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    XhoI
    XhoI 25 000 units
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    Average 99 stars, based on 321 article reviews
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    Images

    1) Product Images from "Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers"

    Article Title: Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-8-23

    Structure of the DNA molecules used in this study. A . Structure of the parent c-DNA. B . l-DNA formed by restriction digest of c-DNA with the enzyme Xho I. The size of the linearized plasmid was 4272 bp, C . pcr-DNA (2209 bp) generated by site-specific primers that amplified the region of the c-DNA containing the HTLV promoter, the hOPG open reading frame, and the SV40 Poly-A site. D . Structure of the parent c-DNA used in transfection. E . l-DNA formed by digest of pEGFP-N2 with the enzyme Cla I, which cuts in the vector backbone. F . pcr-DNA (1713 bp) generated by primers that are complementary to the sequence upstream of the CMV promoter and downstream of the polyA site.
    Figure Legend Snippet: Structure of the DNA molecules used in this study. A . Structure of the parent c-DNA. B . l-DNA formed by restriction digest of c-DNA with the enzyme Xho I. The size of the linearized plasmid was 4272 bp, C . pcr-DNA (2209 bp) generated by site-specific primers that amplified the region of the c-DNA containing the HTLV promoter, the hOPG open reading frame, and the SV40 Poly-A site. D . Structure of the parent c-DNA used in transfection. E . l-DNA formed by digest of pEGFP-N2 with the enzyme Cla I, which cuts in the vector backbone. F . pcr-DNA (1713 bp) generated by primers that are complementary to the sequence upstream of the CMV promoter and downstream of the polyA site.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Generated, Amplification, Transfection, Sequencing

    2) Product Images from "Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site"

    Article Title: Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site

    Journal: BMC Research Notes

    doi: 10.1186/s13104-017-2631-8

    Xho I digests of miniprep DNA. Each DNA sample was digested with Xho I and electrophoresed on a 0.8% agarose gel. Lanes 1–7 represent various transformants. Lane 8 is a 1 kb ladder
    Figure Legend Snippet: Xho I digests of miniprep DNA. Each DNA sample was digested with Xho I and electrophoresed on a 0.8% agarose gel. Lanes 1–7 represent various transformants. Lane 8 is a 1 kb ladder

    Techniques Used: Agarose Gel Electrophoresis

    Design of the mutant loxP oligonucleotide. The 13 bp inverted repeats are underlined . The mutated bases in the 8 bp central spacer are shown in shadowed grey font . The Xho I site is also indicated
    Figure Legend Snippet: Design of the mutant loxP oligonucleotide. The 13 bp inverted repeats are underlined . The mutated bases in the 8 bp central spacer are shown in shadowed grey font . The Xho I site is also indicated

    Techniques Used: Mutagenesis

    3) Product Images from "A partial form of recessive STAT1 deficiency in humans"

    Article Title: A partial form of recessive STAT1 deficiency in humans

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI37083

    STAT1 P696S would be associated with an ESE defect. ( A ). Nucleotides from exon 23 are shown, with the C2086 nucleotide in the WT sequence and the C2086T substitution in the P696S sequence shown in red. The horizontal blue and green bars show the significance threshold homology score for the binding of SC35 and SRp40 proteins, respectively. The predicted binding sites of these proteins are shown as rectangles along the length of the nucleotide sequence at the height of the homology score. ( B ) The genomic STAT1 region from nucleotide 36989 to 38523 (NC_000002) was inserted into an exon-trapping vector using Xho I and BamH 1, with or without the C2086T (P696S) nucleotide substitution. The exons are numbered in Roman numerals and shown in gray boxes, with the introns between them in white boxes, with the exception of exon 23, which is shown in a red box. The vector is shown as black boxes. HEK293 and COS-7 cells were transfected with the various constructs, the exon-trapping pSPL3 mock vector (pmock-p), or no vector (–). RNA was isolated, and the various spliced products were amplified and are shown on an agarose gel with GADPH amplification. The various products were isolated and sequenced, and the resulting sequences are also shown with corresponding exons and MW. These results are representative of 2 independent experiments.
    Figure Legend Snippet: STAT1 P696S would be associated with an ESE defect. ( A ). Nucleotides from exon 23 are shown, with the C2086 nucleotide in the WT sequence and the C2086T substitution in the P696S sequence shown in red. The horizontal blue and green bars show the significance threshold homology score for the binding of SC35 and SRp40 proteins, respectively. The predicted binding sites of these proteins are shown as rectangles along the length of the nucleotide sequence at the height of the homology score. ( B ) The genomic STAT1 region from nucleotide 36989 to 38523 (NC_000002) was inserted into an exon-trapping vector using Xho I and BamH 1, with or without the C2086T (P696S) nucleotide substitution. The exons are numbered in Roman numerals and shown in gray boxes, with the introns between them in white boxes, with the exception of exon 23, which is shown in a red box. The vector is shown as black boxes. HEK293 and COS-7 cells were transfected with the various constructs, the exon-trapping pSPL3 mock vector (pmock-p), or no vector (–). RNA was isolated, and the various spliced products were amplified and are shown on an agarose gel with GADPH amplification. The various products were isolated and sequenced, and the resulting sequences are also shown with corresponding exons and MW. These results are representative of 2 independent experiments.

    Techniques Used: Sequencing, Binding Assay, Plasmid Preparation, Transfection, Construct, Isolation, Amplification, Agarose Gel Electrophoresis

    4) Product Images from "A Germination-Specific Endo-?-Mannanase Gene Is Expressed in the Micropylar Endosperm Cap of Tomato Seeds 1"

    Article Title: A Germination-Specific Endo-?-Mannanase Gene Is Expressed in the Micropylar Endosperm Cap of Tomato Seeds 1

    Journal: Plant Physiology

    doi:

    Southern blots of tomato genomic DNA hybridized with full-length cDNAs of LeMAN1 (left) and LeMAN2 (right). Genomic DNA (10 μg) isolated from tomato leaves was digested with Bam HI ( Bam ), Xba I ( Xba ), and Xho I ( Xho ). Bands marked with arrows hybridized more strongly with LeMAN1 , whereas the band marked with an arrowhead hybridized more strongly to LeMAN2 (see text for details).
    Figure Legend Snippet: Southern blots of tomato genomic DNA hybridized with full-length cDNAs of LeMAN1 (left) and LeMAN2 (right). Genomic DNA (10 μg) isolated from tomato leaves was digested with Bam HI ( Bam ), Xba I ( Xba ), and Xho I ( Xho ). Bands marked with arrows hybridized more strongly with LeMAN1 , whereas the band marked with an arrowhead hybridized more strongly to LeMAN2 (see text for details).

