pgem t vector  (Promega)

 
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    Name:
    pGEM T Vector Systems
    Description:
    T vector for simplified cloning of PCR products
    Catalog Number:
    a3600
    Price:
    None
    Category:
    Nucleic Acid Extraction Analysis PCR PCR Cloning
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    Structured Review

    Promega pgem t vector
    Identification of the recombinant plasmid, pTMICL12 by restriction enzyme. 1: <t>pGEM-T/EcoRI/XhoI;</t> 2: DNA size marker λ DNA/BstII; 3: pTMICL12/EcoRI/XhoI; 4: PCR product of D12D DNA.
    T vector for simplified cloning of PCR products
    https://www.bioz.com/result/pgem t vector/product/Promega
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    Images

    1) Product Images from "Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina"

    Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina

    Journal:

    doi: 10.3748/wjg.v12.i21.3373

    Identification of the recombinant plasmid, pTMICL12 by restriction enzyme. 1: pGEM-T/EcoRI/XhoI; 2: DNA size marker λ DNA/BstII; 3: pTMICL12/EcoRI/XhoI; 4: PCR product of D12D DNA.
    Figure Legend Snippet: Identification of the recombinant plasmid, pTMICL12 by restriction enzyme. 1: pGEM-T/EcoRI/XhoI; 2: DNA size marker λ DNA/BstII; 3: pTMICL12/EcoRI/XhoI; 4: PCR product of D12D DNA.

    Techniques Used: Recombinant, Plasmid Preparation, Marker, Polymerase Chain Reaction

    2) Product Images from "Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing"

    Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing

    Journal: Journal of molecular biochemistry

    doi:

    WT intron 4 pGEM-T construct
    Figure Legend Snippet: WT intron 4 pGEM-T construct

    Techniques Used: Construct

    3) Product Images from "A gene encoding a protein modified by the phytohormone indoleacetic acid"

    Article Title: A gene encoding a protein modified by the phytohormone indoleacetic acid

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

    doi: 10.1073/pnas.032450399

    ( A ) The mass spectrum of purified IAP1 protein (10 μg) was obtained using a MALDI-TOF instrument. ( B ) The iap1 cDNA was expressed in E. coli . A protein with a predicted molecular mass of 38 kDa (35 kDa plus the his tags that contributed ≈3 kDa to the protein mass) was obtained, and it migrated on SDS/PAGE as expected for a protein of mass 45 kDa. ( C ) The iap1 cDNA in a pGEM-T vector was used for coupled in vitro transcription and translation. A distinct band of the largest protein at ≈42 kDa was produced by this system, even though the largest possible transcript encodes a 35-KDa product. Together, these results demonstrate that secondary structure, or other attributes, of this protein affect the migration and thus the apparent molecular mass of the protein on SDS/PAGE.
    Figure Legend Snippet: ( A ) The mass spectrum of purified IAP1 protein (10 μg) was obtained using a MALDI-TOF instrument. ( B ) The iap1 cDNA was expressed in E. coli . A protein with a predicted molecular mass of 38 kDa (35 kDa plus the his tags that contributed ≈3 kDa to the protein mass) was obtained, and it migrated on SDS/PAGE as expected for a protein of mass 45 kDa. ( C ) The iap1 cDNA in a pGEM-T vector was used for coupled in vitro transcription and translation. A distinct band of the largest protein at ≈42 kDa was produced by this system, even though the largest possible transcript encodes a 35-KDa product. Together, these results demonstrate that secondary structure, or other attributes, of this protein affect the migration and thus the apparent molecular mass of the protein on SDS/PAGE.

    Techniques Used: Purification, SDS Page, Plasmid Preparation, In Vitro, Produced, Migration

    4) Product Images from "Chromosome-Based Genetic Complementation System for Xylella fastidiosa ▿"

    Article Title: Chromosome-Based Genetic Complementation System for Xylella fastidiosa ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00024-09

    Restriction maps of the pAX1 series vectors. The parental plasmid pAX1 is a derivative of pGEM-T (Table ), which contains a unique XbaI site that is flanked by DNA homologous to the intergenic region between PD702 and PD703. The DNA
    Figure Legend Snippet: Restriction maps of the pAX1 series vectors. The parental plasmid pAX1 is a derivative of pGEM-T (Table ), which contains a unique XbaI site that is flanked by DNA homologous to the intergenic region between PD702 and PD703. The DNA

    Techniques Used: Plasmid Preparation

    5) Product Images from "Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts"

    Article Title: Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1026

    Trans -acting factors specifically binding to the editing sites in the extracts of tobacco chloroplasts. ( A ) UV-crosslinking was performed with a respective RNA probe that was labeled with 32 P at +1 (C to be edited). Lanes 1, without competitor RNA; lanes 2, a 100-fold molar excess of unlabeled probe RNA was added as a competitor; lanes 3, a 100-fold molar excess of control RNA that was a 161 nt transcript of a pGEM-T vector was added as a competitor. Free indicates the bands of a free probe that migrated in front of the protein bands on SDS–PAGE. ( B ) Comparison of the electrophoretic mobilities of p95s binding to ndhB-9 (lane 1) and ndhF-1 (lane 2).
    Figure Legend Snippet: Trans -acting factors specifically binding to the editing sites in the extracts of tobacco chloroplasts. ( A ) UV-crosslinking was performed with a respective RNA probe that was labeled with 32 P at +1 (C to be edited). Lanes 1, without competitor RNA; lanes 2, a 100-fold molar excess of unlabeled probe RNA was added as a competitor; lanes 3, a 100-fold molar excess of control RNA that was a 161 nt transcript of a pGEM-T vector was added as a competitor. Free indicates the bands of a free probe that migrated in front of the protein bands on SDS–PAGE. ( B ) Comparison of the electrophoretic mobilities of p95s binding to ndhB-9 (lane 1) and ndhF-1 (lane 2).

    Techniques Used: Binding Assay, Labeling, Plasmid Preparation, SDS Page

    6) Product Images from "Octamer binding protein 2 (Oct2) regulates PD-L2 gene expression in B-1 cells through lineage-specific activity of a unique, intronic promoter"

    Article Title: Octamer binding protein 2 (Oct2) regulates PD-L2 gene expression in B-1 cells through lineage-specific activity of a unique, intronic promoter

    Journal: Genes and immunity

    doi: 10.1038/gene.2009.68

    PD-L2 transcripts differ between unstimulated B-1 cells and IL-4-stimulated macrophages in terms of start site. PD-L2 transcription start sites were identified by 5′ RACE as described in Materials and methods. RNA was obtained from sort-purified PD-L2 + B-1 cells and from IL-4-stimulated macrophages. After amplification of 5′ PD-L2 cDNA by nested PCR: (a) PCR products were separated by electrophoresis on 2% agarose gels and visualized with 2 μg/ml ethidium bromide; and, (b) PCR products were TA-cloned into the pGEM-T vector, after which the insert was verified by sequencing and the transcription start site was determined.
    Figure Legend Snippet: PD-L2 transcripts differ between unstimulated B-1 cells and IL-4-stimulated macrophages in terms of start site. PD-L2 transcription start sites were identified by 5′ RACE as described in Materials and methods. RNA was obtained from sort-purified PD-L2 + B-1 cells and from IL-4-stimulated macrophages. After amplification of 5′ PD-L2 cDNA by nested PCR: (a) PCR products were separated by electrophoresis on 2% agarose gels and visualized with 2 μg/ml ethidium bromide; and, (b) PCR products were TA-cloned into the pGEM-T vector, after which the insert was verified by sequencing and the transcription start site was determined.

    Techniques Used: Purification, Amplification, Nested PCR, Polymerase Chain Reaction, Electrophoresis, Clone Assay, Plasmid Preparation, Sequencing

    7) Product Images from "Two Alternative Promoters Direct Neuron-Specific Expression of the Rat Microtubule-Associated Protein 1B Gene"

    Article Title: Two Alternative Promoters Direct Neuron-Specific Expression of the Rat Microtubule-Associated Protein 1B Gene

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.16-16-05026.1996

    Determination of MAP1B gene transcription initiation sites by RNase protection assay. In the RNase protection assay ( A ), 20 μg of total RNA from adult and postnatal day 4 rat DRG ( lanes 1 , 2 ), adult and postnatal day 4 rat brain cortex ( lanes 3 , 4 ), adult and postnatal day 4 rat liver ( lanes 5 , 6 ), PC12 ( lane 7 ), NB2A ( lane 8 ), 3T3 ( lane 9 ), and L6 ( lane 10 ) cells were hybridized with 32 P-labeled riboprobe spanning nt −383 to +100 in the MAP1B gene and 52 nt of the pGEM-T vector sequence, and then treated with a mixture of RNase A and RNase T1. The protected fragments and their size (nt) are indicated by arrowheads . The size of molecular weight markers (nt) is indicated to the left . A diagram ( B ) shows the structure of the MAP1B gene antisense riboprobe and the three protected fragments.
    Figure Legend Snippet: Determination of MAP1B gene transcription initiation sites by RNase protection assay. In the RNase protection assay ( A ), 20 μg of total RNA from adult and postnatal day 4 rat DRG ( lanes 1 , 2 ), adult and postnatal day 4 rat brain cortex ( lanes 3 , 4 ), adult and postnatal day 4 rat liver ( lanes 5 , 6 ), PC12 ( lane 7 ), NB2A ( lane 8 ), 3T3 ( lane 9 ), and L6 ( lane 10 ) cells were hybridized with 32 P-labeled riboprobe spanning nt −383 to +100 in the MAP1B gene and 52 nt of the pGEM-T vector sequence, and then treated with a mixture of RNase A and RNase T1. The protected fragments and their size (nt) are indicated by arrowheads . The size of molecular weight markers (nt) is indicated to the left . A diagram ( B ) shows the structure of the MAP1B gene antisense riboprobe and the three protected fragments.

    Techniques Used: Rnase Protection Assay, Labeling, Plasmid Preparation, Sequencing, Molecular Weight

    8) Product Images from "Identification of a Secondary Promoter within the Human B Cell Receptor Component Gene hCD79b *"

    Article Title: Identification of a Secondary Promoter within the Human B Cell Receptor Component Gene hCD79b *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.461988

    A secondary promoter for the hCD79b is markedly enhanced subsequent to deletion of the primary promoter, and its mRNA encodes an N-terminally deleted CD79b protein. A , 5′-RACE assay analyses of B cell mRNA from the wild-type CD/hGH BAC revealed two sites of polymerase II transcription initiation. Each RNA sample was reverse transcribed using hCD79b -specific exon 6 primer ( 1 ), and the 3′-ends of these cDNAs were extended with poly(G). Poly(G)-tailed cDNAs were then amplified with exon 3 primer ( 2 ) and poly(C) adaptor primer and cloned into pGEM-T vector for sequencing. Each triangle indicates a transcription start site from each sequenced clone in the 50-bp window. The total numbers of cDNAs containing an additional non-templated terminal C (corresponding to the 5′-cap structure) are shown, and the total numbers of cDNAs mapping to the indicated position (with and without the additional C) are included in parentheses . The position of the 0.7-kb deletion that removes the primary promoter and exon 1 is indicated by the dotted line. B , hCD79b proteins were detected by human-specific rabbit monoclonal antibody that recognizes the N terminus of the protein ( left ; ab134103) and a rabbit monoclonal antibody that detects the C terminus of both the mouse and human CD79b ( right ; ab134147). The inability to detect an hCD79b protein in lymphocytes from the CD Δ 0.7/hGH BAC transgene with the N terminus-specific antibody ( left ) was consistent with the exon 1-truncated structure of the mRNA initiated from the secondary promoter. The antibody recognizing the C terminus of both mouse and human CD79b revealed a smaller hCD79b (∼30 kDa) in the protein extract from the CD Δ 0.7/hGH BAC lymphocytes ( arrow ), supporting the presence of a protein encoded by the mRNA originating from the secondary promoter. Ribosomal protein, L7a (30 kDa), detected using rabbit polyclonal antibody, served as an internal loading control.
    Figure Legend Snippet: A secondary promoter for the hCD79b is markedly enhanced subsequent to deletion of the primary promoter, and its mRNA encodes an N-terminally deleted CD79b protein. A , 5′-RACE assay analyses of B cell mRNA from the wild-type CD/hGH BAC revealed two sites of polymerase II transcription initiation. Each RNA sample was reverse transcribed using hCD79b -specific exon 6 primer ( 1 ), and the 3′-ends of these cDNAs were extended with poly(G). Poly(G)-tailed cDNAs were then amplified with exon 3 primer ( 2 ) and poly(C) adaptor primer and cloned into pGEM-T vector for sequencing. Each triangle indicates a transcription start site from each sequenced clone in the 50-bp window. The total numbers of cDNAs containing an additional non-templated terminal C (corresponding to the 5′-cap structure) are shown, and the total numbers of cDNAs mapping to the indicated position (with and without the additional C) are included in parentheses . The position of the 0.7-kb deletion that removes the primary promoter and exon 1 is indicated by the dotted line. B , hCD79b proteins were detected by human-specific rabbit monoclonal antibody that recognizes the N terminus of the protein ( left ; ab134103) and a rabbit monoclonal antibody that detects the C terminus of both the mouse and human CD79b ( right ; ab134147). The inability to detect an hCD79b protein in lymphocytes from the CD Δ 0.7/hGH BAC transgene with the N terminus-specific antibody ( left ) was consistent with the exon 1-truncated structure of the mRNA initiated from the secondary promoter. The antibody recognizing the C terminus of both mouse and human CD79b revealed a smaller hCD79b (∼30 kDa) in the protein extract from the CD Δ 0.7/hGH BAC lymphocytes ( arrow ), supporting the presence of a protein encoded by the mRNA originating from the secondary promoter. Ribosomal protein, L7a (30 kDa), detected using rabbit polyclonal antibody, served as an internal loading control.

