut-site or single alleles isolated by TOPO cloning from MELK knockout clones in the A375 (melanoma) and DLD1 (colorectal cancer) cell lines. Highlighted regions indicate bases recognized by the gRNA. Of note, in A375 MELK-KO c2 we recovered seven different indel mutations in the MELK gene. We believe that at the time of single-cell sorting, one allele in this cell had acquired a small deletion that did not fully abolish gRNA recognition. Then, during clonal expansion, this allele underwent additional mutagenesis to generate the multiple large indels that we recovered. Additionally, in A375 MELK-KO c1, we identified one allele that had three different single-nucleotide substitutions that generated three independent missense mutations (E15V, T16I, I17L). These mutations occur in a highly-conserved region of the protein, and Ile17 is predicted to form a part of MELK’s ATP-binding domain, likely explaining why this allele is non-functional. ( B ) Western blot analysis of A375 and DLD1 MELK-KO clones using an antibody that recognizes an epitope in the MELK N-terminus (Abcam; Cat. No. ab108529) or an antibody that recognizes an epitope in the MELK C-terminus (Cell Signal; Cat. No. 2274S). ( C ) Proliferation and doubling time analysis of A375 and DLD1 Rosa26 and MELK-KO clones. ( D ) Dose-response curves of the putative MELK inhibitor OTS167 in A375 and DLD1 Rosa26 and MELK-KO clones.
    Figure Legend Snippet: Generation and characterization of MELK-KO clones in A375 and DLD1. ( A ) Sanger sequencing of either the PCR-amplified cut-site or single alleles isolated by TOPO cloning from MELK knockout clones in the A375 (melanoma) and DLD1 (colorectal cancer) cell lines. Highlighted regions indicate bases recognized by the gRNA. Of note, in A375 MELK-KO c2 we recovered seven different indel mutations in the MELK gene. We believe that at the time of single-cell sorting, one allele in this cell had acquired a small deletion that did not fully abolish gRNA recognition. Then, during clonal expansion, this allele underwent additional mutagenesis to generate the multiple large indels that we recovered. Additionally, in A375 MELK-KO c1, we identified one allele that had three different single-nucleotide substitutions that generated three independent missense mutations (E15V, T16I, I17L). These mutations occur in a highly-conserved region of the protein, and Ile17 is predicted to form a part of MELK’s ATP-binding domain, likely explaining why this allele is non-functional. ( B ) Western blot analysis of A375 and DLD1 MELK-KO clones using an antibody that recognizes an epitope in the MELK N-terminus (Abcam; Cat. No. ab108529) or an antibody that recognizes an epitope in the MELK C-terminus (Cell Signal; Cat. No. 2274S). ( C ) Proliferation and doubling time analysis of A375 and DLD1 Rosa26 and MELK-KO clones. ( D ) Dose-response curves of the putative MELK inhibitor OTS167 in A375 and DLD1 Rosa26 and MELK-KO clones.

    Techniques Used: Clone Assay, Sequencing, Polymerase Chain Reaction, Amplification, Isolation, Knock-Out, FACS, Mutagenesis, Generated, Binding Assay, Functional Assay, Western Blot

    13) Product Images from "Identification of Differentially Expressed Genes in Papillary Thyroid Cancers"

    Article Title: Identification of Differentially Expressed Genes in Papillary Thyroid Cancers

    Journal: Yonsei Medical Journal

    doi: 10.3349/ymj.2009.50.1.60

    The mRNAs of the 10 DEGs showing distinct results were extracted, amplified using the TOPO® TA Cloning kit and were sequenced. DEGs, differentially expressed genes; TA, Taq -amplified.
    Figure Legend Snippet: The mRNAs of the 10 DEGs showing distinct results were extracted, amplified using the TOPO® TA Cloning kit and were sequenced. DEGs, differentially expressed genes; TA, Taq -amplified.

    Techniques Used: Amplification, TA Cloning

    14) Product Images from "Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines"

    Article Title: Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-6-2

    Plasmid pLBS-GFP-Em R constructed to transform chicken Lactobacillus . The promoter, leader peptide, C-terminal anchoring and terminator sequences of lbs gene of L. crispatus strain F5.7 were PCR amplified, cloned and assembled by overlapping restriction fragments onto pCR2.1-TOPO vector. The genes erm AM and gfp mut2 were also PCR amplified from other plasmids and used as selective marker and reporter gene, respectively.
    Figure Legend Snippet: Plasmid pLBS-GFP-Em R constructed to transform chicken Lactobacillus . The promoter, leader peptide, C-terminal anchoring and terminator sequences of lbs gene of L. crispatus strain F5.7 were PCR amplified, cloned and assembled by overlapping restriction fragments onto pCR2.1-TOPO vector. The genes erm AM and gfp mut2 were also PCR amplified from other plasmids and used as selective marker and reporter gene, respectively.

    Techniques Used: Plasmid Preparation, Construct, Polymerase Chain Reaction, Amplification, Clone Assay, Marker

    15) Product Images from "Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development"

    Article Title: Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gng095

    The gene disruption method. ( A ) Map of the plasmid EZTN:tet r -bs r . The EZTN:tet r -bs r plasmid generates a transposable blasticidin resistance (bs r ) cassette that can be used to create Dictyostelium gene disruptants. In order to create EZTN:tet r -bs r , the bacterial tet r cassette, isolated from pBR322, and the Dictyostelium Actin15Bsr cassette, isolated from Actin15ΔBamBsr, were cloned into the EZ::TNpMOD2 vector (Epicentre, USA) as indicated. The 19 bp Tn 5 transposon recognition sequences are represented as triangles and their sequence is shown below the map. The plasmid has PvuII sites flanking the transposable element and the transposable blasticidin resistance cassette is usually generated by PvuII digestion of the construct. ( B ) Diagrammatic representation of the scheme for gene disruption. A fragment of genomic DNA is isolated by PCR and inserted, by topoisomerase cloning, into pCR2.1-TOPO, a vector that confers resistance to ampicillin and kanamycin. In vitro transposition is then performed using transposon DNA prepared as in (A). The resultant DNA molecules are recovered by transformation into E.coli using a triple drug selection, with ampicillin, kanamycin and tetracycline.
    Figure Legend Snippet: The gene disruption method. ( A ) Map of the plasmid EZTN:tet r -bs r . The EZTN:tet r -bs r plasmid generates a transposable blasticidin resistance (bs r ) cassette that can be used to create Dictyostelium gene disruptants. In order to create EZTN:tet r -bs r , the bacterial tet r cassette, isolated from pBR322, and the Dictyostelium Actin15Bsr cassette, isolated from Actin15ΔBamBsr, were cloned into the EZ::TNpMOD2 vector (Epicentre, USA) as indicated. The 19 bp Tn 5 transposon recognition sequences are represented as triangles and their sequence is shown below the map. The plasmid has PvuII sites flanking the transposable element and the transposable blasticidin resistance cassette is usually generated by PvuII digestion of the construct. ( B ) Diagrammatic representation of the scheme for gene disruption. A fragment of genomic DNA is isolated by PCR and inserted, by topoisomerase cloning, into pCR2.1-TOPO, a vector that confers resistance to ampicillin and kanamycin. In vitro transposition is then performed using transposon DNA prepared as in (A). The resultant DNA molecules are recovered by transformation into E.coli using a triple drug selection, with ampicillin, kanamycin and tetracycline.