    Techniques Used: Isolation

    5) Product Images from "Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection"

    Article Title: Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2008.0792

    Strategy to generate insertion vector for rapid selection of rVVs. ( A ) The sequence of the mutated HIV clade C env gene, termed envC , was obtained by PCR (blue box) and cloned into the Xho I and Bam HI sites of the vector pIRES2-GFP. In the second step, the sequence of the expression cassette envC –IRES–GFP was PCR amplified and cloned into vector pCS59 (sites Xho I– Eco RI) under the control of strong synthetic early/late VV promoter (red box). Gray boxes, flanking sequences of the tk gene used for targeting the insertion vector and thereby disrupt the normal VV tk gene. As a consequence, rVVs containing the desired insert are no longer able to express thymidine kinase, and thus cells infected with rVVs become resistant to the toxic effects of BrdU. ( B ) Experimental protocol to accelerate the generation and purification of rVVs. First, 293T cells are exposed to wt VV, followed by transfection of the insertion vector encoding the gene of interest ( envC in our example). After 12 h, the GFP-positive cells are selected using flow cytometry. As an alternative to FACS sorting, rVVs can also be isolated by picking of green 293T cell plaques under a fluorescent microscope. After freezing and thawing, the GFP-positive 293T cell lysates are used to infect 143B Tk − cells in the presence of BrdU (the latter prevents replication of wt VV). To obtain pure clonal rVV stocks, several rounds of plaque purification can be performed. Finally, the amplified rVVs are collected and frozen in aliquots.
    Figure Legend Snippet: Strategy to generate insertion vector for rapid selection of rVVs. ( A ) The sequence of the mutated HIV clade C env gene, termed envC , was obtained by PCR (blue box) and cloned into the Xho I and Bam HI sites of the vector pIRES2-GFP. In the second step, the sequence of the expression cassette envC –IRES–GFP was PCR amplified and cloned into vector pCS59 (sites Xho I– Eco RI) under the control of strong synthetic early/late VV promoter (red box). Gray boxes, flanking sequences of the tk gene used for targeting the insertion vector and thereby disrupt the normal VV tk gene. As a consequence, rVVs containing the desired insert are no longer able to express thymidine kinase, and thus cells infected with rVVs become resistant to the toxic effects of BrdU. ( B ) Experimental protocol to accelerate the generation and purification of rVVs. First, 293T cells are exposed to wt VV, followed by transfection of the insertion vector encoding the gene of interest ( envC in our example). After 12 h, the GFP-positive cells are selected using flow cytometry. As an alternative to FACS sorting, rVVs can also be isolated by picking of green 293T cell plaques under a fluorescent microscope. After freezing and thawing, the GFP-positive 293T cell lysates are used to infect 143B Tk − cells in the presence of BrdU (the latter prevents replication of wt VV). To obtain pure clonal rVV stocks, several rounds of plaque purification can be performed. Finally, the amplified rVVs are collected and frozen in aliquots.

    Techniques Used: Plasmid Preparation, Selection, Sequencing, Polymerase Chain Reaction, Clone Assay, Expressing, Amplification, Infection, Purification, Transfection, Flow Cytometry, Cytometry, FACS, Isolation, Microscopy

    Strategy to generate insertion vector for rapid selection of rVVs. ( A ) The sequence of the mutated HIV clade C env gene, termed envC , was obtained by PCR (blue box) and cloned into the Xho I and Bam HI sites of the vector pIRES2-GFP. In the second step, the sequence of the expression cassette envC –IRES–GFP was PCR amplified and cloned into vector pCS59 (sites Xho I– Eco RI) under the control of strong synthetic early/late VV promoter (red box). Gray boxes, flanking sequences of the tk gene used for targeting the insertion vector and thereby disrupt the normal VV tk gene. As a consequence, rVVs containing the desired insert are no longer able to express thymidine kinase, and thus cells infected with rVVs become resistant to the toxic effects of BrdU. ( B ) Experimental protocol to accelerate the generation and purification of rVVs. First, 293T cells are exposed to wt VV, followed by transfection of the insertion vector encoding the gene of interest ( envC in our example). After 12 h, the GFP-positive cells are selected using flow cytometry. As an alternative to FACS sorting, rVVs can also be isolated by picking of green 293T cell plaques under a fluorescent microscope. After freezing and thawing, the GFP-positive 293T cell lysates are used to infect 143B Tk − cells in the presence of BrdU (the latter prevents replication of wt VV). To obtain pure clonal rVV stocks, several rounds of plaque purification can be performed. Finally, the amplified rVVs are collected and frozen in aliquots.
    Figure Legend Snippet: Strategy to generate insertion vector for rapid selection of rVVs. ( A ) The sequence of the mutated HIV clade C env gene, termed envC , was obtained by PCR (blue box) and cloned into the Xho I and Bam HI sites of the vector pIRES2-GFP. In the second step, the sequence of the expression cassette envC –IRES–GFP was PCR amplified and cloned into vector pCS59 (sites Xho I– Eco RI) under the control of strong synthetic early/late VV promoter (red box). Gray boxes, flanking sequences of the tk gene used for targeting the insertion vector and thereby disrupt the normal VV tk gene. As a consequence, rVVs containing the desired insert are no longer able to express thymidine kinase, and thus cells infected with rVVs become resistant to the toxic effects of BrdU. ( B ) Experimental protocol to accelerate the generation and purification of rVVs. First, 293T cells are exposed to wt VV, followed by transfection of the insertion vector encoding the gene of interest ( envC in our example). After 12 h, the GFP-positive cells are selected using flow cytometry. As an alternative to FACS sorting, rVVs can also be isolated by picking of green 293T cell plaques under a fluorescent microscope. After freezing and thawing, the GFP-positive 293T cell lysates are used to infect 143B Tk − cells in the presence of BrdU (the latter prevents replication of wt VV). To obtain pure clonal rVV stocks, several rounds of plaque purification can be performed. Finally, the amplified rVVs are collected and frozen in aliquots.

    Techniques Used: Plasmid Preparation, Selection, Sequencing, Polymerase Chain Reaction, Clone Assay, Expressing, Amplification, Infection, Purification, Transfection, Flow Cytometry, Cytometry, FACS, Isolation, Microscopy

    6) Product Images from "Novel Method of Cell-Free In Vitro Synthesis of the Human Fibroblast Growth Factor 1 Gene"

    Article Title: Novel Method of Cell-Free In Vitro Synthesis of the Human Fibroblast Growth Factor 1 Gene

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2010/971340

    Confirmation of assembled plasmid containing the FGF1 gene . The pcDNA3.1/V5-His-TOPO-FGF1 plasmid was cleaved by Hin dIII and Xho I. Lane 1: DNA molecular weight markers. Lane 2: Two digestion fragments of 5433 bp and 561 bp.
    Figure Legend Snippet: Confirmation of assembled plasmid containing the FGF1 gene . The pcDNA3.1/V5-His-TOPO-FGF1 plasmid was cleaved by Hin dIII and Xho I. Lane 1: DNA molecular weight markers. Lane 2: Two digestion fragments of 5433 bp and 561 bp.