    Techniques Used: BAC Assay, Amplification, Clone Assay, Plasmid Preparation, Sequencing

    9) Product Images from "Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene"

    Article Title: Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl1125

    Genomic bisulfite sequencing of hTERT promoter and 5′ exon region (−200 to +100 nucleotide bases around the ATG translational start site). After PCR amplification of bisulfite-modified DNA and cloning into pGEM-T vector, 12 clones of telomerase-positive cell lines (HeLa and SW480), 8 clones of endogenously telomerase-negative cells (BJ and HLF/ hTERT ) and 8 clones from telomerase-positive tumor tissues (breast, bladder and cervix) ( Figure 2 ) were analyzed by sequencing. Clones of each are shown on the figure. Each square represents one CpG site. Filled squares: methylated; open squares: unmethylated. Region A: in this region (−80 to −165 bp), multiple CpG sites were found to be unmethylated in tumors tissues and cell lines. Region B: localization of the CTCF binding site within the first exon of the hTERT gene, most of the CpG sites were found to be methylated in telomerase-positive tumor tissues and cell lines. Percentages of methylated CG are indicated for regions A and B.
    Figure Legend Snippet: Genomic bisulfite sequencing of hTERT promoter and 5′ exon region (−200 to +100 nucleotide bases around the ATG translational start site). After PCR amplification of bisulfite-modified DNA and cloning into pGEM-T vector, 12 clones of telomerase-positive cell lines (HeLa and SW480), 8 clones of endogenously telomerase-negative cells (BJ and HLF/ hTERT ) and 8 clones from telomerase-positive tumor tissues (breast, bladder and cervix) ( Figure 2 ) were analyzed by sequencing. Clones of each are shown on the figure. Each square represents one CpG site. Filled squares: methylated; open squares: unmethylated. Region A: in this region (−80 to −165 bp), multiple CpG sites were found to be unmethylated in tumors tissues and cell lines. Region B: localization of the CTCF binding site within the first exon of the hTERT gene, most of the CpG sites were found to be methylated in telomerase-positive tumor tissues and cell lines. Percentages of methylated CG are indicated for regions A and B.

    Techniques Used: Methylation Sequencing, Polymerase Chain Reaction, Amplification, Modification, Clone Assay, Plasmid Preparation, Sequencing, Methylation, Binding Assay

    CTCF binding to the exogenous hTERT sequence after 5-azadC treatment. (A) Genomic bisulfite sequencing of hTERT promoter and first exon region (−200 to +100 nucleotide bases around the ATG translational start site). After PCR amplification of bisulfite-modified DNA and cloning into pGEM-T vector, 8 representative clones of HeLa and SW480 are shown. Each square represents one CpG site. Filled squares: methylated; open squares: unmethylated. Region A and region B were represented for an easy comparison with Figure 2 , and percentages of methylated CG are indicated for regions A and B. (B) Binding of CTCF to the first exon of hTERT in 5aza-dC cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT exon1 and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (C) Quantitative reverse transcription-PCR analyses of hCTCF and hTERT expression before and after 5azadC treatment. GAPDH expression is used to normalize samples.
    Figure Legend Snippet: CTCF binding to the exogenous hTERT sequence after 5-azadC treatment. (A) Genomic bisulfite sequencing of hTERT promoter and first exon region (−200 to +100 nucleotide bases around the ATG translational start site). After PCR amplification of bisulfite-modified DNA and cloning into pGEM-T vector, 8 representative clones of HeLa and SW480 are shown. Each square represents one CpG site. Filled squares: methylated; open squares: unmethylated. Region A and region B were represented for an easy comparison with Figure 2 , and percentages of methylated CG are indicated for regions A and B. (B) Binding of CTCF to the first exon of hTERT in 5aza-dC cell lines was analyzed by ChIP assay using anti-CTCF antibody. PCR coamplification of the test fragments ( hTERT exon1 and H19) using as template DNA input fraction and DNA recovered from immunoprecipitated fractions bound by the anti-CTCF antibody. (C) Quantitative reverse transcription-PCR analyses of hCTCF and hTERT expression before and after 5azadC treatment. GAPDH expression is used to normalize samples.

    Techniques Used: Binding Assay, Sequencing, Methylation Sequencing, Polymerase Chain Reaction, Amplification, Modification, Clone Assay, Plasmid Preparation, Methylation, Chromatin Immunoprecipitation, Immunoprecipitation, Expressing

    10) Product Images from "Two Gamma Interferon-Activated Site-Like Elements in the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer Are Important for Viral Replication †"

    Article Title: Two Gamma Interferon-Activated Site-Like Elements in the Human Cytomegalovirus Major Immediate-Early Promoter/Enhancer Are Important for Viral Replication †

    Journal:

    doi: 10.1128/JVI.79.8.5035-5046.2005

    Generation of the VRS1 point mutation and rescue viruses. (A, diagram 1) pGEM-lox-zeo was constructed by inserting a loxP/zeo r cassette into pGEM-T. The 131-bp MIEP/E fragment containing the VRS1 mutations then was cloned between the SphI and BamHI sites
    Figure Legend Snippet: Generation of the VRS1 point mutation and rescue viruses. (A, diagram 1) pGEM-lox-zeo was constructed by inserting a loxP/zeo r cassette into pGEM-T. The 131-bp MIEP/E fragment containing the VRS1 mutations then was cloned between the SphI and BamHI sites

    Techniques Used: Mutagenesis, Construct, Clone Assay

    11) Product Images from "Homologue of Macrophage-Activating Lipoprotein in Mycoplasmagallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures"

    Article Title: Homologue of Macrophage-Activating Lipoprotein in Mycoplasmagallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.8.2538-2547.2003

    Schematic diagram of the p47 - tetM gene construct in pGEM-T and clone C46. (A) Depiction of the vector pGEM-T containing the tetM gene ligated into the Spe I sites created in the p47 gene. (B) The location of the p47 - tetM gene construct within the M . gallisepticum strain ts-11 3.894-kbp Bgl II fragment is depicted. The cleavage sites for Bam HI, Bgl II, Hin dIII, Sal I, and Spe I and the locations of oligonucleotides TetFor and TetRev are shown. The positions of nucleotides present in the C46 DNA sequence, but absent from the S6 p47 gene sequence, are indicated by vertical lines.
    Figure Legend Snippet: Schematic diagram of the p47 - tetM gene construct in pGEM-T and clone C46. (A) Depiction of the vector pGEM-T containing the tetM gene ligated into the Spe I sites created in the p47 gene. (B) The location of the p47 - tetM gene construct within the M . gallisepticum strain ts-11 3.894-kbp Bgl II fragment is depicted. The cleavage sites for Bam HI, Bgl II, Hin dIII, Sal I, and Spe I and the locations of oligonucleotides TetFor and TetRev are shown. The positions of nucleotides present in the C46 DNA sequence, but absent from the S6 p47 gene sequence, are indicated by vertical lines.

    Techniques Used: Construct, Plasmid Preparation, Sequencing

    12) Product Images from "The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd"

    Article Title: The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd

    Journal: Nucleic Acids Research

    doi:

    Construction of two tandem pVIII genes. ( A ) Detailed comparison between the wild-type pVIII gene and the recombinant pVIII gene, designated pVIIISTS . In the pVIIISTS gene, the GAC codon for Asp at position 4 of the wild-type pVIII is deleted and replaced by a sequence of 62 bp containing two stop codons and trpA transcription terminator flanked by two Sfi I sites (STS insert). ( B ) pGEM-T(p8STS) construct. The fragment containing the pVIIISTS gene, preceded by a Sna BI restriction site and followed by downstream pIII gene, was introduced into the pGEM-T vector. Black segments are sequences corresponding to the pGEM-T vector. The 62 bp STS insert is indicated by the violet small segment. ( C ) pGEM-T(p88STS) construct. This construct contains the wild-type pVIII and the modified pVIIISTS genes arranged as tandem repeats. The fragment containing the wild-type pVIII gene starting from the Sna BI site and ending just beyond the overlapping promoter/terminator was introduced into the Sna BI site of pGEM-T(p8STS), thus destroying the former site while concomitantly introducing a new Sna BI site upstream to the wild-type pVIII gene. Furthermore, additional unique sites were introduced between the two pVIII genes.
    Figure Legend Snippet: Construction of two tandem pVIII genes. ( A ) Detailed comparison between the wild-type pVIII gene and the recombinant pVIII gene, designated pVIIISTS . In the pVIIISTS gene, the GAC codon for Asp at position 4 of the wild-type pVIII is deleted and replaced by a sequence of 62 bp containing two stop codons and trpA transcription terminator flanked by two Sfi I sites (STS insert). ( B ) pGEM-T(p8STS) construct. The fragment containing the pVIIISTS gene, preceded by a Sna BI restriction site and followed by downstream pIII gene, was introduced into the pGEM-T vector. Black segments are sequences corresponding to the pGEM-T vector. The 62 bp STS insert is indicated by the violet small segment. ( C ) pGEM-T(p88STS) construct. This construct contains the wild-type pVIII and the modified pVIIISTS genes arranged as tandem repeats. The fragment containing the wild-type pVIII gene starting from the Sna BI site and ending just beyond the overlapping promoter/terminator was introduced into the Sna BI site of pGEM-T(p8STS), thus destroying the former site while concomitantly introducing a new Sna BI site upstream to the wild-type pVIII gene. Furthermore, additional unique sites were introduced between the two pVIII genes.

    Techniques Used: Recombinant, Sequencing, Construct, Plasmid Preparation, Modification

    13) Product Images from "Novel Regulator MphX Represses Activation of Phenol Hydroxylase Genes Caused by a XylR/DmpR-Type Regulator MphR in Acinetobacter calcoaceticus"

    Article Title: Novel Regulator MphX Represses Activation of Phenol Hydroxylase Genes Caused by a XylR/DmpR-Type Regulator MphR in Acinetobacter calcoaceticus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017350

    Electrophoretic mobility shift assay. (A) Comparison of the upstream region of the mphK promoter with the corresponding regions of the mop gene cluster of A. calcoaceticus NCIB8250, the xyl upper gene cluster of Pseudomonas putida mt-2 (TOL), and the dmp gene cluster of Pseudomonas sp. CF600. Grey-shaded boxes show the putative upstream activating sequences (UASs) in the mph , mop , xyl , and dmp gene clusters to which the corresponding XylR/DmpR-type regulators bind. (B) gel retardation analysis of His-MphR binding to the mphR - mphK intergenic region. Gel-mobility shift assay was performed as described in the text using a 380-bp fragment (P380) covering the mphR - mphK intergenic region. Lane 1 is a control lane containing 2.5 nM of labeled P380 fragment only. Lane 2–7 contains 2.5 nM of the labeled P380 fragment incubated with 5, 10, 20, 30, 40 and 50 nM His-MphR protein. Lane 8 is containing 2.5 nM of labeled DNA fragment amplified using two primers, IRDye-labeled M13-F and M13-R, and plasmid pGEM T-vector as template, named P150. Lane 9–10 contains 2.5 nM of the labeled P150 fragment incubated with 30 and 50 nM His-MphR protein, respectively. The positions of the free DNA and the MphR-DNA complex are indicated. (C) SDS-PAGE of purified His-MphR protein from E. coli BL21 (pEMR). Lane 1: Molecular markers with their masses indicated; Lane 2: Uninduced of BL21 (pEMR); Lane 3: BL21 (pEMR) induced by 0.1 mM IPTG; Lane 4: BL21 (pEMR) induced by 0.5 mM IPTG; Lane 5: MphR purified by NTA resin and eluted by NTA-60; Lane 6: MphR purified by NTA resin and eluted by NTA-80.
    Figure Legend Snippet: Electrophoretic mobility shift assay. (A) Comparison of the upstream region of the mphK promoter with the corresponding regions of the mop gene cluster of A. calcoaceticus NCIB8250, the xyl upper gene cluster of Pseudomonas putida mt-2 (TOL), and the dmp gene cluster of Pseudomonas sp. CF600. Grey-shaded boxes show the putative upstream activating sequences (UASs) in the mph , mop , xyl , and dmp gene clusters to which the corresponding XylR/DmpR-type regulators bind. (B) gel retardation analysis of His-MphR binding to the mphR - mphK intergenic region. Gel-mobility shift assay was performed as described in the text using a 380-bp fragment (P380) covering the mphR - mphK intergenic region. Lane 1 is a control lane containing 2.5 nM of labeled P380 fragment only. Lane 2–7 contains 2.5 nM of the labeled P380 fragment incubated with 5, 10, 20, 30, 40 and 50 nM His-MphR protein. Lane 8 is containing 2.5 nM of labeled DNA fragment amplified using two primers, IRDye-labeled M13-F and M13-R, and plasmid pGEM T-vector as template, named P150. Lane 9–10 contains 2.5 nM of the labeled P150 fragment incubated with 30 and 50 nM His-MphR protein, respectively. The positions of the free DNA and the MphR-DNA complex are indicated. (C) SDS-PAGE of purified His-MphR protein from E. coli BL21 (pEMR). Lane 1: Molecular markers with their masses indicated; Lane 2: Uninduced of BL21 (pEMR); Lane 3: BL21 (pEMR) induced by 0.1 mM IPTG; Lane 4: BL21 (pEMR) induced by 0.5 mM IPTG; Lane 5: MphR purified by NTA resin and eluted by NTA-60; Lane 6: MphR purified by NTA resin and eluted by NTA-80.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Mobility Shift, Labeling, Incubation, Amplification, Plasmid Preparation, SDS Page, Purification

    14) Product Images from "Effective gene editing by high-fidelity base editor 2 in mouse zygotes"

    Article Title: Effective gene editing by high-fidelity base editor 2 in mouse zygotes