    Techniques Used: Plasmid Preparation, Isolation, Clone Assay, Sequencing, Generated, Construct, Polymerase Chain Reaction, In Vitro, Transformation Assay, Selection

    16) Product Images from "Epigenetic silencing of genes and microRNAs within the imprinted Dlk1-Dio3 region at human chromosome 14.32 in giant cell tumor of bone"

    Article Title: Epigenetic silencing of genes and microRNAs within the imprinted Dlk1-Dio3 region at human chromosome 14.32 in giant cell tumor of bone

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-495

    Identification of a hypermethylated region within the IG-DMR of GCTSCs. Cellular DNA was extracted from GCTSCs (n = 8) and MSCs (n = 8) and DNA fragments covering the Meg3-DMR (44 CpGs) and the IG-DMR (31 CpGs) were amplified by PCR, bisulfite treated, cloned into pCR4-TOPO vector and sequenced. (A, B) Calculated methylation frequencies of all analyzed CpGs within the Meg3-DMR and the IG-DMR of 10 individual clones derived from one GCTSC and one MSC cell line. (C, D) Calculated methylation frequencies within the Meg3-DMR and the IG-DMR of eight different GCTSC and MSC cell lines. (E) Methylation analysis restricted to the first 13 CpGs analyzed within the IG-DMR. Data are presented as mean ± SD. (*p
    Figure Legend Snippet: Identification of a hypermethylated region within the IG-DMR of GCTSCs. Cellular DNA was extracted from GCTSCs (n = 8) and MSCs (n = 8) and DNA fragments covering the Meg3-DMR (44 CpGs) and the IG-DMR (31 CpGs) were amplified by PCR, bisulfite treated, cloned into pCR4-TOPO vector and sequenced. (A, B) Calculated methylation frequencies of all analyzed CpGs within the Meg3-DMR and the IG-DMR of 10 individual clones derived from one GCTSC and one MSC cell line. (C, D) Calculated methylation frequencies within the Meg3-DMR and the IG-DMR of eight different GCTSC and MSC cell lines. (E) Methylation analysis restricted to the first 13 CpGs analyzed within the IG-DMR. Data are presented as mean ± SD. (*p

    Techniques Used: Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Methylation, Derivative Assay

    17) Product Images from "Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells"

    Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021186

    A strong initial IFN response is induced an IRF-3 response by TLR3 but is not enough to clear HCV. A) Viral replication in Huh7.5 cells stably transfected with TOPO (control) TLR3 or TLR7 infected with HCV MOI of 0.1 and collected every 2–3 days for RNA isolation (total 75 days). The HCV copy numbers from each time points were calculated by real time RT-PCR and compared against an HCV standard curve. B) IFNβ mRNA expression of the experiment described in part A. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. C) IP-10 and RANTES mRNA expression of the experiment described in part A. Methodology as described for part B.
    Figure Legend Snippet: A strong initial IFN response is induced an IRF-3 response by TLR3 but is not enough to clear HCV. A) Viral replication in Huh7.5 cells stably transfected with TOPO (control) TLR3 or TLR7 infected with HCV MOI of 0.1 and collected every 2–3 days for RNA isolation (total 75 days). The HCV copy numbers from each time points were calculated by real time RT-PCR and compared against an HCV standard curve. B) IFNβ mRNA expression of the experiment described in part A. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. C) IP-10 and RANTES mRNA expression of the experiment described in part A. Methodology as described for part B.

    Techniques Used: Stable Transfection, Transfection, Infection, Isolation, Quantitative RT-PCR, Expressing

    18) Product Images from "Detection of Circulating hcmv-miR-UL112-3p in Patients with Glioblastoma, Rheumatoid Arthritis, Diabetes Mellitus and Healthy Controls"

    Article Title: Detection of Circulating hcmv-miR-UL112-3p in Patients with Glioblastoma, Rheumatoid Arthritis, Diabetes Mellitus and Healthy Controls

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113740

    Confirmation of a cloned hcmv-miRUL112-3p TaqMan PCR amplicon from in vitro infected HCMV lung fibroblasts (Positive Control): Alignment of sequences (from above) shows the PCR2.1 TOPO empty plasmid, hcmv-miR-UL112 sequence from miRBase database and last two sequences confirms the PCR product was inserted into the PCR2.1 TOPO plasmid. The lower part represents the chromatogram for the recombinant plasmid.
    Figure Legend Snippet: Confirmation of a cloned hcmv-miRUL112-3p TaqMan PCR amplicon from in vitro infected HCMV lung fibroblasts (Positive Control): Alignment of sequences (from above) shows the PCR2.1 TOPO empty plasmid, hcmv-miR-UL112 sequence from miRBase database and last two sequences confirms the PCR product was inserted into the PCR2.1 TOPO plasmid. The lower part represents the chromatogram for the recombinant plasmid.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, In Vitro, Infection, Positive Control, Plasmid Preparation, Sequencing, Recombinant

    19) Product Images from "Combination of RNAseq and SNP nanofluidic array reveals the center of genetic diversity of cacao pathogen Moniliophthora roreri in the upper Magdalena Valley of Colombia and its clonality"

    Article Title: Combination of RNAseq and SNP nanofluidic array reveals the center of genetic diversity of cacao pathogen Moniliophthora roreri in the upper Magdalena Valley of Colombia and its clonality

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.00850

    Sanger sequencing of partial genomic DNA flanking the putative heterozygous SNP marker 485_1_1286 as called by SAMtools mpileup and bcftools with default parameters from M. roreri isolates C13. PCR-amplicons were cloned into pCR4-TOPO vector followed by transformation into DH5-alpha Escherichia coli and 9–11 clones from each amplicon were randomly selected to be sequenced from both ends using M13F and M13R primers. ClustalW2 ( Larkin et al., 2007 ) was used to compare the DNA sequences of each clone and a distance tree of 100 bootstrapped data sets was generated by using the Phylogeny.fr program ( http://phylogeny.lirmm.fr/ ) and the neighbor-joining method.
    Figure Legend Snippet: Sanger sequencing of partial genomic DNA flanking the putative heterozygous SNP marker 485_1_1286 as called by SAMtools mpileup and bcftools with default parameters from M. roreri isolates C13. PCR-amplicons were cloned into pCR4-TOPO vector followed by transformation into DH5-alpha Escherichia coli and 9–11 clones from each amplicon were randomly selected to be sequenced from both ends using M13F and M13R primers. ClustalW2 ( Larkin et al., 2007 ) was used to compare the DNA sequences of each clone and a distance tree of 100 bootstrapped data sets was generated by using the Phylogeny.fr program ( http://phylogeny.lirmm.fr/ ) and the neighbor-joining method.