    Techniques Used: Plasmid Preparation, Molecular Weight

    7) Product Images from "Transcription-induced formation of extrachromosomal DNA during yeast ageing"

    Article Title: Transcription-induced formation of extrachromosomal DNA during yeast ageing

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000471

    Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of CUP1 eccDNA and rDNA-derived ERCs in wild-type, mus81 Δ, yen1 Δ, and slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in Fig 1B . (B) Quantification of CUP1 eccDNA and rDNA-derived ERCs in wild-type and mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in Fig 1B , n = 4. (C) REC-seq analysis of mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy P GAL1 -3HA cup1 tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications. P GAL1 -3HA wild-type, sae2 Δ, and mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1 plasmid to complement the loss of active chromosomal CUP1 genes, labelled as CUP1 plasmid. This plasmid contains an Xho I site and is hence linearised by Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA; n = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on P GAL1 -3HA wild-type and rad52 Δ cells along with BY4741 wild-type and rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4 to 10 1 cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in S1 Data and S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.
    Figure Legend Snippet: Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of CUP1 eccDNA and rDNA-derived ERCs in wild-type, mus81 Δ, yen1 Δ, and slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in Fig 1B . (B) Quantification of CUP1 eccDNA and rDNA-derived ERCs in wild-type and mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in Fig 1B , n = 4. (C) REC-seq analysis of mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy P GAL1 -3HA cup1 tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications. P GAL1 -3HA wild-type, sae2 Δ, and mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1 plasmid to complement the loss of active chromosomal CUP1 genes, labelled as CUP1 plasmid. This plasmid contains an Xho I site and is hence linearised by Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA; n = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on P GAL1 -3HA wild-type and rad52 Δ cells along with BY4741 wild-type and rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4 to 10 1 cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in S1 Data and S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.

    Techniques Used: Southern Blot, Derivative Assay, Plasmid Preparation, Transformation Assay, Colony Assay, DNA Sequencing

    8) Product Images from "Colonization of Vitis vinifera by a Green Fluorescence Protein-Labeled, gfp-Marked Strain of Xylophilus ampelinus, the Causal Agent of Bacterial Necrosis of Grapevine"

    Article Title: Colonization of Vitis vinifera by a Green Fluorescence Protein-Labeled, gfp-Marked Strain of Xylophilus ampelinus, the Causal Agent of Bacterial Necrosis of Grapevine

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.69.4.1904-1912.2003

    Southern blot of Xho I digested DNA of X. ampelinus strains on a nylon membrane hybridized with the DIG-labeled gfp -probe. Lanes: 1, CFBP2098; 2, 2098:: gfp 2; 3, 2098:: gfp 3; 4, 2098:: gfp 4; 5, plasmid pUT gfp. There are two Xho I sites in pUT gfp. One is located in the npt II gene, borne by the mini- gfp transposon. Consequently, the gfp probe hybridized one DNA fragment per insertion. There is one insertion of the mini- gfp transposon in 2098:: gfp 2 and 2098:: gfp 3 and two insertions in 2098:: gfp 4.
    Figure Legend Snippet: Southern blot of Xho I digested DNA of X. ampelinus strains on a nylon membrane hybridized with the DIG-labeled gfp -probe. Lanes: 1, CFBP2098; 2, 2098:: gfp 2; 3, 2098:: gfp 3; 4, 2098:: gfp 4; 5, plasmid pUT gfp. There are two Xho I sites in pUT gfp. One is located in the npt II gene, borne by the mini- gfp transposon. Consequently, the gfp probe hybridized one DNA fragment per insertion. There is one insertion of the mini- gfp transposon in 2098:: gfp 2 and 2098:: gfp 3 and two insertions in 2098:: gfp 4.

    Techniques Used: Southern Blot, Labeling, Plasmid Preparation

    9) Product Images from "Stabilization of Dicentric Translocations through Secondary Rearrangements Mediated by Multiple Mechanisms in S. cerevisiae"

    Article Title: Stabilization of Dicentric Translocations through Secondary Rearrangements Mediated by Multiple Mechanisms in S. cerevisiae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006389

    Degradation of chromosome ends in strains containing mec1 and tlc1 mutations. (A) aGCH analysis of representative chromosomes with signal decay at their ends is presented. (B) The qPCR validation of the decrease in the amount of genomic DNA on the right arm of chromosome XVI in the U1 GCR strain using primer pairs specific to YPR133W , YPR144C and YPR194C loci . The relative amounts of genomic DNA determined by qPCR at the YPR133W , YPR144W and YPR194C loci in the U1 strain are expressed relative to the signal obtained with each primer pair using RDKY3615 control DNA. (C) Telomere lengths in a RDKY3615 Can s 5FOA s strain, a mec1 sml1 tlc1 Can1 r 5FOA r post-senescence strain, a mec1 sml1 lig4 tlc1 Can1 s 5FOA s post-senescence strain, mec1 sml1 tlc1 GCR strains (D4, U1 and D5) and mec1 sml1 lig4 tlc1 GCR strains (D6, D7, D9 and D10) were analyzed by Southern blot with a poly(C 1-3 /TG 1–3 ) radiolabelled probe hybridized to Xho I-digested genomic DNA. (D) Chromosomes with at least one degraded end are listed. The chromosome number is followed by the standard SGD nucleotide coordinates of the chromosome region that has a negative log 2 ratio.
    Figure Legend Snippet: Degradation of chromosome ends in strains containing mec1 and tlc1 mutations. (A) aGCH analysis of representative chromosomes with signal decay at their ends is presented. (B) The qPCR validation of the decrease in the amount of genomic DNA on the right arm of chromosome XVI in the U1 GCR strain using primer pairs specific to YPR133W , YPR144C and YPR194C loci . The relative amounts of genomic DNA determined by qPCR at the YPR133W , YPR144W and YPR194C loci in the U1 strain are expressed relative to the signal obtained with each primer pair using RDKY3615 control DNA. (C) Telomere lengths in a RDKY3615 Can s 5FOA s strain, a mec1 sml1 tlc1 Can1 r 5FOA r post-senescence strain, a mec1 sml1 lig4 tlc1 Can1 s 5FOA s post-senescence strain, mec1 sml1 tlc1 GCR strains (D4, U1 and D5) and mec1 sml1 lig4 tlc1 GCR strains (D6, D7, D9 and D10) were analyzed by Southern blot with a poly(C 1-3 /TG 1–3 ) radiolabelled probe hybridized to Xho I-digested genomic DNA. (D) Chromosomes with at least one degraded end are listed. The chromosome number is followed by the standard SGD nucleotide coordinates of the chromosome region that has a negative log 2 ratio.

    Techniques Used: Real-time Polymerase Chain Reaction, Southern Blot

    10) Product Images from "A Small Peptide Modeled after the NRAGE Repeat Domain Inhibits XIAP-TAB1-TAK1 Signaling for NF-?B Activation and Apoptosis in P19 Cells"

    Article Title: A Small Peptide Modeled after the NRAGE Repeat Domain Inhibits XIAP-TAB1-TAK1 Signaling for NF-?B Activation and Apoptosis in P19 Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020659

    Design of the NRAGE peptide. (A) A list of the 25 hexamers in the order in which they appear in the NRAGE repeat domain partitioned into 4 distinguishing groups. A repeat consensus sequence was determined for each group and was then resolved into a single final consensus sequence as indicated by the red amino acids which was repeated 4 times for the NRAGE peptide. (B) Schematic of nucleotides comprising the NRAGE peptide including a Xho I site and a Kozak sequence to facilitate excision and transcription respectively.
    Figure Legend Snippet: Design of the NRAGE peptide. (A) A list of the 25 hexamers in the order in which they appear in the NRAGE repeat domain partitioned into 4 distinguishing groups. A repeat consensus sequence was determined for each group and was then resolved into a single final consensus sequence as indicated by the red amino acids which was repeated 4 times for the NRAGE peptide. (B) Schematic of nucleotides comprising the NRAGE peptide including a Xho I site and a Kozak sequence to facilitate excision and transcription respectively.