    Journal: Protein & Cell

    doi: 10.1007/s13238-017-0418-2

    Generation of base-edited mice using HF2-BE2 . (A) Representative sequencing chromatographs of the PCR amplicons of target sites from the founder mice are shown here. WT, wild-type embryo. Edited, embryo edited by HF2-BE2, with the successfully edited base indicated by red arrowheads. (B) PCR amplicons of gRNA-1 target sites from the genomic DNA of selected founder mice were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. (C) PCR amplicons of gRNA-2 target sites from the genomic DNA of selected founder mice were subcloned into pGEM-T vectors and sequenced. (D) Founder pups (10 days old) from the gRNA-2 group
    Figure Legend Snippet: Generation of base-edited mice using HF2-BE2 . (A) Representative sequencing chromatographs of the PCR amplicons of target sites from the founder mice are shown here. WT, wild-type embryo. Edited, embryo edited by HF2-BE2, with the successfully edited base indicated by red arrowheads. (B) PCR amplicons of gRNA-1 target sites from the genomic DNA of selected founder mice were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. (C) PCR amplicons of gRNA-2 target sites from the genomic DNA of selected founder mice were subcloned into pGEM-T vectors and sequenced. (D) Founder pups (10 days old) from the gRNA-2 group

    Techniques Used: Mouse Assay, Sequencing, Polymerase Chain Reaction, Clone Assay

    Examination of proximal-site deamination by HF2-BE2 . (A) Design of truncated gRNA variants of gRNA-2 (gRNA-2-T1 and gRNA-2-T2). The codon to be modified is underlined, with the nucleotide to be deaminated in red. PAM is in green. (B) Representative sequencing chromatographs of the PCR amplicons of target sites from the embryos are shown here. Bases successfully edited are indicated by red arrowheads. (C) PCR amplicons of target sites from embryos were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. (D) Statistical analysis of base editing by HF2-BE2 and BE2 in mouse embryos. n.s., not significant, P values > 0.05. ** P
    Figure Legend Snippet: Examination of proximal-site deamination by HF2-BE2 . (A) Design of truncated gRNA variants of gRNA-2 (gRNA-2-T1 and gRNA-2-T2). The codon to be modified is underlined, with the nucleotide to be deaminated in red. PAM is in green. (B) Representative sequencing chromatographs of the PCR amplicons of target sites from the embryos are shown here. Bases successfully edited are indicated by red arrowheads. (C) PCR amplicons of target sites from embryos were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. (D) Statistical analysis of base editing by HF2-BE2 and BE2 in mouse embryos. n.s., not significant, P values > 0.05. ** P

    Techniques Used: Modification, Sequencing, Polymerase Chain Reaction, Clone Assay

    HF2-BE2 edits target bases efficiently in human cells . (A) Schematic representation of HF2-BE2. Point mutations that inactivate Cas9 nuclease activity are shown in red, and mutations that enhance its fidelity are shown in black. (B) The HF2-BE2 expression vector was respectively co-transfected into 293T cells with gRNA expression vectors targeting HEK293 site 3 and RNF2 (Komor et al., 2016 ). Genomic DNA was extracted from the edited cell populations for PCR amplification of the target sites. Sequencing chromatographs of the PCR amplicons are shown. WT, untransfected wild-type control cells. Edited, base-edited cells. Red arrowheads, successfully edited base. (C) The PCR amplicons from (B) were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations
    Figure Legend Snippet: HF2-BE2 edits target bases efficiently in human cells . (A) Schematic representation of HF2-BE2. Point mutations that inactivate Cas9 nuclease activity are shown in red, and mutations that enhance its fidelity are shown in black. (B) The HF2-BE2 expression vector was respectively co-transfected into 293T cells with gRNA expression vectors targeting HEK293 site 3 and RNF2 (Komor et al., 2016 ). Genomic DNA was extracted from the edited cell populations for PCR amplification of the target sites. Sequencing chromatographs of the PCR amplicons are shown. WT, untransfected wild-type control cells. Edited, base-edited cells. Red arrowheads, successfully edited base. (C) The PCR amplicons from (B) were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations

    Techniques Used: Activity Assay, Expressing, Plasmid Preparation, Transfection, Polymerase Chain Reaction, Amplification, Sequencing, Clone Assay

    HF2-BE2 mediates efficient base editing of Tyr in mouse embryos . (A) Schematic representation of the Tyr locus and gRNA target sites. gRNA target sequences are also shown. The codon to be modified is underlined, with the nucleotide to be deaminated in red. The protospacer adjacent motif (PAM) is in green. (B) The two gRNAs were respectively co-injected into 1-cell zygotes with HF2-BE2 mRNA, and the embryos were analyzed 48 h later. Representative sequencing chromatographs of the PCR amplicons of target sites are shown here. WT, wild-type embryo. Edited, embryos edited by HF2-BE2 with the successfully edited base indicated by red arrowheads. (C) PCR amplicons of gRNA-1 target site from the genomic DNA of selected embryos were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. Purple, insertions. Dash, deletions. (D) PCR amplicons of gRNA-2 target site from the genomic DNA of selected embryos were subcloned into pGEM-T vectors and sequenced. (E) Summary of base editing by HF2-BE2 in mouse embryos. a, this biallelic mutant embryo is homozygous
    Figure Legend Snippet: HF2-BE2 mediates efficient base editing of Tyr in mouse embryos . (A) Schematic representation of the Tyr locus and gRNA target sites. gRNA target sequences are also shown. The codon to be modified is underlined, with the nucleotide to be deaminated in red. The protospacer adjacent motif (PAM) is in green. (B) The two gRNAs were respectively co-injected into 1-cell zygotes with HF2-BE2 mRNA, and the embryos were analyzed 48 h later. Representative sequencing chromatographs of the PCR amplicons of target sites are shown here. WT, wild-type embryo. Edited, embryos edited by HF2-BE2 with the successfully edited base indicated by red arrowheads. (C) PCR amplicons of gRNA-1 target site from the genomic DNA of selected embryos were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. Purple, insertions. Dash, deletions. (D) PCR amplicons of gRNA-2 target site from the genomic DNA of selected embryos were subcloned into pGEM-T vectors and sequenced. (E) Summary of base editing by HF2-BE2 in mouse embryos. a, this biallelic mutant embryo is homozygous

    Techniques Used: Modification, Injection, Sequencing, Polymerase Chain Reaction, Clone Assay, Mutagenesis

    15) Product Images from "Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid"

    Article Title: Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid

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

    doi: 10.1073/pnas.1323725111

    Inactivation of slr1573 in the Synechocystis 6803 sam8 transformant. ( A ) Plasmid construction for slr1573 knockout. The spectinomycin-resistance cassette was flanked by upstream (UP) and downstream (DW) regions of slr1573 and cloned into the pGEM-T plasmid.
    Figure Legend Snippet: Inactivation of slr1573 in the Synechocystis 6803 sam8 transformant. ( A ) Plasmid construction for slr1573 knockout. The spectinomycin-resistance cassette was flanked by upstream (UP) and downstream (DW) regions of slr1573 and cloned into the pGEM-T plasmid.

    Techniques Used: Plasmid Preparation, Knock-Out, Clone Assay

    16) Product Images from "Inactivation of an ABC Transporter Gene, mcyH, Results in Loss of Microcystin Production in the Cyanobacterium Microcystis aeruginosa PCC 7806"

    Article Title: Inactivation of an ABC Transporter Gene, mcyH, Results in Loss of Microcystin Production in the Cyanobacterium Microcystis aeruginosa PCC 7806

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.11.6370-6378.2004

    (A) Construction of plasmid p5K8. A diagram illustrating the region of the mcy cluster amplified by primers mcyH SKF and mcyH SKR and cloned into pGEM-T to give rise to plasmid p5K8 is shown. (B) Construction of plasmids p5K8BsmI-3 and p5K8BsmI-2. A schematic diagram depicting the partial deletion of mcyH in p5K8, resulting in the knockout constructs p5K8BsmI-3 and p5K8BsmI-2, is shown. The directions of transcription of the mcyH and Cm r genes are indicated by (−/−) and (−/+), respectively.
    Figure Legend Snippet: (A) Construction of plasmid p5K8. A diagram illustrating the region of the mcy cluster amplified by primers mcyH SKF and mcyH SKR and cloned into pGEM-T to give rise to plasmid p5K8 is shown. (B) Construction of plasmids p5K8BsmI-3 and p5K8BsmI-2. A schematic diagram depicting the partial deletion of mcyH in p5K8, resulting in the knockout constructs p5K8BsmI-3 and p5K8BsmI-2, is shown. The directions of transcription of the mcyH and Cm r genes are indicated by (−/−) and (−/+), respectively.

    Techniques Used: Plasmid Preparation, Amplification, Clone Assay, Knock-Out, Construct

    17) Product Images from "Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing"

    Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing

    Journal: Journal of molecular biochemistry

    doi:

    WT intron 4 pGEM-T construct
    Figure Legend Snippet: WT intron 4 pGEM-T construct

    Techniques Used: Construct

    18) Product Images from "Effective gene editing by high-fidelity base editor 2 in mouse zygotes"

    Article Title: Effective gene editing by high-fidelity base editor 2 in mouse zygotes

    Journal: Protein & Cell

    doi: 10.1007/s13238-017-0418-2

    Generation of base-edited mice using HF2-BE2 . (A) Representative sequencing chromatographs of the PCR amplicons of target sites from the founder mice are shown here. WT, wild-type embryo. Edited, embryo edited by HF2-BE2, with the successfully edited base indicated by red arrowheads. (B) PCR amplicons of gRNA-1 target sites from the genomic DNA of selected founder mice were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. (C) PCR amplicons of gRNA-2 target sites from the genomic DNA of selected founder mice were subcloned into pGEM-T vectors and sequenced. (D) Founder pups (10 days old) from the gRNA-2 group
    Figure Legend Snippet: Generation of base-edited mice using HF2-BE2 . (A) Representative sequencing chromatographs of the PCR amplicons of target sites from the founder mice are shown here. WT, wild-type embryo. Edited, embryo edited by HF2-BE2, with the successfully edited base indicated by red arrowheads. (B) PCR amplicons of gRNA-1 target sites from the genomic DNA of selected founder mice were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. (C) PCR amplicons of gRNA-2 target sites from the genomic DNA of selected founder mice were subcloned into pGEM-T vectors and sequenced. (D) Founder pups (10 days old) from the gRNA-2 group

    Techniques Used: Mouse Assay, Sequencing, Polymerase Chain Reaction, Clone Assay

    Examination of proximal-site deamination by HF2-BE2 . (A) Design of truncated gRNA variants of gRNA-2 (gRNA-2-T1 and gRNA-2-T2). The codon to be modified is underlined, with the nucleotide to be deaminated in red. PAM is in green. (B) Representative sequencing chromatographs of the PCR amplicons of target sites from the embryos are shown here. Bases successfully edited are indicated by red arrowheads. (C) PCR amplicons of target sites from embryos were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. (D) Statistical analysis of base editing by HF2-BE2 and BE2 in mouse embryos. n.s., not significant, P values > 0.05. ** P
    Figure Legend Snippet: Examination of proximal-site deamination by HF2-BE2 . (A) Design of truncated gRNA variants of gRNA-2 (gRNA-2-T1 and gRNA-2-T2). The codon to be modified is underlined, with the nucleotide to be deaminated in red. PAM is in green. (B) Representative sequencing chromatographs of the PCR amplicons of target sites from the embryos are shown here. Bases successfully edited are indicated by red arrowheads. (C) PCR amplicons of target sites from embryos were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. (D) Statistical analysis of base editing by HF2-BE2 and BE2 in mouse embryos. n.s., not significant, P values > 0.05. ** P

    Techniques Used: Modification, Sequencing, Polymerase Chain Reaction, Clone Assay

    HF2-BE2 edits target bases efficiently in human cells . (A) Schematic representation of HF2-BE2. Point mutations that inactivate Cas9 nuclease activity are shown in red, and mutations that enhance its fidelity are shown in black. (B) The HF2-BE2 expression vector was respectively co-transfected into 293T cells with gRNA expression vectors targeting HEK293 site 3 and RNF2 (Komor et al., 2016 ). Genomic DNA was extracted from the edited cell populations for PCR amplification of the target sites. Sequencing chromatographs of the PCR amplicons are shown. WT, untransfected wild-type control cells. Edited, base-edited cells. Red arrowheads, successfully edited base. (C) The PCR amplicons from (B) were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations
    Figure Legend Snippet: HF2-BE2 edits target bases efficiently in human cells . (A) Schematic representation of HF2-BE2. Point mutations that inactivate Cas9 nuclease activity are shown in red, and mutations that enhance its fidelity are shown in black. (B) The HF2-BE2 expression vector was respectively co-transfected into 293T cells with gRNA expression vectors targeting HEK293 site 3 and RNF2 (Komor et al., 2016 ). Genomic DNA was extracted from the edited cell populations for PCR amplification of the target sites. Sequencing chromatographs of the PCR amplicons are shown. WT, untransfected wild-type control cells. Edited, base-edited cells. Red arrowheads, successfully edited base. (C) The PCR amplicons from (B) were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations

    Techniques Used: Activity Assay, Expressing, Plasmid Preparation, Transfection, Polymerase Chain Reaction, Amplification, Sequencing, Clone Assay