    Techniques Used: Sequencing, Marker, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transformation Assay, Amplification, Generated

    20) Product Images from "An enhanced method for sequence walking and paralog mining: TOPO® Vector-Ligation PCR"

    Article Title: An enhanced method for sequence walking and paralog mining: TOPO® Vector-Ligation PCR

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-61

    Recombinant vector with primer sites and alternate insert orientations . For sequencing, the products generated by TVL-PCR must be cloned into the TOPO vector. Two possible insert orientations are possible, as shown. M13F or M13R primers must be used for sequencing because they provide unique priming sites in the recombinant plasmid. The T3 or T7 primers are unsuitable for sequencing because they are represented in both the vector and the insert.
    Figure Legend Snippet: Recombinant vector with primer sites and alternate insert orientations . For sequencing, the products generated by TVL-PCR must be cloned into the TOPO vector. Two possible insert orientations are possible, as shown. M13F or M13R primers must be used for sequencing because they provide unique priming sites in the recombinant plasmid. The T3 or T7 primers are unsuitable for sequencing because they are represented in both the vector and the insert.

    Techniques Used: Recombinant, Plasmid Preparation, Sequencing, Generated, Polymerase Chain Reaction, Clone Assay

    Overview of TVL-PCR . A . A pool of chimeric templates is generated using a ligation reaction that joins end-repaired genomic restriction fragments to TOPO vectors. Ligation is required only between one end of each genomic fragment and a vector molecule. B . A chimeric template molecule contains two priming sites (G1 and G2) within the genomic fragment and two appropriately oriented priming sites (M13F and T3, or M13R and T7) within the conjoined vector. C . In the first round of TVL-PCR, the G1 genomic primer is paired with a vector primer. In the fully nested approach an M13_ primer is used. Because the orientation of ligation is unknown, the G1 primer must be paired in separate reactions with M13F and with M13R. In the semi-nested approach, the G1 primer is paired with a T_ primer. D . In the second round of TVL-PCR, the G2 genomic primer is paired with either the T3 or the T7 primer, as appropriate. The T3 primer must be used if either the M13F or T3 primer was previously used in the first round of TVL-PCR, while the T7 primer must be used if either the M13R or T7 primer was previously used.
    Figure Legend Snippet: Overview of TVL-PCR . A . A pool of chimeric templates is generated using a ligation reaction that joins end-repaired genomic restriction fragments to TOPO vectors. Ligation is required only between one end of each genomic fragment and a vector molecule. B . A chimeric template molecule contains two priming sites (G1 and G2) within the genomic fragment and two appropriately oriented priming sites (M13F and T3, or M13R and T7) within the conjoined vector. C . In the first round of TVL-PCR, the G1 genomic primer is paired with a vector primer. In the fully nested approach an M13_ primer is used. Because the orientation of ligation is unknown, the G1 primer must be paired in separate reactions with M13F and with M13R. In the semi-nested approach, the G1 primer is paired with a T_ primer. D . In the second round of TVL-PCR, the G2 genomic primer is paired with either the T3 or the T7 primer, as appropriate. The T3 primer must be used if either the M13F or T3 primer was previously used in the first round of TVL-PCR, while the T7 primer must be used if either the M13R or T7 primer was previously used.

    Techniques Used: Polymerase Chain Reaction, Generated, Ligation, Plasmid Preparation

    21) Product Images from "Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool"

    Article Title: Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool

    Journal: Biotechnology and Bioengineering

    doi: 10.1002/bit.25233

    Analysis of generated indels in COSMC. A : TOPO™ TA-based sequence analysis of COSMC. Genomic DNA was extracted from CHO-K1 cells 5 days after transfection with plasmids encoding Cas9 and sgRNA against COSMC. PCR amplicons covering the sgRNA-target sites in COSMC were TOPO™ TA-cloned and Sanger sequenced. Between 21 and 32 sequences were obtained for each sgRNA. The percentages of wt and indel sequences are illustrated in the bar plot and shown in the table. B : Alignment of TOPO™ sequence traces. Genomic DNA from cells transfected with Cas9 + sgRNA1_C (replicate #1) were subjected to TOPO™ cloning as described in panel A. The sequence traces are aligned to the CHO-K1 genomic sequence. The red arrow indicates the genomic target site of sgRNA1_C. The numbers denote the position (bp) in the open reading frame of COSMC. Green, red and orange colors indicate insertions, deletions and substitutions, respectively. C : Targeted deep sequencing analysis of COSMC. The same extracted genomic DNA as described for panel A was used as template for the MiSeq analysis. Between 200,000 and 700,000 sequences were obtained for each sgRNA. The percentages of wt and indel sequences are illustrated in the bar plot and shown in the table.
    Figure Legend Snippet: Analysis of generated indels in COSMC. A : TOPO™ TA-based sequence analysis of COSMC. Genomic DNA was extracted from CHO-K1 cells 5 days after transfection with plasmids encoding Cas9 and sgRNA against COSMC. PCR amplicons covering the sgRNA-target sites in COSMC were TOPO™ TA-cloned and Sanger sequenced. Between 21 and 32 sequences were obtained for each sgRNA. The percentages of wt and indel sequences are illustrated in the bar plot and shown in the table. B : Alignment of TOPO™ sequence traces. Genomic DNA from cells transfected with Cas9 + sgRNA1_C (replicate #1) were subjected to TOPO™ cloning as described in panel A. The sequence traces are aligned to the CHO-K1 genomic sequence. The red arrow indicates the genomic target site of sgRNA1_C. The numbers denote the position (bp) in the open reading frame of COSMC. Green, red and orange colors indicate insertions, deletions and substitutions, respectively. C : Targeted deep sequencing analysis of COSMC. The same extracted genomic DNA as described for panel A was used as template for the MiSeq analysis. Between 200,000 and 700,000 sequences were obtained for each sgRNA. The percentages of wt and indel sequences are illustrated in the bar plot and shown in the table.

    Techniques Used: Generated, Sequencing, Transfection, Polymerase Chain Reaction, Clone Assay

    22) Product Images from "Downregulation of ALAS1 by nicarbazin treatment underlies the reduced synthesis of protoporphyrin IX in shell gland of laying hens"

    Article Title: Downregulation of ALAS1 by nicarbazin treatment underlies the reduced synthesis of protoporphyrin IX in shell gland of laying hens

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-06527-y

    Schematic diagram of single-insert plasmid pCR TM 4-TOPO (3956 nucleotides). The diagram shows site into which specific fragments of chicken ND4 or GAPDH genes were inserted. The fragments in the study were flanked by restriction and priming sites.
    Figure Legend Snippet: Schematic diagram of single-insert plasmid pCR TM 4-TOPO (3956 nucleotides). The diagram shows site into which specific fragments of chicken ND4 or GAPDH genes were inserted. The fragments in the study were flanked by restriction and priming sites.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction

    23) Product Images from "Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs)"