    Techniques Used: Sequencing

    11) Product Images from "Cdc73 suppresses genome instability by mediating telomere homeostasis"

    Article Title: Cdc73 suppresses genome instability by mediating telomere homeostasis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007170

    Loss of CDC73 results in a telomere defect. a. Southern blot of Xho I-digested genomic DNA isolated from strains of the indicated genotypes derived by sporulation of appropriate diploids and analyzed with a telomere-specific probe immediately after sporulation and genotyping. The dashed line corresponds to wild-type telomere length. b. Strains were serially propagated on non-selective media for > 20 restreaks and then tested by telomere Southern blot as above. c. Strains of the indicated genotypes were obtained by sporulation of heterozygous diploids and analyzed by pulse field gel electrophoresis. Wild-type chromosome sizes are labeled (left). Chromosome bands with new sizes are indicated with solid triangles, and missing bands are indicated with open triangles. Decreased band intensity and increased smearing can be seen in strains that were shown to undergo senescence. d. TPE was assayed by plating 10-fold serial dilutions of cdc73Δ , tel1Δ , and yku80Δ single and double mutant strains on selective media or selective media containing 5FOA. Loss of telomeric silencing is indicated by increased sensitivity to 5FOA.
    Figure Legend Snippet: Loss of CDC73 results in a telomere defect. a. Southern blot of Xho I-digested genomic DNA isolated from strains of the indicated genotypes derived by sporulation of appropriate diploids and analyzed with a telomere-specific probe immediately after sporulation and genotyping. The dashed line corresponds to wild-type telomere length. b. Strains were serially propagated on non-selective media for > 20 restreaks and then tested by telomere Southern blot as above. c. Strains of the indicated genotypes were obtained by sporulation of heterozygous diploids and analyzed by pulse field gel electrophoresis. Wild-type chromosome sizes are labeled (left). Chromosome bands with new sizes are indicated with solid triangles, and missing bands are indicated with open triangles. Decreased band intensity and increased smearing can be seen in strains that were shown to undergo senescence. d. TPE was assayed by plating 10-fold serial dilutions of cdc73Δ , tel1Δ , and yku80Δ single and double mutant strains on selective media or selective media containing 5FOA. Loss of telomeric silencing is indicated by increased sensitivity to 5FOA.

    Techniques Used: Southern Blot, Isolation, Derivative Assay, Nucleic Acid Electrophoresis, Labeling, Mutagenesis

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

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

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0001777

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

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

    13) Product Images from "Conjugative Botulinum Neurotoxin-Encoding Plasmids in Clostridium botulinum"

    Article Title: Conjugative Botulinum Neurotoxin-Encoding Plasmids in Clostridium botulinum

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0011087

    Confirmation of plasmid pCLJ-Erm transfer from C. botulinum strain 657BaCT4 to strain LNT01. ( A ) Ethidium bromide stained PFGE of C. botulinum strains: LNT01 (Lanes 1 and 7), wild type strain 657Ba (Lanes 2 and 8); 657BaCT4 (Lanes 3 and 9) and LNT01 transconjugants (pCLJ-Erm) (Lanes 4–6 and 10–12); Lanes 1–6, nondigested DNA samples; Lanes 7–12, Xho I digested DNAsamples. Lambda PFG Marker (Lane M), New England Biolabs. The position of the pCLJ plasmid is indicated with an arrow. Southern hybridization with: ( B ) the ermB probe and ( C ) the bont/bvB probe. PFGE conditions: 6V/cm, 12°C, 1–26 s pulse time, 24 h.
    Figure Legend Snippet: Confirmation of plasmid pCLJ-Erm transfer from C. botulinum strain 657BaCT4 to strain LNT01. ( A ) Ethidium bromide stained PFGE of C. botulinum strains: LNT01 (Lanes 1 and 7), wild type strain 657Ba (Lanes 2 and 8); 657BaCT4 (Lanes 3 and 9) and LNT01 transconjugants (pCLJ-Erm) (Lanes 4–6 and 10–12); Lanes 1–6, nondigested DNA samples; Lanes 7–12, Xho I digested DNAsamples. Lambda PFG Marker (Lane M), New England Biolabs. The position of the pCLJ plasmid is indicated with an arrow. Southern hybridization with: ( B ) the ermB probe and ( C ) the bont/bvB probe. PFGE conditions: 6V/cm, 12°C, 1–26 s pulse time, 24 h.

    Techniques Used: Plasmid Preparation, Staining, Marker, Hybridization

    14) Product Images from "Repair of peripheral nerve defects with chemically extracted acellular nerve allografts loaded with neurotrophic factors-transfected bone marrow mesenchymal stem cells"

    Article Title: Repair of peripheral nerve defects with chemically extracted acellular nerve allografts loaded with neurotrophic factors-transfected bone marrow mesenchymal stem cells

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.165523

    Identification of recombinant plasmid pIRES-BDNF-CNTF by enzymatic digestion. 1: DNA marker; 2: recombinant plasmid pIRES-BDNF-CNTF was digested by Xba I and Sal I, Xho I and Mlu I simutaneously; 3: recombinant plasmid pIRES-BDNF-CNTF was digested by Xho I and Mlu I; 4: recombinant plasmid pIRES-BDNF-CNTF was digested by Xba I and Sal I; 5: empty vector pIRES was digested by Xho I and Mlu I; 6: M: DNA marker. Enzymatic digestion products were visualized by gel electrophoresis using a 1.5% agarose gel. A 6.1-kb-sized vector band, a 774-bp-sized BDNF mRNA band and a 597-bp-sized CNTF band appeared, confirming the double-gene recombinant plasmid is pIRES-BDNF-CNTF. BDNF: Brain-derived neurotrophic factor; CNTF: ciliary neurotrophic factor.
    Figure Legend Snippet: Identification of recombinant plasmid pIRES-BDNF-CNTF by enzymatic digestion. 1: DNA marker; 2: recombinant plasmid pIRES-BDNF-CNTF was digested by Xba I and Sal I, Xho I and Mlu I simutaneously; 3: recombinant plasmid pIRES-BDNF-CNTF was digested by Xho I and Mlu I; 4: recombinant plasmid pIRES-BDNF-CNTF was digested by Xba I and Sal I; 5: empty vector pIRES was digested by Xho I and Mlu I; 6: M: DNA marker. Enzymatic digestion products were visualized by gel electrophoresis using a 1.5% agarose gel. A 6.1-kb-sized vector band, a 774-bp-sized BDNF mRNA band and a 597-bp-sized CNTF band appeared, confirming the double-gene recombinant plasmid is pIRES-BDNF-CNTF. BDNF: Brain-derived neurotrophic factor; CNTF: ciliary neurotrophic factor.