    HF2-BE2 mediates efficient base editing of Tyr in mouse embryos . (A) Schematic representation of the Tyr locus and gRNA target sites. gRNA target sequences are also shown. The codon to be modified is underlined, with the nucleotide to be deaminated in red. The protospacer adjacent motif (PAM) is in green. (B) The two gRNAs were respectively co-injected into 1-cell zygotes with HF2-BE2 mRNA, and the embryos were analyzed 48 h later. Representative sequencing chromatographs of the PCR amplicons of target sites are shown here. WT, wild-type embryo. Edited, embryos edited by HF2-BE2 with the successfully edited base indicated by red arrowheads. (C) PCR amplicons of gRNA-1 target site from the genomic DNA of selected embryos were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. Purple, insertions. Dash, deletions. (D) PCR amplicons of gRNA-2 target site from the genomic DNA of selected embryos were subcloned into pGEM-T vectors and sequenced. (E) Summary of base editing by HF2-BE2 in mouse embryos. a, this biallelic mutant embryo is homozygous
    Figure Legend Snippet: HF2-BE2 mediates efficient base editing of Tyr in mouse embryos . (A) Schematic representation of the Tyr locus and gRNA target sites. gRNA target sequences are also shown. The codon to be modified is underlined, with the nucleotide to be deaminated in red. The protospacer adjacent motif (PAM) is in green. (B) The two gRNAs were respectively co-injected into 1-cell zygotes with HF2-BE2 mRNA, and the embryos were analyzed 48 h later. Representative sequencing chromatographs of the PCR amplicons of target sites are shown here. WT, wild-type embryo. Edited, embryos edited by HF2-BE2 with the successfully edited base indicated by red arrowheads. (C) PCR amplicons of gRNA-1 target site from the genomic DNA of selected embryos were subcloned into pGEM-T vectors and sequenced. The number of clones for each sequence pattern is indicated. Underlined, gRNA target regions. Green, PAM sequence. Red, point mutations. Purple, insertions. Dash, deletions. (D) PCR amplicons of gRNA-2 target site from the genomic DNA of selected embryos were subcloned into pGEM-T vectors and sequenced. (E) Summary of base editing by HF2-BE2 in mouse embryos. a, this biallelic mutant embryo is homozygous

    Techniques Used: Modification, Injection, Sequencing, Polymerase Chain Reaction, Clone Assay, Mutagenesis

    19) Product Images from "Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit"

    Article Title: Identification and Characterization of an Alternatively Spliced Isoform of the Human Protein Phosphatase 2Aα Catalytic Subunit

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.283341

    Identification and cloning of a novel splicing isoform of the PP2A catalytic subunit α. A , PCR amplification of PP2Ac cDNAs from three human cDNA libraries (leukocyte, fetal brain, and bone marrow) results in two distinct bands, corresponding to the canonical and to a novel shorter isoform ( PP2Ac α 2 ). Clone 3.4 and PPA2c α indicate the control reactions containing the short and the canonical cDNA forms, respectively. B , RT-PCR amplification of PP2Ac cDNAs from immortalized cell lines results in a single band corresponding to the canonical isoform. C , diagram of the PP2Acα cDNAs showing the unique XhoI restriction site located at exon 5, which is missing in PP2Acα2. D and E , analysis of PP2Acα cDNA amplification by RT-PCR from two immortalized cell lines (HEK293 and L540). Following one round of PCR amplification, the PCR product was digested with XhoI to deplete the samples from the predominant PP2Acα amplification product. The cDNA containing exon 5 is digested with XhoI, producing two fragments (638 and 292 bp), whereas the shorter cDNA is not digested. E , the undigested PCR product was extracted from the gel and purified ( left ) and used as template in a second round of PCR amplification using PP2Acα-specific primers. This amplification resulted in two bands of similar intensity. The shorter band was cloned into pGEM-T easy vector and sequenced, which confirmed its identity as the shorter PP2Acα2 isoform.
    Figure Legend Snippet: Identification and cloning of a novel splicing isoform of the PP2A catalytic subunit α. A , PCR amplification of PP2Ac cDNAs from three human cDNA libraries (leukocyte, fetal brain, and bone marrow) results in two distinct bands, corresponding to the canonical and to a novel shorter isoform ( PP2Ac α 2 ). Clone 3.4 and PPA2c α indicate the control reactions containing the short and the canonical cDNA forms, respectively. B , RT-PCR amplification of PP2Ac cDNAs from immortalized cell lines results in a single band corresponding to the canonical isoform. C , diagram of the PP2Acα cDNAs showing the unique XhoI restriction site located at exon 5, which is missing in PP2Acα2. D and E , analysis of PP2Acα cDNA amplification by RT-PCR from two immortalized cell lines (HEK293 and L540). Following one round of PCR amplification, the PCR product was digested with XhoI to deplete the samples from the predominant PP2Acα amplification product. The cDNA containing exon 5 is digested with XhoI, producing two fragments (638 and 292 bp), whereas the shorter cDNA is not digested. E , the undigested PCR product was extracted from the gel and purified ( left ) and used as template in a second round of PCR amplification using PP2Acα-specific primers. This amplification resulted in two bands of similar intensity. The shorter band was cloned into pGEM-T easy vector and sequenced, which confirmed its identity as the shorter PP2Acα2 isoform.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Purification, Plasmid Preparation

    20) Product Images from "The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd"

    Article Title: The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd

    Journal: Nucleic Acids Research

    doi:

    Construction of two tandem pVIII genes. ( A ) Detailed comparison between the wild-type pVIII gene and the recombinant pVIII gene, designated pVIIISTS . In the pVIIISTS gene, the GAC codon for Asp at position 4 of the wild-type pVIII is deleted and replaced by a sequence of 62 bp containing two stop codons and trpA transcription terminator flanked by two Sfi I sites (STS insert). ( B ) pGEM-T(p8STS) construct. The fragment containing the pVIIISTS gene, preceded by a Sna BI restriction site and followed by downstream pIII gene, was introduced into the pGEM-T vector. Black segments are sequences corresponding to the pGEM-T vector. The 62 bp STS insert is indicated by the violet small segment. ( C ) pGEM-T(p88STS) construct. This construct contains the wild-type pVIII and the modified pVIIISTS genes arranged as tandem repeats. The fragment containing the wild-type pVIII gene starting from the Sna BI site and ending just beyond the overlapping promoter/terminator was introduced into the Sna BI site of pGEM-T(p8STS), thus destroying the former site while concomitantly introducing a new Sna BI site upstream to the wild-type pVIII gene. Furthermore, additional unique sites were introduced between the two pVIII genes.
    Figure Legend Snippet: Construction of two tandem pVIII genes. ( A ) Detailed comparison between the wild-type pVIII gene and the recombinant pVIII gene, designated pVIIISTS . In the pVIIISTS gene, the GAC codon for Asp at position 4 of the wild-type pVIII is deleted and replaced by a sequence of 62 bp containing two stop codons and trpA transcription terminator flanked by two Sfi I sites (STS insert). ( B ) pGEM-T(p8STS) construct. The fragment containing the pVIIISTS gene, preceded by a Sna BI restriction site and followed by downstream pIII gene, was introduced into the pGEM-T vector. Black segments are sequences corresponding to the pGEM-T vector. The 62 bp STS insert is indicated by the violet small segment. ( C ) pGEM-T(p88STS) construct. This construct contains the wild-type pVIII and the modified pVIIISTS genes arranged as tandem repeats. The fragment containing the wild-type pVIII gene starting from the Sna BI site and ending just beyond the overlapping promoter/terminator was introduced into the Sna BI site of pGEM-T(p8STS), thus destroying the former site while concomitantly introducing a new Sna BI site upstream to the wild-type pVIII gene. Furthermore, additional unique sites were introduced between the two pVIII genes.

    Techniques Used: Recombinant, Sequencing, Construct, Plasmid Preparation, Modification

    21) Product Images from "Characterization of Transcripts Expressed from Human Herpesvirus 6A Strain GS Immediate-Early Region B U16-U17 Open Reading Frames"

    Article Title: Characterization of Transcripts Expressed from Human Herpesvirus 6A Strain GS Immediate-Early Region B U16-U17 Open Reading Frames

    Journal: Journal of Virology

    doi:

    (A) Reactivity of monoclonal antibody against the B701 (U16) protein. Lane 1, reactivities of antibodies in the Western blot reaction with the B701 protein expressed in the pET-3b vector. The 20K protein and the higher-molecular-weight dimeric and/or multimeric forms of B701 protein recognized are indicated by the arrowheads. (B) SDS-PAGE analysis of in vitro-expressed polypeptides from the 1.8-kb cDNA insert. In vitro-synthesized mRNA, transcribed from the cDNA insert in the pGEM-T plasmid, was translated in vitro using rabbit reticulocyte lysate. Lanes 1, 4, and 7, translation reaction components without the addition of RNA. Lanes 2, 6, and 9, translation from RNA transcribed with SP6 RNA polymerase. Lanes 3, 5, and 8, translation from RNA transcribed with T7 RNA polymerase. Lanes 1 to 3, total translated products. Lanes 4 to 6, immunoprecipitations of translated products using a monoclonal antibody against the B701 (U16) protein. Lanes 7 to 9, immunoprecipitations of translation products using polyclonal rabbit antisera raised against the B701 protein. Samples were analyzed on 12% acrylamide cross-linked with bisacrylamide, and standard molecular mass markers were included in parallel lanes. The numbers indicate approximate molecular masses (in kilodaltons) of the prominent polypeptides identified.
    Figure Legend Snippet: (A) Reactivity of monoclonal antibody against the B701 (U16) protein. Lane 1, reactivities of antibodies in the Western blot reaction with the B701 protein expressed in the pET-3b vector. The 20K protein and the higher-molecular-weight dimeric and/or multimeric forms of B701 protein recognized are indicated by the arrowheads. (B) SDS-PAGE analysis of in vitro-expressed polypeptides from the 1.8-kb cDNA insert. In vitro-synthesized mRNA, transcribed from the cDNA insert in the pGEM-T plasmid, was translated in vitro using rabbit reticulocyte lysate. Lanes 1, 4, and 7, translation reaction components without the addition of RNA. Lanes 2, 6, and 9, translation from RNA transcribed with SP6 RNA polymerase. Lanes 3, 5, and 8, translation from RNA transcribed with T7 RNA polymerase. Lanes 1 to 3, total translated products. Lanes 4 to 6, immunoprecipitations of translated products using a monoclonal antibody against the B701 (U16) protein. Lanes 7 to 9, immunoprecipitations of translation products using polyclonal rabbit antisera raised against the B701 protein. Samples were analyzed on 12% acrylamide cross-linked with bisacrylamide, and standard molecular mass markers were included in parallel lanes. The numbers indicate approximate molecular masses (in kilodaltons) of the prominent polypeptides identified.

    Techniques Used: Western Blot, Positron Emission Tomography, Plasmid Preparation, Molecular Weight, SDS Page, In Vitro, Synthesized

    22) Product Images from "Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning"

    Article Title: Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

    Journal: Viruses

    doi: 10.3390/v7122935

    Strategy for constructing intron-less and intron-containing infectious full-length cDNA clone of PLDMV-DF using In-Fusion cloning. ( A ) Schematic representation of the genomic structure of PLDMV; ( B ) Two genomic fragments overlapping the complete genome (fragments I and II) and fragment III of pGEM-T vector containing a T7 promoter were fused to generate the pT7-PLDMV vector by In-Fusion cloning. Arrows indicated the primers used in construction of pT7-PLDMV ( Table 1 ); ( C ) Two genomic fragments overlapping the complete genome (fragments IV and V), the intron 2 (220 bp) of the NiR gene from Phaseolus vulgaris and fragment III of the pGEM-T vector containing a T7 promoter were fused to generate the PLDMV-DF-In2 vector by In-Fusion cloning. Arrows indicated the primers used in construction of PLDMV-DF-In2 ( Table 1 ).
    Figure Legend Snippet: Strategy for constructing intron-less and intron-containing infectious full-length cDNA clone of PLDMV-DF using In-Fusion cloning. ( A ) Schematic representation of the genomic structure of PLDMV; ( B ) Two genomic fragments overlapping the complete genome (fragments I and II) and fragment III of pGEM-T vector containing a T7 promoter were fused to generate the pT7-PLDMV vector by In-Fusion cloning. Arrows indicated the primers used in construction of pT7-PLDMV ( Table 1 ); ( C ) Two genomic fragments overlapping the complete genome (fragments IV and V), the intron 2 (220 bp) of the NiR gene from Phaseolus vulgaris and fragment III of the pGEM-T vector containing a T7 promoter were fused to generate the PLDMV-DF-In2 vector by In-Fusion cloning. Arrows indicated the primers used in construction of PLDMV-DF-In2 ( Table 1 ).

    Techniques Used: Clone Assay, Plasmid Preparation

    23) Product Images from "Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli"

    Article Title: Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-9-54

    Scheme for construction of the hydrogenase 1 expression vector . Full-length hya operon was cloned into pGEM-T to generate (A) pGemT-EcH3, and two subunit genes ( hya A hya B) comprising the core enzyme cloned into pET-21b and pTrcHisC expression vectors to generate (B) pET-EcHAB for the analysis of hydrogen production in recombinant BL21 and (C) pTrc-EcH1ABHis for the analysis of protein expression with His 6 -tag, respectively. His 6 -tag was fused to each subunit gene in pTrc-EcH1ABHis for facile purification of hydrogenase (Refer to Materials and Methods for scheme of His 6 -tag fusion).
    Figure Legend Snippet: Scheme for construction of the hydrogenase 1 expression vector . Full-length hya operon was cloned into pGEM-T to generate (A) pGemT-EcH3, and two subunit genes ( hya A hya B) comprising the core enzyme cloned into pET-21b and pTrcHisC expression vectors to generate (B) pET-EcHAB for the analysis of hydrogen production in recombinant BL21 and (C) pTrc-EcH1ABHis for the analysis of protein expression with His 6 -tag, respectively. His 6 -tag was fused to each subunit gene in pTrc-EcH1ABHis for facile purification of hydrogenase (Refer to Materials and Methods for scheme of His 6 -tag fusion).

    Techniques Used: Expressing, Plasmid Preparation, Clone Assay, Positron Emission Tomography, Recombinant, Purification

    24) Product Images from "Expression of Canonical SOS Genes Is Not under LexA Repression in Bdellovibrio bacteriovorus"

    Article Title: Expression of Canonical SOS Genes Is Not under LexA Repression in Bdellovibrio bacteriovorus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5367-5375.2005

    Electrophoretic mobility shift assay (EMSA) of the B. bacteriovorus lexA promoter in the absence (lane 1) or presence (lane 2) of 80 nM purified B. bacteriovorus LexA protein. To determine the specificity of LexA binding, a 300-fold molar excess of either unlabeled lexA promoter (lane 3) or pGEM-T plasmid DNA (lane 4) was used as a specific or nonspecific competitor fragment, respectively.
    Figure Legend Snippet: Electrophoretic mobility shift assay (EMSA) of the B. bacteriovorus lexA promoter in the absence (lane 1) or presence (lane 2) of 80 nM purified B. bacteriovorus LexA protein. To determine the specificity of LexA binding, a 300-fold molar excess of either unlabeled lexA promoter (lane 3) or pGEM-T plasmid DNA (lane 4) was used as a specific or nonspecific competitor fragment, respectively.