    Article Title: Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063074

    Using two TALEN pairs to delete the entire human miR-155 hairpin sequence. (A) TALEN pairs targeting miR-155 were designed and constructed (called TALEN C). The upper panel shows a schematic of the TALEN A pair binding sites. The lower panel shows the sequence alignments comparing Wt and TALEN C mutated miR-155. The left and right TALEN binding sites are highlighted in yellow and the miR-155 seed region is boxed in red. (B) Schematic of the binding sites of two TALEN pairs (TALEN A and TALEN C) targeting miR-155. (C–D) Both TALEN A and TALEN C pairs were transfected into 293T cells. The miR-155 locus was amplified by PCR and subjected to TOPO cloning and sequencing. (C) Electrophoresis gel analysis showing deletions in the miR-155 locus. The arrows on the right indicate the two expected PCR products with or without large deletions. (D) Sequence alignments between a Wt clone and two TOPO clones with large deletions. The left and right TALEN binding sites for both TALEN A and TALEN C are highlighted in yellow and the miR-155 hairpin sequence is boxed in red.
    Figure Legend Snippet: Using two TALEN pairs to delete the entire human miR-155 hairpin sequence. (A) TALEN pairs targeting miR-155 were designed and constructed (called TALEN C). The upper panel shows a schematic of the TALEN A pair binding sites. The lower panel shows the sequence alignments comparing Wt and TALEN C mutated miR-155. The left and right TALEN binding sites are highlighted in yellow and the miR-155 seed region is boxed in red. (B) Schematic of the binding sites of two TALEN pairs (TALEN A and TALEN C) targeting miR-155. (C–D) Both TALEN A and TALEN C pairs were transfected into 293T cells. The miR-155 locus was amplified by PCR and subjected to TOPO cloning and sequencing. (C) Electrophoresis gel analysis showing deletions in the miR-155 locus. The arrows on the right indicate the two expected PCR products with or without large deletions. (D) Sequence alignments between a Wt clone and two TOPO clones with large deletions. The left and right TALEN binding sites for both TALEN A and TALEN C are highlighted in yellow and the miR-155 hairpin sequence is boxed in red.

    Techniques Used: Sequencing, Construct, Binding Assay, Transfection, Amplification, Polymerase Chain Reaction, Clone Assay, Electrophoresis

    The miR-155* targeting TALEN pair causes mutations in the miR-155* sequence. 293T cells were transfected with or without plasmids encoding the TALEN pair designed to target the miR-155* region. A GFP expressing plasmid was co-transfected. (A) 48 hours later, GFP+ cells were sorted by FACS and subjected to gDNA extraction. (B–F) The miR-155* targeted region was amplified by PCR and subjected to an HRMA analysis (B–D) or TOPO cloning and sequencing (E,F). (B) Schematic of the HRMA approach. A small region of the genome that includes different lengths of DNA deletions is amplified by PCR. Upon annealing, different types of homoduplex and heteroduplex dsDNA molecules are produced with different melting temperatures. (C) HRMA of the mir-155* PCR amplicons generated using gDNA from Wt (mock transfected 293t cells), Unsorted (293t cells with the TALEN pair transfection), Sorted GFP+ and sorted GFP-(293t cells with the TALEN pair and a GFP plasmid co-transfection followed by FACS sorting). (D) The results of the HRMA analysis are also shown as fluorescence difference plots using the normalized data. The Wt sample is used as the baseline. (E) PCR products from C were cloned into a TOPO vector and the length of the individual DNA fragment was assessed by gel electrophoresis. (F) Sequencing results of TOPO clones from E are shown. They are aligned with the wild-type miR-155* sequence. The left and right TALEN binding sites are highlighted in yellow and the miR-155* region is boxed in red.
    Figure Legend Snippet: The miR-155* targeting TALEN pair causes mutations in the miR-155* sequence. 293T cells were transfected with or without plasmids encoding the TALEN pair designed to target the miR-155* region. A GFP expressing plasmid was co-transfected. (A) 48 hours later, GFP+ cells were sorted by FACS and subjected to gDNA extraction. (B–F) The miR-155* targeted region was amplified by PCR and subjected to an HRMA analysis (B–D) or TOPO cloning and sequencing (E,F). (B) Schematic of the HRMA approach. A small region of the genome that includes different lengths of DNA deletions is amplified by PCR. Upon annealing, different types of homoduplex and heteroduplex dsDNA molecules are produced with different melting temperatures. (C) HRMA of the mir-155* PCR amplicons generated using gDNA from Wt (mock transfected 293t cells), Unsorted (293t cells with the TALEN pair transfection), Sorted GFP+ and sorted GFP-(293t cells with the TALEN pair and a GFP plasmid co-transfection followed by FACS sorting). (D) The results of the HRMA analysis are also shown as fluorescence difference plots using the normalized data. The Wt sample is used as the baseline. (E) PCR products from C were cloned into a TOPO vector and the length of the individual DNA fragment was assessed by gel electrophoresis. (F) Sequencing results of TOPO clones from E are shown. They are aligned with the wild-type miR-155* sequence. The left and right TALEN binding sites are highlighted in yellow and the miR-155* region is boxed in red.

    Techniques Used: Sequencing, Transfection, Expressing, Plasmid Preparation, FACS, Amplification, Polymerase Chain Reaction, Clone Assay, Produced, Generated, Cotransfection, Fluorescence, Nucleic Acid Electrophoresis, Binding Assay

    The miR-155* targeting TALEN pair causes both bi-allelic and mono-allelic mutations in human cells. (A) Schematic of the experimental design. 293T cells were transfected with the TALEN pair targeting the miR-155* region along with a GFP plasmid. 48 hours later, GFP+ cells were sorted by FACS and subjected to single cell cloning. After individual cell clones were expanded, gDNA was extracted from the cell clones. The miR-155* TALEN pair-targeted region was amplified by PCR and subjected to TOPO cloning and sequencing. (B,C) Representative cell clones showing bi-allelic mutations (B) or mono-allelic mutations (C). In the sequence alignment graph, the left and right TALEN binding sites are highlighted in yellow and the miR-155* region is in the red box.
    Figure Legend Snippet: The miR-155* targeting TALEN pair causes both bi-allelic and mono-allelic mutations in human cells. (A) Schematic of the experimental design. 293T cells were transfected with the TALEN pair targeting the miR-155* region along with a GFP plasmid. 48 hours later, GFP+ cells were sorted by FACS and subjected to single cell cloning. After individual cell clones were expanded, gDNA was extracted from the cell clones. The miR-155* TALEN pair-targeted region was amplified by PCR and subjected to TOPO cloning and sequencing. (B,C) Representative cell clones showing bi-allelic mutations (B) or mono-allelic mutations (C). In the sequence alignment graph, the left and right TALEN binding sites are highlighted in yellow and the miR-155* region is in the red box.