    Techniques Used: Recombinant, Plasmid Preparation, Marker, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Derivative Assay

    Identificatiion of PCR products by enzymatic digestion. M: DNA marker; 1: recombinant plasmid pIRES-BDNF (774 bp) by Xho I and Mlu I; 2: recombinant plasmid pIRES-CNTF (597 bp) by Xba I and Sal I; two fragments [ i.e ., target gene CNTF fragment (597 bp) and pIRES vector fragment (approximately 6.1 kb)] were acquired, confirming the product was recombinant plasmid pIRES-CNTF. BDNF: Brain-derived neurotrophic factor; CNTF: ciliary neurotrophic factor.
    Figure Legend Snippet: Identificatiion of PCR products by enzymatic digestion. M: DNA marker; 1: recombinant plasmid pIRES-BDNF (774 bp) by Xho I and Mlu I; 2: recombinant plasmid pIRES-CNTF (597 bp) by Xba I and Sal I; two fragments [ i.e ., target gene CNTF fragment (597 bp) and pIRES vector fragment (approximately 6.1 kb)] were acquired, confirming the product was recombinant plasmid pIRES-CNTF. BDNF: Brain-derived neurotrophic factor; CNTF: ciliary neurotrophic factor.

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

    Identification of double-gene recon by PCR. M: DNA marker; 1: double-gene recon 1 BDNF mRNA amplification product (788 bp); 2: double-gene recon 1 CNTF mRNA amplification product (611 bp); 3: double-gene recon 2 BDNF mRNA amplification product (774 bp); 4: double-gene recon 2 CNTF mRNA amplification product (597 bp). Recombinant plasmid pIRES-BDNF was digested by Xho I and Mlu I, and two fragments, a 774-bp-long target gene BDNF fragment and an approximately 1.6-kb-long pIRES vector fragment, were acquired, confirming the product was recombinant plasmid pIRES-BDNF. BDNF: Brain-derived neurotrophic factor. CNTF: ciliary neurotrophic factor.
    Figure Legend Snippet: Identification of double-gene recon by PCR. M: DNA marker; 1: double-gene recon 1 BDNF mRNA amplification product (788 bp); 2: double-gene recon 1 CNTF mRNA amplification product (611 bp); 3: double-gene recon 2 BDNF mRNA amplification product (774 bp); 4: double-gene recon 2 CNTF mRNA amplification product (597 bp). Recombinant plasmid pIRES-BDNF was digested by Xho I and Mlu I, and two fragments, a 774-bp-long target gene BDNF fragment and an approximately 1.6-kb-long pIRES vector fragment, were acquired, confirming the product was recombinant plasmid pIRES-BDNF. BDNF: Brain-derived neurotrophic factor. CNTF: ciliary neurotrophic factor.

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

    15) Product Images from "Characterization of Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus and Protective Efficacy against H1 Viruses in Mice"

    Article Title: Characterization of Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus and Protective Efficacy against H1 Viruses in Mice

    Journal: Viruses

    doi: 10.3390/v9080209

    The recombinant pCI-neo-HA identification by Nhe I/Xho I digestion. Lane 1 and 4: DNA molecular weight marker; lane 2: empty pCI-neo plasmid; lane 3: pCI-neo-HA plasmid digested with Nhe I and Xho I.
    Figure Legend Snippet: The recombinant pCI-neo-HA identification by Nhe I/Xho I digestion. Lane 1 and 4: DNA molecular weight marker; lane 2: empty pCI-neo plasmid; lane 3: pCI-neo-HA plasmid digested with Nhe I and Xho I.

    Techniques Used: Recombinant, Molecular Weight, Marker, Plasmid Preparation

    16) Product Images from "Role of Pathogenicity Determinant Protein C (PdpC) in Determining the Virulence of the Francisella tularensis Subspecies tularensis SCHU"

    Article Title: Role of Pathogenicity Determinant Protein C (PdpC) in Determining the Virulence of the Francisella tularensis Subspecies tularensis SCHU

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089075

    Construction of pdpC knockout mutants. The pdpC mutants were constructed using the TargeTron gene knockout system (Sigma-Aldrich) and pKEK1140 plasmid. (A) pKEK1140 plasmids were optimized for use in Francisella tularensis with the TargeTron gene knockout system. This plasmid consists of a plasmid origin for temperature-sensitive replication in F. tularensis (Ft ori), a plasmid origin in E. coli (p15A ori), an antibiotic resistance gene (Kan), FTN1451 promoter for Kan expressed in F. tularensis (FTN1451p), the RNP ( ltr B L1 and LtrA) driven by the F. tularensis groEL promoter (groELp), and a lacZ ′ within ltr B L1 . To target specific F. tularensis genes for group II intron insertion, the ltrB L1 intron in pKEK1140 was retargeted by replacing lacZ′, which is located between the Xho I and BsrG I sites, with a 350-bp PCR product [27] . (B) The insertion in each pdpC mutant derived from SCHU P9 was confirmed by PCR. In the left panel, genomic DNA extracted from SCHU P9, 538 ins, 1119 ins, and 2013 ins mutants was amplified using sense (pdpC-435F) and antisense (pdpC-2204R) primers. In the middle and right panel, genomic DNA was amplified using insertion-and pdpC -specific primer pairs (pdpC-435F/EBS universal and EBS universal/pdpC-2204R). These amplicons and molecular weight marker were electrophoresed in a 0.7% agarose gel. (C) Summary of pdpC mutants generated in this study. ltrB introns were inserted into SCHU P9 pdpC positions 538|539, 1119|1120, and 2013|2014. The resultant mutants were designated 538 ins, 1119 ins, and 2013 ins mutants, respectively. Both duplicated pdpC were mutated by pKEK1140 plasmid in all mutants. (D) SCHU P5, SCHU P9, 538 ins, 1119 ins, and 2013 ins were grown in CDM and the lysates were subjected to western blot analysis was performed using anti-PdpC polyclonal antibody [20] . Intact PdpC was observed only in SCHU P9. Western blot analysis demonstrated that truncated forms of PdpC proteins with predicted molecular size (538 ins, approximately 20 kDa; 1119 ins, approximately 45 kDa; 2013 ins, approximately 75 kDa) were expressed in three pdpC mutants obtained.
    Figure Legend Snippet: Construction of pdpC knockout mutants. The pdpC mutants were constructed using the TargeTron gene knockout system (Sigma-Aldrich) and pKEK1140 plasmid. (A) pKEK1140 plasmids were optimized for use in Francisella tularensis with the TargeTron gene knockout system. This plasmid consists of a plasmid origin for temperature-sensitive replication in F. tularensis (Ft ori), a plasmid origin in E. coli (p15A ori), an antibiotic resistance gene (Kan), FTN1451 promoter for Kan expressed in F. tularensis (FTN1451p), the RNP ( ltr B L1 and LtrA) driven by the F. tularensis groEL promoter (groELp), and a lacZ ′ within ltr B L1 . To target specific F. tularensis genes for group II intron insertion, the ltrB L1 intron in pKEK1140 was retargeted by replacing lacZ′, which is located between the Xho I and BsrG I sites, with a 350-bp PCR product [27] . (B) The insertion in each pdpC mutant derived from SCHU P9 was confirmed by PCR. In the left panel, genomic DNA extracted from SCHU P9, 538 ins, 1119 ins, and 2013 ins mutants was amplified using sense (pdpC-435F) and antisense (pdpC-2204R) primers. In the middle and right panel, genomic DNA was amplified using insertion-and pdpC -specific primer pairs (pdpC-435F/EBS universal and EBS universal/pdpC-2204R). These amplicons and molecular weight marker were electrophoresed in a 0.7% agarose gel. (C) Summary of pdpC mutants generated in this study. ltrB introns were inserted into SCHU P9 pdpC positions 538|539, 1119|1120, and 2013|2014. The resultant mutants were designated 538 ins, 1119 ins, and 2013 ins mutants, respectively. Both duplicated pdpC were mutated by pKEK1140 plasmid in all mutants. (D) SCHU P5, SCHU P9, 538 ins, 1119 ins, and 2013 ins were grown in CDM and the lysates were subjected to western blot analysis was performed using anti-PdpC polyclonal antibody [20] . Intact PdpC was observed only in SCHU P9. Western blot analysis demonstrated that truncated forms of PdpC proteins with predicted molecular size (538 ins, approximately 20 kDa; 1119 ins, approximately 45 kDa; 2013 ins, approximately 75 kDa) were expressed in three pdpC mutants obtained.