    Techniques Used: Electrophoretic Mobility Shift Assay, Purification, Binding Assay, Plasmid Preparation

    Effect of a 300-fold molar excess of B. bacteriovorus Bd0386 promoter (lane 3), lexA promoter (lane 4), or pGEM-T plasmid DNA (lane 5), used as nonspecific DNA, in the electrophoretic mobility of Bd0386 DIG-labeled promoter in presence of purified LexA protein. The migration of this fragment without any additional DNA (lane 2) or in absence of LexA protein (lane 1) is also presented as a positive or negative control, respectively.
    Figure Legend Snippet: Effect of a 300-fold molar excess of B. bacteriovorus Bd0386 promoter (lane 3), lexA promoter (lane 4), or pGEM-T plasmid DNA (lane 5), used as nonspecific DNA, in the electrophoretic mobility of Bd0386 DIG-labeled promoter in presence of purified LexA protein. The migration of this fragment without any additional DNA (lane 2) or in absence of LexA protein (lane 1) is also presented as a positive or negative control, respectively.

    Techniques Used: Plasmid Preparation, Labeling, Purification, Migration, Negative Control

    25) Product Images from "Dual Qualitative-Quantitative Nested PCR for Detection of JC Virus in Cerebrospinal Fluid: High Potential for Evaluation and Monitoring of Progressive Multifocal Leukoencephalopathy in AIDS Patients Receiving Highly Active Antiretroviral Therapy"

    Article Title: Dual Qualitative-Quantitative Nested PCR for Detection of JC Virus in Cerebrospinal Fluid: High Potential for Evaluation and Monitoring of Progressive Multifocal Leukoencephalopathy in AIDS Patients Receiving Highly Active Antiretroviral Therapy

    Journal: Journal of Clinical Microbiology

    doi:

    (a) Scheme of construction of the recombinant plasmids used as internal controls: pGemJC, pGemJCins, and pGembla. Cloning vector pGem-T is also included. The origin of the different DNA fragments used for cloning and their respective lengths are indicated. The region of JCV selected for cloning is defined between coordinates 3546 and 4001. pUC18 MCS, multicloning site from pUC18 vector. (b) Amplicons derived from PCR amplification of control plasmids: pGemJC (277 bp), pGemJCins (334 bp), and pGembla (539 bp). Products of pGembla and pGemJC when coamplified in the same reaction are shown in the last lane. Left lane, 100-bp ladder as a molecular weight marker.
    Figure Legend Snippet: (a) Scheme of construction of the recombinant plasmids used as internal controls: pGemJC, pGemJCins, and pGembla. Cloning vector pGem-T is also included. The origin of the different DNA fragments used for cloning and their respective lengths are indicated. The region of JCV selected for cloning is defined between coordinates 3546 and 4001. pUC18 MCS, multicloning site from pUC18 vector. (b) Amplicons derived from PCR amplification of control plasmids: pGemJC (277 bp), pGemJCins (334 bp), and pGembla (539 bp). Products of pGembla and pGemJC when coamplified in the same reaction are shown in the last lane. Left lane, 100-bp ladder as a molecular weight marker.

    Techniques Used: Recombinant, Clone Assay, Plasmid Preparation, Derivative Assay, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    26) Product Images from "Structural and Functional Significance of the FGL Sequence of the Periplasmic Chaperone Caf1M of Yersinia pestis"

    Article Title: Structural and Functional Significance of the FGL Sequence of the Periplasmic Chaperone Caf1M of Yersinia pestis

    Journal: Journal of Bacteriology

    doi:

    Structure and products of recombinant caf plasmids. Details of plasmids are given in Materials and Methods. With the exception of p12R, which carries the original subcloned caf operon in pUC19, and pGEM-T caf1A , all plasmids are pTrc99A (Pharmacia) derivatives and carry lacI q . Only restriction sites used in manipulation of caf genes are shown. B , Bam HI; Bg , Bgl II; Bs , Bsp HI; H , Hpa I; N , Nco I; P , Pst I; S , Sal I; Sm , Sma I. Brackets ({}) indicate corresponding plasmids encoding the deletion mutant chaperone, dCaf1M, and the level of product from each respective plasmid. The unique Bgl II site (Bg in parentheses) created at the site of deletion within caf1M is also shown. The Product columns summarize the amount of each caf gene product detectable following induction in E. coli ) (Caf1A). For periplasmic chaperone and subunit, +++ corresponds to approximately 20 to 40 μg/ml/OD 600 culture in the final osmotic shock fraction. l, location of subunit. Surface location, s, was identified by a positive reaction in the quantitative immunofluorescence assay, with only low levels present in the osmotic shock fraction; periplasmic location, p, was identified by a negative reaction (i.e., no higher than background, or
    Figure Legend Snippet: Structure and products of recombinant caf plasmids. Details of plasmids are given in Materials and Methods. With the exception of p12R, which carries the original subcloned caf operon in pUC19, and pGEM-T caf1A , all plasmids are pTrc99A (Pharmacia) derivatives and carry lacI q . Only restriction sites used in manipulation of caf genes are shown. B , Bam HI; Bg , Bgl II; Bs , Bsp HI; H , Hpa I; N , Nco I; P , Pst I; S , Sal I; Sm , Sma I. Brackets ({}) indicate corresponding plasmids encoding the deletion mutant chaperone, dCaf1M, and the level of product from each respective plasmid. The unique Bgl II site (Bg in parentheses) created at the site of deletion within caf1M is also shown. The Product columns summarize the amount of each caf gene product detectable following induction in E. coli ) (Caf1A). For periplasmic chaperone and subunit, +++ corresponds to approximately 20 to 40 μg/ml/OD 600 culture in the final osmotic shock fraction. l, location of subunit. Surface location, s, was identified by a positive reaction in the quantitative immunofluorescence assay, with only low levels present in the osmotic shock fraction; periplasmic location, p, was identified by a negative reaction (i.e., no higher than background, or

    Techniques Used: Recombinant, Mutagenesis, Plasmid Preparation, Immunofluorescence

    27) Product Images from "Screening of hepatocyte proteins binding with C-terminally truncated surface antigen middle protein of hepatitis B virus (MHBst167) by a yeast two-hybrid system"

    Article Title: Screening of hepatocyte proteins binding with C-terminally truncated surface antigen middle protein of hepatitis B virus (MHBst167) by a yeast two-hybrid system

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2014.2356

    Sequence of the pGEM-T-MHBst 167 by direct sequencing.
    Figure Legend Snippet: Sequence of the pGEM-T-MHBst 167 by direct sequencing.

    Techniques Used: Sequencing

    Construction and identification of plasmids by agarose gel electrophoresis. (A) Polymerase chain reaction product MHBst 167 (501 bp). (B) Restriction enzyme digestion of pGBKT7-MHBst 167 plasmids by Eco RI/ Bam HI (pGBKT7, 7,300 bp; MHBst 167 , 501 bp). (C) Restriction enzyme digestion of (Lane 1) pGBKT7-MHBst 167 and (Lane 2) pGADT7-MHBst 167 by Eco RI/ Bam HI. (D) Restriction enzyme digestion of pGEM-T-MHBst 167 plasmids by Eco RI/ Bam HI. M, DL2000 DNA marker; bp, base pairs.
    Figure Legend Snippet: Construction and identification of plasmids by agarose gel electrophoresis. (A) Polymerase chain reaction product MHBst 167 (501 bp). (B) Restriction enzyme digestion of pGBKT7-MHBst 167 plasmids by Eco RI/ Bam HI (pGBKT7, 7,300 bp; MHBst 167 , 501 bp). (C) Restriction enzyme digestion of (Lane 1) pGBKT7-MHBst 167 and (Lane 2) pGADT7-MHBst 167 by Eco RI/ Bam HI. (D) Restriction enzyme digestion of pGEM-T-MHBst 167 plasmids by Eco RI/ Bam HI. M, DL2000 DNA marker; bp, base pairs.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    28) Product Images from "Control of Filament Formation in Candida albicans by Polyamine Levels"

    Article Title: Control of Filament Formation in Candida albicans by Polyamine Levels

    Journal: Infection and Immunity

    doi:

    Restriction maps of the plasmids used in this work. pSGS3 contains a 2,217-bp Xba I fragment carrying the ODC gene of C. albicans (CaODC) in pGEM-T. pAHH1 is a plasmid containing the hisG-URA3-hisG cassette disrupting the ODC open reading frame. pAHH7 and pAHH8 are derivatives of pYPB1-ADHp7 constructed by insertion of the Candida ODC open reading frame under the control of the promoter of ADH1 and by insertion of the Candida ODC gene under the control of its own promoter (thus deleting the ADH promoter), respectively.
    Figure Legend Snippet: Restriction maps of the plasmids used in this work. pSGS3 contains a 2,217-bp Xba I fragment carrying the ODC gene of C. albicans (CaODC) in pGEM-T. pAHH1 is a plasmid containing the hisG-URA3-hisG cassette disrupting the ODC open reading frame. pAHH7 and pAHH8 are derivatives of pYPB1-ADHp7 constructed by insertion of the Candida ODC open reading frame under the control of the promoter of ADH1 and by insertion of the Candida ODC gene under the control of its own promoter (thus deleting the ADH promoter), respectively.

    Techniques Used: Plasmid Preparation, Construct

    29) Product Images from "Homologue of Macrophage-Activating Lipoprotein in Mycoplasmagallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures"

    Article Title: Homologue of Macrophage-Activating Lipoprotein in Mycoplasmagallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.8.2538-2547.2003

    Schematic diagram of the p47 - tetM gene construct in pGEM-T and clone C46. (A) Depiction of the vector pGEM-T containing the tetM gene ligated into the Spe I sites created in the p47 gene. (B) The location of the p47 - tetM gene construct within the M . gallisepticum strain ts-11 3.894-kbp Bgl II fragment is depicted. The cleavage sites for Bam HI, Bgl II, Hin dIII, Sal I, and Spe I and the locations of oligonucleotides TetFor and TetRev are shown. The positions of nucleotides present in the C46 DNA sequence, but absent from the S6 p47 gene sequence, are indicated by vertical lines.
    Figure Legend Snippet: Schematic diagram of the p47 - tetM gene construct in pGEM-T and clone C46. (A) Depiction of the vector pGEM-T containing the tetM gene ligated into the Spe I sites created in the p47 gene. (B) The location of the p47 - tetM gene construct within the M . gallisepticum strain ts-11 3.894-kbp Bgl II fragment is depicted. The cleavage sites for Bam HI, Bgl II, Hin dIII, Sal I, and Spe I and the locations of oligonucleotides TetFor and TetRev are shown. The positions of nucleotides present in the C46 DNA sequence, but absent from the S6 p47 gene sequence, are indicated by vertical lines.

    Techniques Used: Construct, Plasmid Preparation, Sequencing

    30) Product Images from "Characterization of Transcripts Expressed from Human Herpesvirus 6A Strain GS Immediate-Early Region B U16-U17 Open Reading Frames"

    Article Title: Characterization of Transcripts Expressed from Human Herpesvirus 6A Strain GS Immediate-Early Region B U16-U17 Open Reading Frames

    Journal: Journal of Virology

    doi:

    (A) Reactivity of monoclonal antibody against the B701 (U16) protein. Lane 1, reactivities of antibodies in the Western blot reaction with the B701 protein expressed in the pET-3b vector. The 20K protein and the higher-molecular-weight dimeric and/or multimeric forms of B701 protein recognized are indicated by the arrowheads. (B) SDS-PAGE analysis of in vitro-expressed polypeptides from the 1.8-kb cDNA insert. In vitro-synthesized mRNA, transcribed from the cDNA insert in the pGEM-T plasmid, was translated in vitro using rabbit reticulocyte lysate. Lanes 1, 4, and 7, translation reaction components without the addition of RNA. Lanes 2, 6, and 9, translation from RNA transcribed with SP6 RNA polymerase. Lanes 3, 5, and 8, translation from RNA transcribed with T7 RNA polymerase. Lanes 1 to 3, total translated products. Lanes 4 to 6, immunoprecipitations of translated products using a monoclonal antibody against the B701 (U16) protein. Lanes 7 to 9, immunoprecipitations of translation products using polyclonal rabbit antisera raised against the B701 protein. Samples were analyzed on 12% acrylamide cross-linked with bisacrylamide, and standard molecular mass markers were included in parallel lanes. The numbers indicate approximate molecular masses (in kilodaltons) of the prominent polypeptides identified.
    Figure Legend Snippet: (A) Reactivity of monoclonal antibody against the B701 (U16) protein. Lane 1, reactivities of antibodies in the Western blot reaction with the B701 protein expressed in the pET-3b vector. The 20K protein and the higher-molecular-weight dimeric and/or multimeric forms of B701 protein recognized are indicated by the arrowheads. (B) SDS-PAGE analysis of in vitro-expressed polypeptides from the 1.8-kb cDNA insert. In vitro-synthesized mRNA, transcribed from the cDNA insert in the pGEM-T plasmid, was translated in vitro using rabbit reticulocyte lysate. Lanes 1, 4, and 7, translation reaction components without the addition of RNA. Lanes 2, 6, and 9, translation from RNA transcribed with SP6 RNA polymerase. Lanes 3, 5, and 8, translation from RNA transcribed with T7 RNA polymerase. Lanes 1 to 3, total translated products. Lanes 4 to 6, immunoprecipitations of translated products using a monoclonal antibody against the B701 (U16) protein. Lanes 7 to 9, immunoprecipitations of translation products using polyclonal rabbit antisera raised against the B701 protein. Samples were analyzed on 12% acrylamide cross-linked with bisacrylamide, and standard molecular mass markers were included in parallel lanes. The numbers indicate approximate molecular masses (in kilodaltons) of the prominent polypeptides identified.