    Techniques Used: Transfection, Plasmid Preparation, FACS, Clone Assay, Amplification, Polymerase Chain Reaction, Sequencing, Binding Assay

    24) Product Images from "A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division"

    Article Title: A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

    Journal: eLife

    doi: 10.7554/eLife.08828

    Spire1C contains a previously uncharacterized alternate exon of 58 amino acids. ( A ) Spire1 ExonC was found to be present in the tissues listed. *PCR from cDNA did not yield conclusive results, but the ExonC-containing gene was originally amplified from mouse brain cDNA. ( B ) Sequencing results from TOPO pCR2.1 vector using the M13 reverse primer. ExonC sequence is shown in red. ( C ) Translated protein sequence from red portion in C . ( D ) Representative immunoblots comparing commercial antibodies to Spire1 used to detect endogenous Spire1 in HeLa and PC12 cell lysates, 25 μg per lane. ( E ) Affinity purified Spire1 C-terminal antibody and ExonC antisera (both generated in this study) tested on HeLa and PC12 cell lysates, as in ( A ). Strikingly different protein bands are observed for the same cell type with different antibodies and between cell types with the same antibody. Molecular weight protein markers are the same size for each blot, though the positions vary slightly (as indicated). ( F ) The Phyre server was used to predict any secondary structural elements in ExonC. Phyre compiles structure predictions from three different algorithms (psipred, jnet, and sspro) to form a consensus prediction and probability for each structural element. Probability ranges from 1–10, with 10 being the most probable. Yellow amino acids are small/polar, green are hydrophobic, red are charged, and purple are aromatic + cysteine. DOI: http://dx.doi.org/10.7554/eLife.08828.006
    Figure Legend Snippet: Spire1C contains a previously uncharacterized alternate exon of 58 amino acids. ( A ) Spire1 ExonC was found to be present in the tissues listed. *PCR from cDNA did not yield conclusive results, but the ExonC-containing gene was originally amplified from mouse brain cDNA. ( B ) Sequencing results from TOPO pCR2.1 vector using the M13 reverse primer. ExonC sequence is shown in red. ( C ) Translated protein sequence from red portion in C . ( D ) Representative immunoblots comparing commercial antibodies to Spire1 used to detect endogenous Spire1 in HeLa and PC12 cell lysates, 25 μg per lane. ( E ) Affinity purified Spire1 C-terminal antibody and ExonC antisera (both generated in this study) tested on HeLa and PC12 cell lysates, as in ( A ). Strikingly different protein bands are observed for the same cell type with different antibodies and between cell types with the same antibody. Molecular weight protein markers are the same size for each blot, though the positions vary slightly (as indicated). ( F ) The Phyre server was used to predict any secondary structural elements in ExonC. Phyre compiles structure predictions from three different algorithms (psipred, jnet, and sspro) to form a consensus prediction and probability for each structural element. Probability ranges from 1–10, with 10 being the most probable. Yellow amino acids are small/polar, green are hydrophobic, red are charged, and purple are aromatic + cysteine. DOI: http://dx.doi.org/10.7554/eLife.08828.006

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Plasmid Preparation, Western Blot, Affinity Purification, Generated, Molecular Weight

    25) Product Images from "Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]"

    Article Title: Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]

    Journal: AMB Express

    doi: 10.1186/2191-0855-3-6

    Purification profiles of recombinant PhaR. Soluble proteins were subjected to electrophoresis in an SDS (4-15%) polyacrylamide gradient gel and stained with CBB. Lane 1, molecular mass standard proteins; lane 2, soluble protein fraction of E. coli BL21 (DE3) harboring pET100/D-TOPO-PhaR Re .; lane 3, PhaR purified by nickel-nitrilotriacetic acid agarose (5 μg).
    Figure Legend Snippet: Purification profiles of recombinant PhaR. Soluble proteins were subjected to electrophoresis in an SDS (4-15%) polyacrylamide gradient gel and stained with CBB. Lane 1, molecular mass standard proteins; lane 2, soluble protein fraction of E. coli BL21 (DE3) harboring pET100/D-TOPO-PhaR Re .; lane 3, PhaR purified by nickel-nitrilotriacetic acid agarose (5 μg).

    Techniques Used: Purification, Recombinant, Electrophoresis, Staining

    26) Product Images from "Combination of RNAseq and SNP nanofluidic array reveals the center of genetic diversity of cacao pathogen Moniliophthora roreri in the upper Magdalena Valley of Colombia and its clonality"

    Article Title: Combination of RNAseq and SNP nanofluidic array reveals the center of genetic diversity of cacao pathogen Moniliophthora roreri in the upper Magdalena Valley of Colombia and its clonality

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.00850

    Sanger sequencing of partial genomic DNA flanking the putative heterozygous SNP marker 485_1_1286 as called by SAMtools mpileup and bcftools with default parameters from M. roreri isolates C13. PCR-amplicons were cloned into pCR4-TOPO vector followed by transformation into DH5-alpha Escherichia coli and 9–11 clones from each amplicon were randomly selected to be sequenced from both ends using M13F and M13R primers. ClustalW2 ( Larkin et al., 2007 ) was used to compare the DNA sequences of each clone and a distance tree of 100 bootstrapped data sets was generated by using the Phylogeny.fr program ( http://phylogeny.lirmm.fr/ ) and the neighbor-joining method.
    Figure Legend Snippet: Sanger sequencing of partial genomic DNA flanking the putative heterozygous SNP marker 485_1_1286 as called by SAMtools mpileup and bcftools with default parameters from M. roreri isolates C13. PCR-amplicons were cloned into pCR4-TOPO vector followed by transformation into DH5-alpha Escherichia coli and 9–11 clones from each amplicon were randomly selected to be sequenced from both ends using M13F and M13R primers. ClustalW2 ( Larkin et al., 2007 ) was used to compare the DNA sequences of each clone and a distance tree of 100 bootstrapped data sets was generated by using the Phylogeny.fr program ( http://phylogeny.lirmm.fr/ ) and the neighbor-joining method.

    Techniques Used: Sequencing, Marker, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Transformation Assay, Amplification, Generated

    27) Product Images from "STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells"

    Article Title: STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-8-67

    (A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents
    Figure Legend Snippet: (A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents "%" distribution.

    Techniques Used: Chromatin Immunoprecipitation, Clone Assay, Sonication, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Immunoprecipitation, Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Genomic Sequencing

    28) Product Images from "Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing"

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv738

    Comparison of MethylC-seq and 4mC-TAB-seq. ( A ) MethylC-seq converts C and a portion of 4mC to T. The remaining 4mC and almost all 5mC will be read as C. ( B ) 4mC-TAB-seq converts C, 5mC and a portion of 4mC to T, whereas about half of 4mC will be exclusively read as C. ( c ) Properties of 4mC and 5mC under different treatment conditions. Untreated or Tet-treated 4mC/5mC-containing model DNA is applied to either standard or optimized bisulfite treatment condition. Samples were PCR amplified, subcloned into TOPO vector and Sanger sequenced to quantify the number of 4mC or 5mC being read as C.
    Figure Legend Snippet: Comparison of MethylC-seq and 4mC-TAB-seq. ( A ) MethylC-seq converts C and a portion of 4mC to T. The remaining 4mC and almost all 5mC will be read as C. ( B ) 4mC-TAB-seq converts C, 5mC and a portion of 4mC to T, whereas about half of 4mC will be exclusively read as C. ( c ) Properties of 4mC and 5mC under different treatment conditions. Untreated or Tet-treated 4mC/5mC-containing model DNA is applied to either standard or optimized bisulfite treatment condition. Samples were PCR amplified, subcloned into TOPO vector and Sanger sequenced to quantify the number of 4mC or 5mC being read as C.