    Techniques Used: Knock-Out, Construct, Gene Knockout, Plasmid Preparation, Polymerase Chain Reaction, Mutagenesis, Derivative Assay, Amplification, Molecular Weight, Marker, Agarose Gel Electrophoresis, Generated, Western Blot

    17) Product Images from "One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1"

    Article Title: One-Step Recovery of scFv Clones from High-Throughput Sequencing-Based Screening of Phage Display Libraries Challenged to Cells Expressing Native Claudin-1

    Journal: BioMed Research International

    doi: 10.1155/2015/703213

    Library screening and analysis of sequences. (a) The panel shows the DNA fragments, gel-purified from sublibraries, after the indicated selection cycles. The corresponding plasmid preparations were digested with Nco I and Xho I restriction endonucleases, to release the DNA fragments encoding for the VH regions of the scFv fragments. The fragments were bar-coded and subjected to high-throughput sequencing, as described in the text. SM, size marker. (b) The chart reports the entropy values for the populations of fragments originating from the indicated selection cycles, after sequencing. (c) The reported values indicate the total number of clones, and the relative representation of the most abundant clone, within the corresponding selection cycles. (d) The chart indicates the relative distribution of clones, according to the number of counts observed, within the indicated ranges, for each of the 4 selection cycles. Cycles 3 and 4 show similar distributions.
    Figure Legend Snippet: Library screening and analysis of sequences. (a) The panel shows the DNA fragments, gel-purified from sublibraries, after the indicated selection cycles. The corresponding plasmid preparations were digested with Nco I and Xho I restriction endonucleases, to release the DNA fragments encoding for the VH regions of the scFv fragments. The fragments were bar-coded and subjected to high-throughput sequencing, as described in the text. SM, size marker. (b) The chart reports the entropy values for the populations of fragments originating from the indicated selection cycles, after sequencing. (c) The reported values indicate the total number of clones, and the relative representation of the most abundant clone, within the corresponding selection cycles. (d) The chart indicates the relative distribution of clones, according to the number of counts observed, within the indicated ranges, for each of the 4 selection cycles. Cycles 3 and 4 show similar distributions.

    Techniques Used: Library Screening, Purification, Selection, Plasmid Preparation, Next-Generation Sequencing, Marker, Sequencing, Clone Assay

    18) Product Images from "Distinct Requirements for Evoked and Spontaneous Release of Neurotransmitter Are Revealed by Mutations in theDrosophila Gene neuronal-synaptobrevin"

    Article Title: Distinct Requirements for Evoked and Spontaneous Release of Neurotransmitter Are Revealed by Mutations in theDrosophila Gene neuronal-synaptobrevin

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-06-02028.1998

    Restriction mapping of excision alleles. A , Restriction map of the n-syb locus ( top ), the excision allele n-syb ΔF33-8 ( middle ), and the excision allele n-syb ΔF33B ( bottom ). The restriction site abbreviations are the following: Eco RI, E ; Xho I, X ; Pst I, P . Deficiencies are indicated by a dashed line , PCR primers are shown as arrows , and exons are shown as boxes ( shaded boxes are coding regions, and unshaded boxes are noncoding regions). Probes used in B are diagrammed below the wild-type n-syb and the n-syb ΔF33B loci. B , Genomic Southern blots of DNA from n-syb ΔF33B heterozygote. Lanes 1–4 are all digested with Xho I and are hybridized with probes 1–4 , respectively. Mutant (−) and wild-type (+) bands are indicated. In lane 1 , probe 1 hybridized to both the wild-type and mutant Xho I restriction fragments. The larger size of the mutant band results from upstream n-syb sequences and the remainder of the P-element that failed to excise fully. In lane 2 , probe 2 recognized the same wild-type band as in lane 1 . The higher molecular weight mutant band failed to hybridize as the 3′ half of exon 1, and all of exons 2, 3, and 4 are deleted in the mutant. In lane 3 , probe 3 hybridized to a wild-type band of ∼1 kb and the high molecular weight mutant band from lane 1 . Probe 3 hybridized to the mutant band because the Xho I restriction site between exons 4 and 5 was deleted, but the deletion did not extend to exon 5. In lane 4 , probe 4 hybridized to a single unaltered band because the probe is outside the deleted region.
    Figure Legend Snippet: Restriction mapping of excision alleles. A , Restriction map of the n-syb locus ( top ), the excision allele n-syb ΔF33-8 ( middle ), and the excision allele n-syb ΔF33B ( bottom ). The restriction site abbreviations are the following: Eco RI, E ; Xho I, X ; Pst I, P . Deficiencies are indicated by a dashed line , PCR primers are shown as arrows , and exons are shown as boxes ( shaded boxes are coding regions, and unshaded boxes are noncoding regions). Probes used in B are diagrammed below the wild-type n-syb and the n-syb ΔF33B loci. B , Genomic Southern blots of DNA from n-syb ΔF33B heterozygote. Lanes 1–4 are all digested with Xho I and are hybridized with probes 1–4 , respectively. Mutant (−) and wild-type (+) bands are indicated. In lane 1 , probe 1 hybridized to both the wild-type and mutant Xho I restriction fragments. The larger size of the mutant band results from upstream n-syb sequences and the remainder of the P-element that failed to excise fully. In lane 2 , probe 2 recognized the same wild-type band as in lane 1 . The higher molecular weight mutant band failed to hybridize as the 3′ half of exon 1, and all of exons 2, 3, and 4 are deleted in the mutant. In lane 3 , probe 3 hybridized to a wild-type band of ∼1 kb and the high molecular weight mutant band from lane 1 . Probe 3 hybridized to the mutant band because the Xho I restriction site between exons 4 and 5 was deleted, but the deletion did not extend to exon 5. In lane 4 , probe 4 hybridized to a single unaltered band because the probe is outside the deleted region.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Molecular Weight