    Techniques Used: Western Blot, Positron Emission Tomography, Plasmid Preparation, Molecular Weight, SDS Page, In Vitro, Synthesized

    31) Product Images from "Novel Regulator MphX Represses Activation of Phenol Hydroxylase Genes Caused by a XylR/DmpR-Type Regulator MphR in Acinetobacter calcoaceticus"

    Article Title: Novel Regulator MphX Represses Activation of Phenol Hydroxylase Genes Caused by a XylR/DmpR-Type Regulator MphR in Acinetobacter calcoaceticus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017350

    Electrophoretic mobility shift assay. (A) Comparison of the upstream region of the mphK promoter with the corresponding regions of the mop gene cluster of A. calcoaceticus NCIB8250, the xyl upper gene cluster of Pseudomonas putida mt-2 (TOL), and the dmp gene cluster of Pseudomonas sp. CF600. Grey-shaded boxes show the putative upstream activating sequences (UASs) in the mph , mop , xyl , and dmp gene clusters to which the corresponding XylR/DmpR-type regulators bind. (B) gel retardation analysis of His-MphR binding to the mphR - mphK intergenic region. Gel-mobility shift assay was performed as described in the text using a 380-bp fragment (P380) covering the mphR - mphK intergenic region. Lane 1 is a control lane containing 2.5 nM of labeled P380 fragment only. Lane 2–7 contains 2.5 nM of the labeled P380 fragment incubated with 5, 10, 20, 30, 40 and 50 nM His-MphR protein. Lane 8 is containing 2.5 nM of labeled DNA fragment amplified using two primers, IRDye-labeled M13-F and M13-R, and plasmid pGEM T-vector as template, named P150. Lane 9–10 contains 2.5 nM of the labeled P150 fragment incubated with 30 and 50 nM His-MphR protein, respectively. The positions of the free DNA and the MphR-DNA complex are indicated. (C) SDS-PAGE of purified His-MphR protein from E. coli BL21 (pEMR). Lane 1: Molecular markers with their masses indicated; Lane 2: Uninduced of BL21 (pEMR); Lane 3: BL21 (pEMR) induced by 0.1 mM IPTG; Lane 4: BL21 (pEMR) induced by 0.5 mM IPTG; Lane 5: MphR purified by NTA resin and eluted by NTA-60; Lane 6: MphR purified by NTA resin and eluted by NTA-80.
    Figure Legend Snippet: Electrophoretic mobility shift assay. (A) Comparison of the upstream region of the mphK promoter with the corresponding regions of the mop gene cluster of A. calcoaceticus NCIB8250, the xyl upper gene cluster of Pseudomonas putida mt-2 (TOL), and the dmp gene cluster of Pseudomonas sp. CF600. Grey-shaded boxes show the putative upstream activating sequences (UASs) in the mph , mop , xyl , and dmp gene clusters to which the corresponding XylR/DmpR-type regulators bind. (B) gel retardation analysis of His-MphR binding to the mphR - mphK intergenic region. Gel-mobility shift assay was performed as described in the text using a 380-bp fragment (P380) covering the mphR - mphK intergenic region. Lane 1 is a control lane containing 2.5 nM of labeled P380 fragment only. Lane 2–7 contains 2.5 nM of the labeled P380 fragment incubated with 5, 10, 20, 30, 40 and 50 nM His-MphR protein. Lane 8 is containing 2.5 nM of labeled DNA fragment amplified using two primers, IRDye-labeled M13-F and M13-R, and plasmid pGEM T-vector as template, named P150. Lane 9–10 contains 2.5 nM of the labeled P150 fragment incubated with 30 and 50 nM His-MphR protein, respectively. The positions of the free DNA and the MphR-DNA complex are indicated. (C) SDS-PAGE of purified His-MphR protein from E. coli BL21 (pEMR). Lane 1: Molecular markers with their masses indicated; Lane 2: Uninduced of BL21 (pEMR); Lane 3: BL21 (pEMR) induced by 0.1 mM IPTG; Lane 4: BL21 (pEMR) induced by 0.5 mM IPTG; Lane 5: MphR purified by NTA resin and eluted by NTA-60; Lane 6: MphR purified by NTA resin and eluted by NTA-80.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Mobility Shift, Labeling, Incubation, Amplification, Plasmid Preparation, SDS Page, Purification

    32) Product Images from "Human glycolipid transfer protein (GLTP) genes: organization, transcriptional status and evolution"

    Article Title: Human glycolipid transfer protein (GLTP) genes: organization, transcriptional status and evolution

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-9-72

    Transcriptional Status of Human GLTP Genes . A . Agarose gel electrophoresis patterns of PCR products (37 cycles) obtained with 'universal GLTP primers' from different cells. Odd numbered lanes = RT-PCR; Even numbered lanes = No-RT PCR controls (PCR with no reverse transcriptase); Lanes 1 2 = glioma cells; Lanes 3 4 = human skin fibroblasts; Lanes 5 6 = human HBL100 breast cancer cells; Lanes 7 8 = Gaucher cells; Lanes 9 10 = human T47D breast cancer cells; Lanes 11 12 = human IMR32 neuroblastoma cells; Lanes 13 14 = human HTB126 breast cancer cells; Lanes 15 16 = human Caov3 ovarian cancer cells. β-actin transcription is shown in subtending panel at 30 PCR cycles. B . Agarose gel electrophoresis patterns obtained after Bsp HI restriction digestion of pGEM-T clones containing GLTP ORFs originating from 5-exon/4-intron GLTP (Lane 1) or from intronless GLTP (Lane 2). Std = molecular weight standards.
    Figure Legend Snippet: Transcriptional Status of Human GLTP Genes . A . Agarose gel electrophoresis patterns of PCR products (37 cycles) obtained with 'universal GLTP primers' from different cells. Odd numbered lanes = RT-PCR; Even numbered lanes = No-RT PCR controls (PCR with no reverse transcriptase); Lanes 1 2 = glioma cells; Lanes 3 4 = human skin fibroblasts; Lanes 5 6 = human HBL100 breast cancer cells; Lanes 7 8 = Gaucher cells; Lanes 9 10 = human T47D breast cancer cells; Lanes 11 12 = human IMR32 neuroblastoma cells; Lanes 13 14 = human HTB126 breast cancer cells; Lanes 15 16 = human Caov3 ovarian cancer cells. β-actin transcription is shown in subtending panel at 30 PCR cycles. B . Agarose gel electrophoresis patterns obtained after Bsp HI restriction digestion of pGEM-T clones containing GLTP ORFs originating from 5-exon/4-intron GLTP (Lane 1) or from intronless GLTP (Lane 2). Std = molecular weight standards.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Molecular Weight

    33) Product Images from "The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex"

    Article Title: The autoregulatory translational control element of poly(A)-binding protein mRNA forms a heteromeric ribonucleoprotein complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki1014

    Affinity chromatography of ARS RNA-binding proteins. In vitro synthesized ARS RNA was covalently linked to agarose beads and incubated with HeLa cell extract. The bound polypeptides were eluted, resolved on 10% SDS–PAGE and visualized by silver staining. Lane 1, pGEM-T and lane 2, ARS RNA bound protein fractions. The polypeptides specific for the ARS RNA affinity chromatography are shown by arrows. The polypeptides with bold arrows were seen in the UV crosslinking experiments.
    Figure Legend Snippet: Affinity chromatography of ARS RNA-binding proteins. In vitro synthesized ARS RNA was covalently linked to agarose beads and incubated with HeLa cell extract. The bound polypeptides were eluted, resolved on 10% SDS–PAGE and visualized by silver staining. Lane 1, pGEM-T and lane 2, ARS RNA bound protein fractions. The polypeptides specific for the ARS RNA affinity chromatography are shown by arrows. The polypeptides with bold arrows were seen in the UV crosslinking experiments.

    Techniques Used: Affinity Chromatography, RNA Binding Assay, In Vitro, Synthesized, Incubation, SDS Page, Silver Staining

    Formation of ARS RNA–protein complex. ( A ) RNA–protein crosslinking by UV. The in vitro synthesized [ 32 P]-labeled ARS RNA (≈3 ng, 3 × 10 5 c.p.m.) was incubated with different cell extract (≈60 µg protein) in the flat cap of a 0.2 ml PCR tube. Following the UV treatment, the samples were treated with RNaseA/RNase T1, analyzed by 10% SDS–PAGE and autoradiographed as described under experimental procedures. Cell extracts from mouse NIH3T3 fibroblasts (lane 2) and C2 myoblasts (lane 3), and human HEK293 (lane 4) and HeLa cells (lane 5) were used for these studies. One sample containing HeLa cell extract (lane 1) was analyzed without UV treatment as a control. Approximately 300 ng of the non-radioactive pGEM-T (lane 6), ARS (lane 7) or poly(A) 50 RNA (lane 8) was used for competition studies. ( B ) Analysis of RNP complex by REMSA. The in vitro synthesized [ 32 P]-labeled ARS RNA (≈1 ng, 1 × 10 5 c.p.m.) was incubated with different cell extracts (20 µg) as indicated above each lane. The unbound RNA was digested with RNaseT1 and subjected to electrophoresis in a 2% agarose as described in the experimental procedures. Lane 1, radioactive ARS RNA incubated without the cell extract; lanes 2–5, radioactive ARS RNA incubated with NIH3T3, C2, HEK293 and HeLa cell extracts, respectively. Lanes 6–8, competition with 100 ng of non-radioactive pGEM-T, ARS, or poly(A) 50 RNA, respectively. Lane 9, cell extract was pre-incubated with the PABP antibody and precleared with protein A-sepharose beads before being used for REMSA. Approximately 20 µg of PABP deficient cell extract was used for REMSA. Lane 10, cell extract was similarly treated with the GFP antibody (BD Biosciences) before being used for REMSA. Lane 11, radiolabeled ARS RNA (≈ 1 ng, 1 × 10 5 c.p.m.) incubated with ≈2 ng of purified 6×His-PABP. ( C ) Analysis of RNP complexes by SDS–PAGE. RNP complex formation was initiated as described above and the samples were irradiated by UV before being resolved in a 2% agarose gel. The RNP bands (as shown in Figure 1B ) were excised from the gel, treated with RNaseA/RNase T1, and analyzed by 10% SDS–PAGE as described in the Materials and Methods. Lane 1, ARS RNA and HeLa extracts treated with UV and analyzed before gel purification. Lane 2, polypeptides from the gel purified slower migrating ARC. Lane 3, polypeptides from the faster migrating minor complex.
    Figure Legend Snippet: Formation of ARS RNA–protein complex. ( A ) RNA–protein crosslinking by UV. The in vitro synthesized [ 32 P]-labeled ARS RNA (≈3 ng, 3 × 10 5 c.p.m.) was incubated with different cell extract (≈60 µg protein) in the flat cap of a 0.2 ml PCR tube. Following the UV treatment, the samples were treated with RNaseA/RNase T1, analyzed by 10% SDS–PAGE and autoradiographed as described under experimental procedures. Cell extracts from mouse NIH3T3 fibroblasts (lane 2) and C2 myoblasts (lane 3), and human HEK293 (lane 4) and HeLa cells (lane 5) were used for these studies. One sample containing HeLa cell extract (lane 1) was analyzed without UV treatment as a control. Approximately 300 ng of the non-radioactive pGEM-T (lane 6), ARS (lane 7) or poly(A) 50 RNA (lane 8) was used for competition studies. ( B ) Analysis of RNP complex by REMSA. The in vitro synthesized [ 32 P]-labeled ARS RNA (≈1 ng, 1 × 10 5 c.p.m.) was incubated with different cell extracts (20 µg) as indicated above each lane. The unbound RNA was digested with RNaseT1 and subjected to electrophoresis in a 2% agarose as described in the experimental procedures. Lane 1, radioactive ARS RNA incubated without the cell extract; lanes 2–5, radioactive ARS RNA incubated with NIH3T3, C2, HEK293 and HeLa cell extracts, respectively. Lanes 6–8, competition with 100 ng of non-radioactive pGEM-T, ARS, or poly(A) 50 RNA, respectively. Lane 9, cell extract was pre-incubated with the PABP antibody and precleared with protein A-sepharose beads before being used for REMSA. Approximately 20 µg of PABP deficient cell extract was used for REMSA. Lane 10, cell extract was similarly treated with the GFP antibody (BD Biosciences) before being used for REMSA. Lane 11, radiolabeled ARS RNA (≈ 1 ng, 1 × 10 5 c.p.m.) incubated with ≈2 ng of purified 6×His-PABP. ( C ) Analysis of RNP complexes by SDS–PAGE. RNP complex formation was initiated as described above and the samples were irradiated by UV before being resolved in a 2% agarose gel. The RNP bands (as shown in Figure 1B ) were excised from the gel, treated with RNaseA/RNase T1, and analyzed by 10% SDS–PAGE as described in the Materials and Methods. Lane 1, ARS RNA and HeLa extracts treated with UV and analyzed before gel purification. Lane 2, polypeptides from the gel purified slower migrating ARC. Lane 3, polypeptides from the faster migrating minor complex.