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation

    29) Product Images from "The rK39 Antigen from an Iranian Strain of Leishmania infantum: Detection of Anti-Leishmania Antibodies in Humans and Dogs"

    Article Title: The rK39 Antigen from an Iranian Strain of Leishmania infantum: Detection of Anti-Leishmania Antibodies in Humans and Dogs

    Journal: Iranian Journal of Parasitology

    doi:

    PCR results of screening of cloned plasmids after ligation into pCR®-TOPO® with M13 primers Left to right: M, molecular-weight standard 1kbp; lanes 3, 4,7,8 cloned plasmids for k39, respectively; lanes 2, 5,6 non-cloned plasmids; lane 9, Negative control
    Figure Legend Snippet: PCR results of screening of cloned plasmids after ligation into pCR®-TOPO® with M13 primers Left to right: M, molecular-weight standard 1kbp; lanes 3, 4,7,8 cloned plasmids for k39, respectively; lanes 2, 5,6 non-cloned plasmids; lane 9, Negative control

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Ligation, Molecular Weight, Negative Control

    30) Product Images from "Ohgata, the Single Drosophila Ortholog of Human Cereblon, Regulates Insulin Signaling-dependent Organismic Growth *"

    Article Title: Ohgata, the Single Drosophila Ortholog of Human Cereblon, Regulates Insulin Signaling-dependent Organismic Growth *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.757823

    Generating an ohgt mutant. A , a scheme representing the ohgt locus and the target site. A 20-bp-long sequence within exon 2 was chosen for targeting by the CRISPR-Cas9 system. The predicted cleavage site is indicated by a vertical bar . Two nucleotides deleted by Cas9 activity are indicated by horizontal bars (-). The protospacer adjacent motif (PAM) sequence is shown in green. The putative stop codon after the frameshift is shown in red. (B) A scheme to recover flies with ohgt Ex2 allele. Microinjection survivors were crossed with TM3-GFP balancer flies and subsequently sacrificed to perform the HRMA-based mutagenesis screening. The F 1 progenies from the cross were again crossed to the same strain, and further HRMA was performed to confirm germ line transmission of the mutation. The amplicon was inserted into pCRII-TOPO vector and subjected to molecular characterization. C , HRMA of an F 1 mutant candidate. Melt curves of ohgt Ex2 / TM3-GFP ( orange ) and TM3-GFP balancer ( gray ) can be easily distinguished due to heteroduplex formation in candidate-derived sample. D , change in relative fluorescence units ( RFU ) relative to TM3-GFP balancer emphasizes the change of melt curve shown in C. E , total lysates were prepared from control ( w *), homozygous ohgt Ex2 ( Ex2/Ex2 ), and transheterozygous ohgt Ex2 / ohgt Df ( Ex2/Df ) third instar larvae. Proteins from each lysate were subjected to Western blotting with antibodies to the indicated proteins.
    Figure Legend Snippet: Generating an ohgt mutant. A , a scheme representing the ohgt locus and the target site. A 20-bp-long sequence within exon 2 was chosen for targeting by the CRISPR-Cas9 system. The predicted cleavage site is indicated by a vertical bar . Two nucleotides deleted by Cas9 activity are indicated by horizontal bars (-). The protospacer adjacent motif (PAM) sequence is shown in green. The putative stop codon after the frameshift is shown in red. (B) A scheme to recover flies with ohgt Ex2 allele. Microinjection survivors were crossed with TM3-GFP balancer flies and subsequently sacrificed to perform the HRMA-based mutagenesis screening. The F 1 progenies from the cross were again crossed to the same strain, and further HRMA was performed to confirm germ line transmission of the mutation. The amplicon was inserted into pCRII-TOPO vector and subjected to molecular characterization. C , HRMA of an F 1 mutant candidate. Melt curves of ohgt Ex2 / TM3-GFP ( orange ) and TM3-GFP balancer ( gray ) can be easily distinguished due to heteroduplex formation in candidate-derived sample. D , change in relative fluorescence units ( RFU ) relative to TM3-GFP balancer emphasizes the change of melt curve shown in C. E , total lysates were prepared from control ( w *), homozygous ohgt Ex2 ( Ex2/Ex2 ), and transheterozygous ohgt Ex2 / ohgt Df ( Ex2/Df ) third instar larvae. Proteins from each lysate were subjected to Western blotting with antibodies to the indicated proteins.

    Techniques Used: Mutagenesis, Sequencing, CRISPR, Activity Assay, Transmission Assay, Amplification, Plasmid Preparation, Derivative Assay, Fluorescence, Western Blot

    31) Product Images from "The C/ebp-Atf response element (CARE) location reveals two distinct Atf4-dependent, elongation-mediated mechanisms for transcriptional induction of aminoacyl-tRNA synthetase genes in response to amino acid limitation"

    Article Title: The C/ebp-Atf response element (CARE) location reveals two distinct Atf4-dependent, elongation-mediated mechanisms for transcriptional induction of aminoacyl-tRNA synthetase genes in response to amino acid limitation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw667

    Transcription of SARS mRNA starts at the annotated proximal promoter. Human hepatocellular carcinoma (HepG2) cells were incubated for 8 h in DMEM or DMEM + 5 mM HisOH to activate the AAR. The total RNA was subjected to RLM-5′ RACE analysis, as described in the Materials and Methods section. PCR products amplified with a 5′ RACE primer and a gene-specific primer were cloned into the pCRII-TOPO TA vector and the resulting clones were randomly selected for sequencing analysis. The top panel is the SARS gene structure; the grey boxes are the first three exons and the dotted oval is the approximate location of the CARE element. The arrow indicates the annotated transcription start site. The gene-specific primer used for PCR is labeled with a thick line under Exon 3. In the lower panels showing sequence, the annotated transcription start site is indicated with a ‘+1’ and the bold ATG represents the translation start codon. The underlined sequence is the location of the gene-specific primer. The multiple arrows inside the sequences are the actual transcription start sites established by the analysis of the clones obtained by the RLM-5′ RACE experiments.
    Figure Legend Snippet: Transcription of SARS mRNA starts at the annotated proximal promoter. Human hepatocellular carcinoma (HepG2) cells were incubated for 8 h in DMEM or DMEM + 5 mM HisOH to activate the AAR. The total RNA was subjected to RLM-5′ RACE analysis, as described in the Materials and Methods section. PCR products amplified with a 5′ RACE primer and a gene-specific primer were cloned into the pCRII-TOPO TA vector and the resulting clones were randomly selected for sequencing analysis. The top panel is the SARS gene structure; the grey boxes are the first three exons and the dotted oval is the approximate location of the CARE element. The arrow indicates the annotated transcription start site. The gene-specific primer used for PCR is labeled with a thick line under Exon 3. In the lower panels showing sequence, the annotated transcription start site is indicated with a ‘+1’ and the bold ATG represents the translation start codon. The underlined sequence is the location of the gene-specific primer. The multiple arrows inside the sequences are the actual transcription start sites established by the analysis of the clones obtained by the RLM-5′ RACE experiments.