    19) Product Images from "Enhanced production of recombinant serratiopeptidase in Escherichia coli and its characterization as a potential biosimilar to native biotherapeutic counterpart"

    Article Title: Enhanced production of recombinant serratiopeptidase in Escherichia coli and its characterization as a potential biosimilar to native biotherapeutic counterpart

    Journal: Microbial Cell Factories

    doi: 10.1186/s12934-019-1267-x

    Recombinant cloning and development of mature serratiopeptidase specific expression construct. a Representative Agarose gel (1.2%) showing amplification of ~ 1500 bp gene fragment, particular to the size of mature serratiopeptidase gene (M-PCR). The gene cloned in the pET23b(+) vector having Amp r for selection. When digested with single restriction enzyme; i.e. Nde I and two different enzymes; i.e., Nde I and Xho I. b Representative agarose gel (1.2%) shows a linear fragment (pMSrp-SD) of ~ 5000 bp and two fragments equal to the size of plasmid backbone ~ 3600 bp and insert gene (MSrp) ~ 1500 bp in Lane (pMSrp-DD) respectively confirming the successful insertion/ligation of gene and construction of recombinant plasmid
    Figure Legend Snippet: Recombinant cloning and development of mature serratiopeptidase specific expression construct. a Representative Agarose gel (1.2%) showing amplification of ~ 1500 bp gene fragment, particular to the size of mature serratiopeptidase gene (M-PCR). The gene cloned in the pET23b(+) vector having Amp r for selection. When digested with single restriction enzyme; i.e. Nde I and two different enzymes; i.e., Nde I and Xho I. b Representative agarose gel (1.2%) shows a linear fragment (pMSrp-SD) of ~ 5000 bp and two fragments equal to the size of plasmid backbone ~ 3600 bp and insert gene (MSrp) ~ 1500 bp in Lane (pMSrp-DD) respectively confirming the successful insertion/ligation of gene and construction of recombinant plasmid

    Techniques Used: Recombinant, Clone Assay, Expressing, Construct, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Selection, Ligation

    20) Product Images from "Adenovirus Type 4 Respiratory Infections among Civilian Adults, Northeastern United States, 2011–2015 1"

    Article Title: Adenovirus Type 4 Respiratory Infections among Civilian Adults, Northeastern United States, 2011–2015 1

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid2402.171407

    In silico restriction enzyme analysis of human adenovirus type 4 genomes representing the spectrum of genetic variability of the 36 isolates characterized in study of acute respiratory infection detected in the northeastern United States, 2011–2015. We generated restriction enzyme profiles for the completely sequenced genomes obtained in this study and from reference sequences available in GenBank using Geneious Pro ( 31 ). 4p4 MIL is isolate NHR90339, 4a1 MIL is isolate NHRC3, and 4a2 MIL is isolate 42606; 4a Sma I v is isolate NY11 (GenBank accession no. KY996449) and 4a Sma I/ Xho I v is isolate NY24 (GenBank accession no. KY996446). MIL, military isolate; v, variant; Vac, vaccine strain.
    Figure Legend Snippet: In silico restriction enzyme analysis of human adenovirus type 4 genomes representing the spectrum of genetic variability of the 36 isolates characterized in study of acute respiratory infection detected in the northeastern United States, 2011–2015. We generated restriction enzyme profiles for the completely sequenced genomes obtained in this study and from reference sequences available in GenBank using Geneious Pro ( 31 ). 4p4 MIL is isolate NHR90339, 4a1 MIL is isolate NHRC3, and 4a2 MIL is isolate 42606; 4a Sma I v is isolate NY11 (GenBank accession no. KY996449) and 4a Sma I/ Xho I v is isolate NY24 (GenBank accession no. KY996446). MIL, military isolate; v, variant; Vac, vaccine strain.

    Techniques Used: In Silico, Infection, Generated, Variant Assay

    21) Product Images from "A Nucleolus-Predominant piggyBac Transposase, NP-mPB, Mediates Elevated Transposition Efficiency in Mammalian Cells"

    Article Title: A Nucleolus-Predominant piggyBac Transposase, NP-mPB, Mediates Elevated Transposition Efficiency in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089396

    Schematic representation of expression constructs for PBase variants, PBase fusion proteins, and the dual fluorescent UGm transposon used in this study. (A) The mPB and NP-mPB coding sequences of mouse codon-optimized PBase were cloned into the pTriEx-HTNC plasmid by replacing the Cre cassette between Spe I and Xho I. The mPB and NP-mPB coding sequences were preceded by a hexa-histidine encoding sequence (6× His tag). NP-mPB included an additional nucleolus-predominant (NP) signal peptide. (B) The pTriEx-mPB-tGFP and pTriEx-NP-mPB-tGFP constructs expressed tGFP-fused PBases. The tGFP moiety allowed real-time imaging of the PBase variant subcellular distributions. The pTriEx-mPB-2A-eGFP and pTriEx-NP-mPB-2A-eGFP plasmids expressed PBase variants, which were linked to eGFP by the self-cleaving 2A peptide. All protein-encoding cassettes were transcriptionally regulated by a hybrid promoter composed of the CMV immediate early enhancer fused to the chicken β-actin promoter (CAG), and followed by a polyadenylation signal sequence (pA). (C) Flanking the 5′ and 3′ inverted terminal repeats (ITRs) (empty arrows), the dual fluorescent transposon ( pXL-T3-Neo-UGm-cHS4X ; UGm ) carried a human ubiquitin C (UBC) promoter-driven H2B-eGFP-2A-mCherry-GPI (Gm) transgene that labeled the transposed cells with a characteristic chromatin EGFP and membrane mCherry dual fluorescence. Additional abbreviations: Neo r , a neomycin phosphotransferase expression cassette providing resistance to G418 selection; 2× Ins, two copies of the insulator sequence from chicken β-globin.
    Figure Legend Snippet: Schematic representation of expression constructs for PBase variants, PBase fusion proteins, and the dual fluorescent UGm transposon used in this study. (A) The mPB and NP-mPB coding sequences of mouse codon-optimized PBase were cloned into the pTriEx-HTNC plasmid by replacing the Cre cassette between Spe I and Xho I. The mPB and NP-mPB coding sequences were preceded by a hexa-histidine encoding sequence (6× His tag). NP-mPB included an additional nucleolus-predominant (NP) signal peptide. (B) The pTriEx-mPB-tGFP and pTriEx-NP-mPB-tGFP constructs expressed tGFP-fused PBases. The tGFP moiety allowed real-time imaging of the PBase variant subcellular distributions. The pTriEx-mPB-2A-eGFP and pTriEx-NP-mPB-2A-eGFP plasmids expressed PBase variants, which were linked to eGFP by the self-cleaving 2A peptide. All protein-encoding cassettes were transcriptionally regulated by a hybrid promoter composed of the CMV immediate early enhancer fused to the chicken β-actin promoter (CAG), and followed by a polyadenylation signal sequence (pA). (C) Flanking the 5′ and 3′ inverted terminal repeats (ITRs) (empty arrows), the dual fluorescent transposon ( pXL-T3-Neo-UGm-cHS4X ; UGm ) carried a human ubiquitin C (UBC) promoter-driven H2B-eGFP-2A-mCherry-GPI (Gm) transgene that labeled the transposed cells with a characteristic chromatin EGFP and membrane mCherry dual fluorescence. Additional abbreviations: Neo r , a neomycin phosphotransferase expression cassette providing resistance to G418 selection; 2× Ins, two copies of the insulator sequence from chicken β-globin.