    Techniques Used: In Vitro, Synthesized, Labeling, Incubation, Polymerase Chain Reaction, SDS Page, Electrophoresis, Purification, Irradiation, Agarose Gel Electrophoresis, Gel Purification

    34) Product Images from "A forward genetic approach to mapping a P-element second site mutation identifies DCP2 as a novel tumor suppressor in Drosophila melanogaster"

    Article Title: A forward genetic approach to mapping a P-element second site mutation identifies DCP2 as a novel tumor suppressor in Drosophila melanogaster

    Journal: bioRxiv

    doi: 10.1101/2020.03.30.016865

    Gel electrophoretogram showing the PCR analysis of the full-length DCP2 (A) and candidate region (B) in the wild type, mutant and the heterozygote. The schematic in C shows the gene arrangement along the chromosome along with the important coordinates. The primers are indicated by red arrows. For amplification of the full – length gene, the wild type amplicon is of 8.6 kb while the mutant amplicon is sized ∼17 kb (A, upper half), whereas the wild type amplicon for the candidate region is of 945 bp while the mutant amplicon is sized ∼8.5 kb (B, upper half). The heterozygote harbors both the alleles (wild type and mutant) and thus shows both the amplicons. The lower half in both A and B shows the the blot of the same probed with the pGEM-T-812 probe which spans the candidate mutated region in DCP2 and is represented by the purple line. D shows the gel electrophoretogram and Southern blot of DCP2 in the wild type and mutant genome. Hin dIII digested genomic DNA showed banding at ∼ 2.1 kb in the wild type genome as against ∼ 10 kb in the mutant genome, the size difference being almost in agreement with the banding profile exemplified by Bam HI digestion, with the wild type genome hybridizing at ∼ 10.2 kb and the mutant at ∼ 18 kb.
    Figure Legend Snippet: Gel electrophoretogram showing the PCR analysis of the full-length DCP2 (A) and candidate region (B) in the wild type, mutant and the heterozygote. The schematic in C shows the gene arrangement along the chromosome along with the important coordinates. The primers are indicated by red arrows. For amplification of the full – length gene, the wild type amplicon is of 8.6 kb while the mutant amplicon is sized ∼17 kb (A, upper half), whereas the wild type amplicon for the candidate region is of 945 bp while the mutant amplicon is sized ∼8.5 kb (B, upper half). The heterozygote harbors both the alleles (wild type and mutant) and thus shows both the amplicons. The lower half in both A and B shows the the blot of the same probed with the pGEM-T-812 probe which spans the candidate mutated region in DCP2 and is represented by the purple line. D shows the gel electrophoretogram and Southern blot of DCP2 in the wild type and mutant genome. Hin dIII digested genomic DNA showed banding at ∼ 2.1 kb in the wild type genome as against ∼ 10 kb in the mutant genome, the size difference being almost in agreement with the banding profile exemplified by Bam HI digestion, with the wild type genome hybridizing at ∼ 10.2 kb and the mutant at ∼ 18 kb.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Amplification, Southern Blot

    35) Product Images from "A Helicobacter hepaticus catalase mutant is hypersensitive to oxidative stress and suffers increased DNA damage"

    Article Title: A Helicobacter hepaticus catalase mutant is hypersensitive to oxidative stress and suffers increased DNA damage

    Journal:

    doi: 10.1099/jmm.0.46891-0

    (a) Organization of the katA gene in the H. hepaticus genome and DNA constructs for generating the katA mutant. A 1200 bp fragment of the katA gene was amplified using the katAF/katAR primer pair and cloned into pGEM-T. A Cm R cassette was then inserted
    Figure Legend Snippet: (a) Organization of the katA gene in the H. hepaticus genome and DNA constructs for generating the katA mutant. A 1200 bp fragment of the katA gene was amplified using the katAF/katAR primer pair and cloned into pGEM-T. A Cm R cassette was then inserted

    Techniques Used: Construct, Mutagenesis, Amplification, Clone Assay

    36) Product Images from "FGFR2 molecular analysis and related clinical findings in one Chinese family with Crouzon Syndrome"

    Article Title: FGFR2 molecular analysis and related clinical findings in one Chinese family with Crouzon Syndrome

    Journal: Molecular Vision

    doi:

    DNA sequence of a part of FGF2 in the affected patients and unaffected individuals. A : A mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. B : Sequence of the normal allele of exon 8 subcloned into the pGEM-T vector used as a control. C : A heterozygous missense mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. The mutation causes the glutarnine 289 codon (CAG) to change to a proline codon (CCG).
    Figure Legend Snippet: DNA sequence of a part of FGF2 in the affected patients and unaffected individuals. A : A mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. B : Sequence of the normal allele of exon 8 subcloned into the pGEM-T vector used as a control. C : A heterozygous missense mutation c.866A > C (Gln289Pro) in exon 8 in the affected individuals. The mutation causes the glutarnine 289 codon (CAG) to change to a proline codon (CCG).

    Techniques Used: Sequencing, Mutagenesis, Plasmid Preparation

    37) Product Images from "Inactivation of an ABC Transporter Gene, mcyH, Results in Loss of Microcystin Production in the Cyanobacterium Microcystis aeruginosa PCC 7806"

    Article Title: Inactivation of an ABC Transporter Gene, mcyH, Results in Loss of Microcystin Production in the Cyanobacterium Microcystis aeruginosa PCC 7806

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.11.6370-6378.2004

    (A) Construction of plasmid p5K8. A diagram illustrating the region of the mcy cluster amplified by primers mcyH SKF and mcyH SKR and cloned into pGEM-T to give rise to plasmid p5K8 is shown. (B) Construction of plasmids p5K8BsmI-3 and p5K8BsmI-2. A schematic diagram depicting the partial deletion of mcyH in p5K8, resulting in the knockout constructs p5K8BsmI-3 and p5K8BsmI-2, is shown. The directions of transcription of the mcyH and Cm r genes are indicated by (−/−) and (−/+), respectively.
    Figure Legend Snippet: (A) Construction of plasmid p5K8. A diagram illustrating the region of the mcy cluster amplified by primers mcyH SKF and mcyH SKR and cloned into pGEM-T to give rise to plasmid p5K8 is shown. (B) Construction of plasmids p5K8BsmI-3 and p5K8BsmI-2. A schematic diagram depicting the partial deletion of mcyH in p5K8, resulting in the knockout constructs p5K8BsmI-3 and p5K8BsmI-2, is shown. The directions of transcription of the mcyH and Cm r genes are indicated by (−/−) and (−/+), respectively.

    Techniques Used: Plasmid Preparation, Amplification, Clone Assay, Knock-Out, Construct

    38) Product Images from "Isolation and Characterization of a Mn(II)-Oxidizing Bacillus Strain from the Demosponge Suberites domuncula"

    Article Title: Isolation and Characterization of a Mn(II)-Oxidizing Bacillus Strain from the Demosponge Suberites domuncula

    Journal: Marine Drugs

    doi: 10.3390/md9010001

    Expression of mnxG gene in BAC-SubDo-03, grown, in absence of Mn (minus), or presence of Mn (plus). As described under “Experimental Section”, and schematically outlined in Figure 2C , the cultures were incubated for 4 hrs, 16 hrs, and 32 hrs ( lanes 1 to 3 ) in the absence of Mn; or, the microorganisms were cultivated in the absence of Mn and then exposed for 0.5 hr, 1 hr, 2 hr or 18 hrs to 100 μm MnCl 2 ( lanes 4 to 7 ). The same quantity of bacteria was taken after the indicated period of incubation. Then, RNA was extracted and the level of transcripts was semi-quantitatively determined by RT-PCR. The molecular size markers (M) are shown in lane 8 . In parallel, amplifications by RT-PCR in an assay supplemented with the mnxG-SubDo-03 containing plasmid (positive control [pc]), or with an empty pGEM-T vector (negative control [nc]). This PCR gel analysis is representative for five separate analyses performed.
    Figure Legend Snippet: Expression of mnxG gene in BAC-SubDo-03, grown, in absence of Mn (minus), or presence of Mn (plus). As described under “Experimental Section”, and schematically outlined in Figure 2C , the cultures were incubated for 4 hrs, 16 hrs, and 32 hrs ( lanes 1 to 3 ) in the absence of Mn; or, the microorganisms were cultivated in the absence of Mn and then exposed for 0.5 hr, 1 hr, 2 hr or 18 hrs to 100 μm MnCl 2 ( lanes 4 to 7 ). The same quantity of bacteria was taken after the indicated period of incubation. Then, RNA was extracted and the level of transcripts was semi-quantitatively determined by RT-PCR. The molecular size markers (M) are shown in lane 8 . In parallel, amplifications by RT-PCR in an assay supplemented with the mnxG-SubDo-03 containing plasmid (positive control [pc]), or with an empty pGEM-T vector (negative control [nc]). This PCR gel analysis is representative for five separate analyses performed.

    Techniques Used: Expressing, BAC Assay, Incubation, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Negative Control, Polymerase Chain Reaction

    39) Product Images from "Role of nucleostemin in growth regulation of gastric cancer, liver cancer and other malignancies"

    Article Title: Role of nucleostemin in growth regulation of gastric cancer, liver cancer and other malignancies

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v10.i9.1246

    Molecular cloning of nucleostemin cDNA fragment. 1, DNA marker: λDNA, Eco RI + Hi nd III; 2, pGEM-T-NS, Bam HI + Hin d III.
    Figure Legend Snippet: Molecular cloning of nucleostemin cDNA fragment. 1, DNA marker: λDNA, Eco RI + Hi nd III; 2, pGEM-T-NS, Bam HI + Hin d III.

    Techniques Used: Molecular Cloning, Marker

    40) Product Images from "Two Alternative Promoters Direct Neuron-Specific Expression of the Rat Microtubule-Associated Protein 1B Gene"

    Article Title: Two Alternative Promoters Direct Neuron-Specific Expression of the Rat Microtubule-Associated Protein 1B Gene

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.16-16-05026.1996

    Determination of MAP1B gene transcription initiation sites by RNase protection assay. In the RNase protection assay ( A ), 20 μg of total RNA from adult and postnatal day 4 rat DRG ( lanes 1 , 2 ), adult and postnatal day 4 rat brain cortex ( lanes 3 , 4 ), adult and postnatal day 4 rat liver ( lanes 5 , 6 ), PC12 ( lane 7 ), NB2A ( lane 8 ), 3T3 ( lane 9 ), and L6 ( lane 10 ) cells were hybridized with 32 P-labeled riboprobe spanning nt −383 to +100 in the MAP1B gene and 52 nt of the pGEM-T vector sequence, and then treated with a mixture of RNase A and RNase T1. The protected fragments and their size (nt) are indicated by arrowheads . The size of molecular weight markers (nt) is indicated to the left . A diagram ( B ) shows the structure of the MAP1B gene antisense riboprobe and the three protected fragments.
    Figure Legend Snippet: Determination of MAP1B gene transcription initiation sites by RNase protection assay. In the RNase protection assay ( A ), 20 μg of total RNA from adult and postnatal day 4 rat DRG ( lanes 1 , 2 ), adult and postnatal day 4 rat brain cortex ( lanes 3 , 4 ), adult and postnatal day 4 rat liver ( lanes 5 , 6 ), PC12 ( lane 7 ), NB2A ( lane 8 ), 3T3 ( lane 9 ), and L6 ( lane 10 ) cells were hybridized with 32 P-labeled riboprobe spanning nt −383 to +100 in the MAP1B gene and 52 nt of the pGEM-T vector sequence, and then treated with a mixture of RNase A and RNase T1. The protected fragments and their size (nt) are indicated by arrowheads . The size of molecular weight markers (nt) is indicated to the left . A diagram ( B ) shows the structure of the MAP1B gene antisense riboprobe and the three protected fragments.

    Techniques Used: Rnase Protection Assay, Labeling, Plasmid Preparation, Sequencing, Molecular Weight

    Related Articles

    Clone Assay:

    Article Title: Chromosome-Based Genetic Complementation System for Xylella fastidiosa ▿
    Article Snippet: .. The resulting 1.6-kb fragment was then cloned into the pGEM-T vector (Promega), creating plasmid pAX1. .. PCR was also used to generate DNA fragments carrying the multiple cloning sites and the four antibiotic resistance cassettes that were inserted into pAX1.

    Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina
    Article Snippet: .. PCR product about 1.2 kb long was gel-purified by electrophoresis and cloned into pGEM-T vector. .. Positive clones were selected on LB plate by color reaction.

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
    Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

    Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing
    Article Snippet: .. To improve the specificity of amplification in order to generate different length variants, the following touchdown PCR program ( ) was used: 95°C for 2 min, 5 cycles of 95°C for 30 s, 70°C for 30 s and 68°C for 30 s, then 5 cycles of 95°C for 30 s, 68°C for 30 s and 68°C for 30 s, and then 5 cycles of 95°C for 30 s, 66°C for 30 s and 68°C for 30 s, followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 68°C for 30 s and a final extension step at 68°C for 5 min. All PCR fragments, ranging from 96 to 437 were separated on agarose gel, purified and cloned into a pGEM-T vector (Promega, WI, USA). .. The recombinant plasmid DNAs were digested, purified and sub-cloned into the EcoR I and Not I sites of pcDNA 3.1 minigene Del-m ( , inset).

    Article Title: Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts
    Article Snippet: .. The amplified fragments were cloned into a pGEM-T vector using the pGEM-T Vector System (Promega). .. From these cloned plasmids, RNA substrates for in vitro editing and UV-crosslinking were prepared as previously described ( ) with slight modifications.

    Article Title: A gene encoding a protein modified by the phytohormone indoleacetic acid
    Article Snippet: .. The iap1 cDNA was cloned into a pGEM-T vector and was used for coupled in vitro transcription and translation using the TnT Quick Coupled System (Promega) with [35 S]methionine. .. GeneRacer Kit (Invitrogen) was used for full-length, RNA ligase-mediated rapid amplification of 5′ ends using RNA isolated 24 days after flowering.