    Techniques Used: Incubation, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Labeling

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    Article Title: Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders
    Article Snippet: .. Cycling conditions were 40 cycles of 94 ° C for 30 s, 50–60 ° C (depending on primer pair) for 45 s, and 68 ° C for 10 min. PCR products were gel-excised and cloned using the TOPO®-TA Cloning® Kit (Invitrogen). .. Clones were screened by PCR amplification of MiSp N and/or C-terminal encoding region, and MiSp positive clones were sequenced with Sp6, T7, M13F, or M13R universal primers.

    Article Title: Single-cell analysis of the fate of c-kit-positive bone marrow cells
    Article Snippet: .. Taq polymerase-amplified PCR products were inserted into the plasmid vector pCR4-TOPO using the TOPO TA Cloning Kit (Invitrogen). .. Subsequently, chemically competent TOP10 E. coli cells were transformed with the vector carrying the PCR products.

    Article Title: CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose
    Article Snippet: .. When the sequencing results were ambiguous, the PCR products were cloned in pTOPO from the TOPO TA Cloning Kit (Thermo Fisher Scientific, Waltham) and 5–10 clones were sequenced individually for each sample. ..

    Article Title: Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells
    Article Snippet: .. For bisulphite sequencing, the PCR products were amplified using primers CIITA-GM1F and CIITA-SEQR, 5′-ACAATCTCRAAACCTCRATTCTC-3′, and cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen), after which the plasmid DNA was purified using a PI system (Kurabo, Tokyo, Japan). .. Sequencing was carried out using a BigDye Terminator Kit and an ABI 3100 DNA sequencer (Applied Biosystems).

    Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens, et al. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens
    Article Snippet: .. Determination of the absence of any genomic polymorphisms was achieved cloning each resulting PCR products in E coli using the TOPO TA cloning kit (Thermo Fisher Scientific). .. Resulting purified plasmids (Plasmid Mini kit, Qiagen) were subjected to Sanger sequencing analyses (Eurofins Genomics).

    Generated:

    Article Title: In vivo dissection of the chromosome condensation machinery
    Article Snippet: .. PCR cloning was performed using the TOPO-TA cloning kit (Invitrogen). rDNA and CEN16 proximal FISH probes were generated as previously described ( ). .. Antibodies used were as follows: mAb YOL1/34 (rat anti-tubulin; Serotec), mAb 12CA5 (anti-HA; BAbCo), mouse antidigoxigenin and pig anti–goat-FITC (Boehringer), goat anti–mouse-FITC (BAbCo), rabbit anti-GFP (CLONTECH Laboratories, Inc.), and goat anti–rat-FITC and CY3 anti–rabbit (Jackson ImmunoResearch Laboratories).

    Sequencing:

    Article Title: CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose
    Article Snippet: .. When the sequencing results were ambiguous, the PCR products were cloned in pTOPO from the TOPO TA Cloning Kit (Thermo Fisher Scientific, Waltham) and 5–10 clones were sequenced individually for each sample. ..

    Fluorescence In Situ Hybridization:

    Article Title: In vivo dissection of the chromosome condensation machinery
    Article Snippet: .. PCR cloning was performed using the TOPO-TA cloning kit (Invitrogen). rDNA and CEN16 proximal FISH probes were generated as previously described ( ). .. Antibodies used were as follows: mAb YOL1/34 (rat anti-tubulin; Serotec), mAb 12CA5 (anti-HA; BAbCo), mouse antidigoxigenin and pig anti–goat-FITC (Boehringer), goat anti–mouse-FITC (BAbCo), rabbit anti-GFP (CLONTECH Laboratories, Inc.), and goat anti–rat-FITC and CY3 anti–rabbit (Jackson ImmunoResearch Laboratories).

    Plasmid Preparation:

    Article Title: SOD1 Is Essential for the Viability of DT40 Cells and Nuclear SOD1 Functions as a Guardian of Genomic DNA
    Article Snippet: .. Plasmid Construction DNA containing SOD1 exons I–V was obtained by PCR from DT40 genomic DNA using the Easy-DNA Kit (Invitrogen, Carlsbad, California, USA) and Ex-Taq polymerase (Takara Bio Inc., Otsu, Shiga, Japan). .. The chicken targeting constructs for SOD1 , SOD1 -blastcidinr and SOD1 -puromycinr , were made by replacing the exons I–III with blastcidin (Bsr) or puromycin (Puro) selection marker cassette.

    Article Title: Single-cell analysis of the fate of c-kit-positive bone marrow cells
    Article Snippet: .. Taq polymerase-amplified PCR products were inserted into the plasmid vector pCR4-TOPO using the TOPO TA Cloning Kit (Invitrogen). .. Subsequently, chemically competent TOP10 E. coli cells were transformed with the vector carrying the PCR products.

    Article Title: Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells
    Article Snippet: .. For bisulphite sequencing, the PCR products were amplified using primers CIITA-GM1F and CIITA-SEQR, 5′-ACAATCTCRAAACCTCRATTCTC-3′, and cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen), after which the plasmid DNA was purified using a PI system (Kurabo, Tokyo, Japan). .. Sequencing was carried out using a BigDye Terminator Kit and an ABI 3100 DNA sequencer (Applied Biosystems).

    Bisulfite Sequencing:

    Article Title: Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells
    Article Snippet: .. For bisulphite sequencing, the PCR products were amplified using primers CIITA-GM1F and CIITA-SEQR, 5′-ACAATCTCRAAACCTCRATTCTC-3′, and cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen), after which the plasmid DNA was purified using a PI system (Kurabo, Tokyo, Japan). .. Sequencing was carried out using a BigDye Terminator Kit and an ABI 3100 DNA sequencer (Applied Biosystems).

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    Thermo Fisher topo ta cloning kit
    Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, <t>PCR</t> analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting <t>TOPO</t> TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1

    Journal: Xenotransplantation

    Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens, et al. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

    doi: 10.1111/xen.12524

    Figure Lengend Snippet: Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1

    Article Snippet: Determination of the absence of any genomic polymorphisms was achieved cloning each resulting PCR products in E coli using the TOPO TA cloning kit (Thermo Fisher Scientific).