    Amplification:

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
    Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

    Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing
    Article Snippet: .. To improve the specificity of amplification in order to generate different length variants, the following touchdown PCR program ( ) was used: 95°C for 2 min, 5 cycles of 95°C for 30 s, 70°C for 30 s and 68°C for 30 s, then 5 cycles of 95°C for 30 s, 68°C for 30 s and 68°C for 30 s, and then 5 cycles of 95°C for 30 s, 66°C for 30 s and 68°C for 30 s, followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 68°C for 30 s and a final extension step at 68°C for 5 min. All PCR fragments, ranging from 96 to 437 were separated on agarose gel, purified and cloned into a pGEM-T vector (Promega, WI, USA). .. The recombinant plasmid DNAs were digested, purified and sub-cloned into the EcoR I and Not I sites of pcDNA 3.1 minigene Del-m ( , inset).

    Article Title: Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts
    Article Snippet: .. The amplified fragments were cloned into a pGEM-T vector using the pGEM-T Vector System (Promega). .. From these cloned plasmids, RNA substrates for in vitro editing and UV-crosslinking were prepared as previously described ( ) with slight modifications.

    Agarose Gel Electrophoresis:

    Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing
    Article Snippet: .. To improve the specificity of amplification in order to generate different length variants, the following touchdown PCR program ( ) was used: 95°C for 2 min, 5 cycles of 95°C for 30 s, 70°C for 30 s and 68°C for 30 s, then 5 cycles of 95°C for 30 s, 68°C for 30 s and 68°C for 30 s, and then 5 cycles of 95°C for 30 s, 66°C for 30 s and 68°C for 30 s, followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 68°C for 30 s and a final extension step at 68°C for 5 min. All PCR fragments, ranging from 96 to 437 were separated on agarose gel, purified and cloned into a pGEM-T vector (Promega, WI, USA). .. The recombinant plasmid DNAs were digested, purified and sub-cloned into the EcoR I and Not I sites of pcDNA 3.1 minigene Del-m ( , inset).

    In Vitro:

    Article Title: A gene encoding a protein modified by the phytohormone indoleacetic acid
    Article Snippet: .. The iap1 cDNA was cloned into a pGEM-T vector and was used for coupled in vitro transcription and translation using the TnT Quick Coupled System (Promega) with [35 S]methionine. .. GeneRacer Kit (Invitrogen) was used for full-length, RNA ligase-mediated rapid amplification of 5′ ends using RNA isolated 24 days after flowering.

    Purification:

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
    Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

    Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing
    Article Snippet: .. To improve the specificity of amplification in order to generate different length variants, the following touchdown PCR program ( ) was used: 95°C for 2 min, 5 cycles of 95°C for 30 s, 70°C for 30 s and 68°C for 30 s, then 5 cycles of 95°C for 30 s, 68°C for 30 s and 68°C for 30 s, and then 5 cycles of 95°C for 30 s, 66°C for 30 s and 68°C for 30 s, followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 68°C for 30 s and a final extension step at 68°C for 5 min. All PCR fragments, ranging from 96 to 437 were separated on agarose gel, purified and cloned into a pGEM-T vector (Promega, WI, USA). .. The recombinant plasmid DNAs were digested, purified and sub-cloned into the EcoR I and Not I sites of pcDNA 3.1 minigene Del-m ( , inset).

    Electrophoresis:

    Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina
    Article Snippet: .. PCR product about 1.2 kb long was gel-purified by electrophoresis and cloned into pGEM-T vector. .. Positive clones were selected on LB plate by color reaction.

    Polymerase Chain Reaction:

    Article Title: Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose
    Article Snippet: .. The 5′ UP nth1 -kanMX-3′ DOWN nth1 , obtained from the third round of PCR, was ligated with pGEMT vector (Promega Corporation, Madison, WI, USA) and transformed into CaCl2 -treated E. coli TOP10F’ according to Hanahan and Meselson. .. The transformed host was screened by blue/white selection.

    Article Title: Octamer binding protein 2 (Oct2) regulates PD-L2 gene expression in B-1 cells through lineage-specific activity of a unique, intronic promoter
    Article Snippet: .. After checking the band pattern, PCR products were TA-cloned into pGEM-T vector (Promega, Madison, WI, USA). ..

    Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina
    Article Snippet: .. PCR product about 1.2 kb long was gel-purified by electrophoresis and cloned into pGEM-T vector. .. Positive clones were selected on LB plate by color reaction.

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
    Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

    Touchdown PCR:

    Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing
    Article Snippet: .. To improve the specificity of amplification in order to generate different length variants, the following touchdown PCR program ( ) was used: 95°C for 2 min, 5 cycles of 95°C for 30 s, 70°C for 30 s and 68°C for 30 s, then 5 cycles of 95°C for 30 s, 68°C for 30 s and 68°C for 30 s, and then 5 cycles of 95°C for 30 s, 66°C for 30 s and 68°C for 30 s, followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 68°C for 30 s and a final extension step at 68°C for 5 min. All PCR fragments, ranging from 96 to 437 were separated on agarose gel, purified and cloned into a pGEM-T vector (Promega, WI, USA). .. The recombinant plasmid DNAs were digested, purified and sub-cloned into the EcoR I and Not I sites of pcDNA 3.1 minigene Del-m ( , inset).

    Sequencing:

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
    Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

    Transformation Assay:

    Article Title: Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose
    Article Snippet: .. The 5′ UP nth1 -kanMX-3′ DOWN nth1 , obtained from the third round of PCR, was ligated with pGEMT vector (Promega Corporation, Madison, WI, USA) and transformed into CaCl2 -treated E. coli TOP10F’ according to Hanahan and Meselson. .. The transformed host was screened by blue/white selection.

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
    Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

    Plasmid Preparation:

    Article Title: Chromosome-Based Genetic Complementation System for Xylella fastidiosa ▿
    Article Snippet: .. The resulting 1.6-kb fragment was then cloned into the pGEM-T vector (Promega), creating plasmid pAX1. .. PCR was also used to generate DNA fragments carrying the multiple cloning sites and the four antibiotic resistance cassettes that were inserted into pAX1.

    Article Title: Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose
    Article Snippet: .. The 5′ UP nth1 -kanMX-3′ DOWN nth1 , obtained from the third round of PCR, was ligated with pGEMT vector (Promega Corporation, Madison, WI, USA) and transformed into CaCl2 -treated E. coli TOP10F’ according to Hanahan and Meselson. .. The transformed host was screened by blue/white selection.

    Article Title: Octamer binding protein 2 (Oct2) regulates PD-L2 gene expression in B-1 cells through lineage-specific activity of a unique, intronic promoter
    Article Snippet: .. After checking the band pattern, PCR products were TA-cloned into pGEM-T vector (Promega, Madison, WI, USA). ..

    Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina
    Article Snippet: .. PCR product about 1.2 kb long was gel-purified by electrophoresis and cloned into pGEM-T vector. .. Positive clones were selected on LB plate by color reaction.

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale
    Article Snippet: .. In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock. .. Positive colonies were selected by blue/white screening followed by colony PCR using M13F/R primers under the following conditions: 5 min at 95°C and 35 cycles of 60 s at 95°C, 60 s at 51°C, and 1 min and 20 s at 72°C and a final extension of 5 min at 72°C.

    Article Title: Two RNA editing sites with cis-acting elements of moderate sequence identity are recognized by an identical site-recognition protein in tobacco chloroplasts
    Article Snippet: .. The amplified fragments were cloned into a pGEM-T vector using the pGEM-T Vector System (Promega). .. From these cloned plasmids, RNA substrates for in vitro editing and UV-crosslinking were prepared as previously described ( ) with slight modifications.

    Article Title: A gene encoding a protein modified by the phytohormone indoleacetic acid
    Article Snippet: .. The iap1 cDNA was cloned into a pGEM-T vector and was used for coupled in vitro transcription and translation using the TnT Quick Coupled System (Promega) with [35 S]methionine. .. GeneRacer Kit (Invitrogen) was used for full-length, RNA ligase-mediated rapid amplification of 5′ ends using RNA isolated 24 days after flowering.

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  • 93
    Promega pgem t easy cloning vector
    (A) Southern blotting of EcoRI-digested total DNA from FD-C-infected and FD-D-infected and healthy (H) periwinkles probed with a DIG-labeled malG gene amplicon obtained through <t>PCR</t> driven by primer pair malG _F/ malG _R (C+, probe positive control represented by <t>pGEM-T-</t> malG1 plasmid). (B) Electrophoresis separation of amplicons obtained following PCR of total DNA from FD-C-infected and FD-D-infected periwinkles with copy-specific primer pairs (002 and 005), according to the draft genome of FD92, and from healthy periwinkle. *, nonspecific PCR product.
    Pgem T Easy Cloning Vector, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgem t easy cloning vector/product/Promega
    Average 93 stars, based on 282 article reviews
    Price from $9.99 to $1999.99
    pgem t easy cloning vector - by Bioz Stars, 2020-08
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    99
    Promega pgem t vector
    Identification of the recombinant plasmid, pTMICL12 by restriction enzyme. 1: <t>pGEM-T/EcoRI/XhoI;</t> 2: DNA size marker λ DNA/BstII; 3: pTMICL12/EcoRI/XhoI; 4: PCR product of D12D DNA.
    Pgem T Vector, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 2577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgem t vector/product/Promega
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    (A) Southern blotting of EcoRI-digested total DNA from FD-C-infected and FD-D-infected and healthy (H) periwinkles probed with a DIG-labeled malG gene amplicon obtained through PCR driven by primer pair malG _F/ malG _R (C+, probe positive control represented by pGEM-T- malG1 plasmid). (B) Electrophoresis separation of amplicons obtained following PCR of total DNA from FD-C-infected and FD-D-infected periwinkles with copy-specific primer pairs (002 and 005), according to the draft genome of FD92, and from healthy periwinkle. *, nonspecific PCR product.

    Journal: Applied and Environmental Microbiology

    Article Title: Genetic Diversity of Flavescence Dorée Phytoplasmas at the Vineyard Scale

    doi: 10.1128/AEM.03123-18

    Figure Lengend Snippet: (A) Southern blotting of EcoRI-digested total DNA from FD-C-infected and FD-D-infected and healthy (H) periwinkles probed with a DIG-labeled malG gene amplicon obtained through PCR driven by primer pair malG _F/ malG _R (C+, probe positive control represented by pGEM-T- malG1 plasmid). (B) Electrophoresis separation of amplicons obtained following PCR of total DNA from FD-C-infected and FD-D-infected periwinkles with copy-specific primer pairs (002 and 005), according to the draft genome of FD92, and from healthy periwinkle. *, nonspecific PCR product.

    Article Snippet: In the case of mixed infections (presence of double peaks in the analyzed pherograms from the sequencing of the original PCR amplicon), purified PCR products were ligated into pGEM-T easy cloning vector following the manufacturer's instructions (pGEM-T clone kit; Promega, Madison, WI) and transformed into Escherichia coli DH5α competent cells by heat shock.

    Techniques: Southern Blot, Infection, Labeling, Amplification, Polymerase Chain Reaction, Positive Control, Plasmid Preparation, Electrophoresis

    Identification of the recombinant plasmid, pTMICL12 by restriction enzyme. 1: pGEM-T/EcoRI/XhoI; 2: DNA size marker λ DNA/BstII; 3: pTMICL12/EcoRI/XhoI; 4: PCR product of D12D DNA.

    Journal:

    Article Title: Cloning and molecular characterization of Δ12-fatty acid desaturase gene from Mortierella isabellina

    doi: 10.3748/wjg.v12.i21.3373

    Figure Lengend Snippet: Identification of the recombinant plasmid, pTMICL12 by restriction enzyme. 1: pGEM-T/EcoRI/XhoI; 2: DNA size marker λ DNA/BstII; 3: pTMICL12/EcoRI/XhoI; 4: PCR product of D12D DNA.

    Article Snippet: PCR product about 1.2 kb long was gel-purified by electrophoresis and cloned into pGEM-T vector.

    Techniques: Recombinant, Plasmid Preparation, Marker, Polymerase Chain Reaction

    WT intron 4 pGEM-T construct

    Journal: Journal of molecular biochemistry

    Article Title: Motifs within the CA-repeat-rich region of Surfactant Protein B (SFTPB) intron 4 differentially affect mRNA splicing

    doi:

    Figure Lengend Snippet: WT intron 4 pGEM-T construct

    Article Snippet: To improve the specificity of amplification in order to generate different length variants, the following touchdown PCR program ( ) was used: 95°C for 2 min, 5 cycles of 95°C for 30 s, 70°C for 30 s and 68°C for 30 s, then 5 cycles of 95°C for 30 s, 68°C for 30 s and 68°C for 30 s, and then 5 cycles of 95°C for 30 s, 66°C for 30 s and 68°C for 30 s, followed by 25 cycles of 94°C for 30 s, 64°C for 30 s and 68°C for 30 s and a final extension step at 68°C for 5 min. All PCR fragments, ranging from 96 to 437 were separated on agarose gel, purified and cloned into a pGEM-T vector (Promega, WI, USA).

    Techniques: Construct

    Disruption of BBA74 by insertion of kanamycin resistance cassette. (A) Schematic diagram of mutant construct. The region of lp54 between BBA73 and BBA76 was amplified by PCR using primers p73F/p76R and cloned into pGEM-T. The insertion of the kanamycin

    Journal:

    Article Title: Comparative Transcriptional Profiling of Borrelia burgdorferi Clinical Isolates Differing in Capacities for Hematogenous Dissemination

    doi: 10.1128/IAI.73.10.6791-6802.2005

    Figure Lengend Snippet: Disruption of BBA74 by insertion of kanamycin resistance cassette. (A) Schematic diagram of mutant construct. The region of lp54 between BBA73 and BBA76 was amplified by PCR using primers p73F/p76R and cloned into pGEM-T. The insertion of the kanamycin

    Article Snippet: A 2,327-bp DNA fragment spanning BBA73 to BBA76 (nucleotides 50601 to 52908 on plasmid lp54; GenBank accession number, ) was amplified by PCR using primers pA73F and pA76R (Table ) as forward and reverse primers, respectively, and cloned into the plasmid vector pGEM-T (Promega).

    Techniques: Mutagenesis, Construct, Amplification, Polymerase Chain Reaction, Clone Assay