    Techniques: Sequencing, Polymerase Chain Reaction, Electrophoresis, Clone Assay, Transfection, Produced, CRISPR

    Sequencing and Western blot analysis for the T 2 lines X‐KO #34‐4‐4 and F‐KO #6‐10‐6 and the F 2 line FX‐KO #20‐4. (a) Mutations in XylT 1 and 2 of X‐KO #34‐4‐4 as identified by Sanger sequencing of PCR amplicons ( XylT 1) and TOPO‐cloned PCR products ( XylT 2). The corresponding wild‐type sequence is shown above, the gRNA target sequences are indicated by coloured boxes, and the PAM sequences are shown in bold. (b) Mutations in FucT 1, 2, 3 and 4 of F‐KO #6‐10‐6. The PAM‐distal mismatch between the gRNA and Fuc T 3 and 4 is highlighted in yellow. (c) Mutations in FucT 1‐4 and XylT 1 and 2 of FX‐KO #20‐4. Five of six genes carry homozygous mutations, with the exception of FucT 3 which has biallelic mutations. (d) Western blot of FX‐KO #20‐4 in comparison to N. benthamiana wild type, F‐KO #6‐10‐6 and X‐KO #34‐4‐4. ~10 µg of total soluble protein were loaded for each sample. Anti‐fucose blot: 1st antibody rabbit‐anti‐α1,3‐fucose (1 : 10 000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed). Anti‐xylose blot: 1st antibody rabbit‐anti‐β1,2‐xylose (1 : 5000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed).

    Journal: Plant Biotechnology Journal

    Article Title: CRISPR/Cas9‐mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β‐1,2‐xylose and core α‐1,3‐fucose

    doi: 10.1111/pbi.12981

    Figure Lengend Snippet: Sequencing and Western blot analysis for the T 2 lines X‐KO #34‐4‐4 and F‐KO #6‐10‐6 and the F 2 line FX‐KO #20‐4. (a) Mutations in XylT 1 and 2 of X‐KO #34‐4‐4 as identified by Sanger sequencing of PCR amplicons ( XylT 1) and TOPO‐cloned PCR products ( XylT 2). The corresponding wild‐type sequence is shown above, the gRNA target sequences are indicated by coloured boxes, and the PAM sequences are shown in bold. (b) Mutations in FucT 1, 2, 3 and 4 of F‐KO #6‐10‐6. The PAM‐distal mismatch between the gRNA and Fuc T 3 and 4 is highlighted in yellow. (c) Mutations in FucT 1‐4 and XylT 1 and 2 of FX‐KO #20‐4. Five of six genes carry homozygous mutations, with the exception of FucT 3 which has biallelic mutations. (d) Western blot of FX‐KO #20‐4 in comparison to N. benthamiana wild type, F‐KO #6‐10‐6 and X‐KO #34‐4‐4. ~10 µg of total soluble protein were loaded for each sample. Anti‐fucose blot: 1st antibody rabbit‐anti‐α1,3‐fucose (1 : 10 000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed). Anti‐xylose blot: 1st antibody rabbit‐anti‐β1,2‐xylose (1 : 5000), 2nd antibody goat‐anti‐rabbit H+L AP‐labelled (1 : 10 000, pre‐absorbed).

    Article Snippet: When the sequencing results were ambiguous, the PCR products were cloned in pTOPO from the TOPO TA Cloning Kit (Thermo Fisher Scientific, Waltham) and 5–10 clones were sequenced individually for each sample.

    Techniques: Sequencing, Western Blot, Polymerase Chain Reaction, Clone Assay

    CRISPR/Cas9 genome editing of NtFT5 in N. tabacum cv. SR1 and identification of induced mutations in T 0 plants. (A) Location of the NtFT5 -specific protospacer and the protospacer adjacent motif (PAM) on the antisense DNA strand in the 147–169 bp region of exon I. Exons are shown as boxes, introns as lines. The underlines indicate triplets corresponding to amino acids. (B) PCR-based screening of three independent transgenic T 0 lines (L1–L3) for mutated allelic variants of NtFT5 . NtFT5 (exon I) was amplified using the primer combination listed in Supplemental Table S1 , single amplicons were transferred to pCRII-TOPO and sequenced (n = 15 per line). Point mutations (mut), insertions and deletions are highlighted in red letters. (C) Sequence alignment of native NtFT5 with the protein variants encoded by the identified mutated alleles. Red letters indicate amino acid substitutions and red stops indicate premature termination. (D) Experimental workflow of NtFT5 knockout analysis under LD conditions. Flowering T 0 plant L2 was chosen for detailed characterization. In the T 1 generation (n = 107) all characterized individuals lacking the cas9 gene (n = 7) were non-flowering and showed a nullizygous Ntft5 – genotype. T 1 plant #78 was representatively selected for backcrossing, and flowering was induced by grafting onto wild-type ( NtFT5 + ) rootstock. After pollination using a wild-type donor, heterozygous Ntft5 – /NtFT5 + plants of the first backcross generation (BC 1 , n = 4) flowered and one of these (#2) was randomly self-fertilized. Detailed phenotyping of non-flowering nullizygous Ntft5 – plants and flowering NtFT5 + and Ntft5 – /NtFT5 + plants was finally carried out in the second backcross generation (BC 2 ).

    Journal: Frontiers in Plant Science

    Article Title: The Major Floral Promoter NtFT5 in Tobacco (Nicotiana tabacum) Is a Promising Target for Crop Improvement

    doi: 10.3389/fpls.2019.01666

    Figure Lengend Snippet: CRISPR/Cas9 genome editing of NtFT5 in N. tabacum cv. SR1 and identification of induced mutations in T 0 plants. (A) Location of the NtFT5 -specific protospacer and the protospacer adjacent motif (PAM) on the antisense DNA strand in the 147–169 bp region of exon I. Exons are shown as boxes, introns as lines. The underlines indicate triplets corresponding to amino acids. (B) PCR-based screening of three independent transgenic T 0 lines (L1–L3) for mutated allelic variants of NtFT5 . NtFT5 (exon I) was amplified using the primer combination listed in Supplemental Table S1 , single amplicons were transferred to pCRII-TOPO and sequenced (n = 15 per line). Point mutations (mut), insertions and deletions are highlighted in red letters. (C) Sequence alignment of native NtFT5 with the protein variants encoded by the identified mutated alleles. Red letters indicate amino acid substitutions and red stops indicate premature termination. (D) Experimental workflow of NtFT5 knockout analysis under LD conditions. Flowering T 0 plant L2 was chosen for detailed characterization. In the T 1 generation (n = 107) all characterized individuals lacking the cas9 gene (n = 7) were non-flowering and showed a nullizygous Ntft5 – genotype. T 1 plant #78 was representatively selected for backcrossing, and flowering was induced by grafting onto wild-type ( NtFT5 + ) rootstock. After pollination using a wild-type donor, heterozygous Ntft5 – /NtFT5 + plants of the first backcross generation (BC 1 , n = 4) flowered and one of these (#2) was randomly self-fertilized. Detailed phenotyping of non-flowering nullizygous Ntft5 – plants and flowering NtFT5 + and Ntft5 – /NtFT5 + plants was finally carried out in the second backcross generation (BC 2 ).

    Article Snippet: The resulting PCR products were adenylated using MangoTaq DNA polymerase (Bioline, London, UK), transferred to pCRII-TOPO using the TOPO TA Cloning kit (Thermo Fisher Scientific) and sequenced.

    Techniques: CRISPR, Polymerase Chain Reaction, Transgenic Assay, Amplification, Sequencing, Knock-Out