    Techniques Used: Expressing, Construct, Clone Assay, Plasmid Preparation, Sequencing, Imaging, Variant Assay, Labeling, Fluorescence, Selection

    22) Product Images from "Prevalence and Infection Load Dynamics of Rickettsia felis in Actively Feeding Cat Fleas"

    Article Title: Prevalence and Infection Load Dynamics of Rickettsia felis in Actively Feeding Cat Fleas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0002805

    Estimation of C. felis 18S rDNA copy number using Southern blot. C. felis genomic DNA (6 µg/enzyme) was digested with Eag I, Eco R I, Pst I, Xba I and Xho I. Uncut gDNA served as a negative control and PCR product of a portion of C. felis 18S rDNA served as a positive control. Genomic DNA was hybridized with Cf 18S bp probe to estimate the number of 18S rDNA gene copies in C. felis . Digestion with each enzyme results in a single digestion product. (*DNA was not completely digested by Pst I. Top band is uncut DNA.)
    Figure Legend Snippet: Estimation of C. felis 18S rDNA copy number using Southern blot. C. felis genomic DNA (6 µg/enzyme) was digested with Eag I, Eco R I, Pst I, Xba I and Xho I. Uncut gDNA served as a negative control and PCR product of a portion of C. felis 18S rDNA served as a positive control. Genomic DNA was hybridized with Cf 18S bp probe to estimate the number of 18S rDNA gene copies in C. felis . Digestion with each enzyme results in a single digestion product. (*DNA was not completely digested by Pst I. Top band is uncut DNA.)

    Techniques Used: Southern Blot, Negative Control, Polymerase Chain Reaction, Positive Control

    23) Product Images from "Cdc73 suppresses genome instability by mediating telomere homeostasis"

    Article Title: Cdc73 suppresses genome instability by mediating telomere homeostasis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007170

    Loss of CDC73 results in a telomere defect. a. Southern blot of Xho I-digested genomic DNA isolated from strains of the indicated genotypes derived by sporulation of appropriate diploids and analyzed with a telomere-specific probe immediately after sporulation and genotyping. The dashed line corresponds to wild-type telomere length. b. Strains were serially propagated on non-selective media for > 20 restreaks and then tested by telomere Southern blot as above. c. Strains of the indicated genotypes were obtained by sporulation of heterozygous diploids and analyzed by pulse field gel electrophoresis. Wild-type chromosome sizes are labeled (left). Chromosome bands with new sizes are indicated with solid triangles, and missing bands are indicated with open triangles. Decreased band intensity and increased smearing can be seen in strains that were shown to undergo senescence. d. TPE was assayed by plating 10-fold serial dilutions of cdc73Δ , tel1Δ , and yku80Δ single and double mutant strains on selective media or selective media containing 5FOA. Loss of telomeric silencing is indicated by increased sensitivity to 5FOA.
    Figure Legend Snippet: Loss of CDC73 results in a telomere defect. a. Southern blot of Xho I-digested genomic DNA isolated from strains of the indicated genotypes derived by sporulation of appropriate diploids and analyzed with a telomere-specific probe immediately after sporulation and genotyping. The dashed line corresponds to wild-type telomere length. b. Strains were serially propagated on non-selective media for > 20 restreaks and then tested by telomere Southern blot as above. c. Strains of the indicated genotypes were obtained by sporulation of heterozygous diploids and analyzed by pulse field gel electrophoresis. Wild-type chromosome sizes are labeled (left). Chromosome bands with new sizes are indicated with solid triangles, and missing bands are indicated with open triangles. Decreased band intensity and increased smearing can be seen in strains that were shown to undergo senescence. d. TPE was assayed by plating 10-fold serial dilutions of cdc73Δ , tel1Δ , and yku80Δ single and double mutant strains on selective media or selective media containing 5FOA. Loss of telomeric silencing is indicated by increased sensitivity to 5FOA.

    Techniques Used: Southern Blot, Isolation, Derivative Assay, Nucleic Acid Electrophoresis, Labeling, Mutagenesis

    24) Product Images from "Identification of Novel Linear Megaplasmids Carrying a ss-Lactamase Gene in Neurotoxigenic Clostridium butyricum Type E Strains"

    Article Title: Identification of Novel Linear Megaplasmids Carrying a ss-Lactamase Gene in Neurotoxigenic Clostridium butyricum Type E Strains

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021706

    Clustering analysis of the PFGE profiles of the ten neurotoxigenic C. butyricum type E strains obtained by Xho I (4a) and Sma I (4b) endonucleases. The similarity of PFGE profiles was evaluated using the Bionumerics software (version 4.0) (Applied Maths, Sint-Martens-Latem, Belgium); a similarity level of ≥ 90% was used to define PFGE groups. The 10 neurotoxigenic C. butyricum type E strains were divided in 6 PFGE groups with both endonucleases.
    Figure Legend Snippet: Clustering analysis of the PFGE profiles of the ten neurotoxigenic C. butyricum type E strains obtained by Xho I (4a) and Sma I (4b) endonucleases. The similarity of PFGE profiles was evaluated using the Bionumerics software (version 4.0) (Applied Maths, Sint-Martens-Latem, Belgium); a similarity level of ≥ 90% was used to define PFGE groups. The 10 neurotoxigenic C. butyricum type E strains were divided in 6 PFGE groups with both endonucleases.

    Techniques Used: Software

    PFGE of Xho I-digested DNA of strains ISS-145/1 (lane 1); ISS-20 (lane 2); ISS-21 (lane 3); ISS-109 (lane 4); ISS-86 (lane 5); ISS-190 (lane 6); KZ-1886 (lane 7); KZ-1890 (lane 8); LCL-063 (lane 9); LCL-155 (lane 10). S.e., XbaI -digested genomic DNA fragments of Salmonella enterica serotype Braenderup strain H9812 (ref. 34). PFGE conditions: 4–40 s pulse at 6 V/cm for 18 h ( 6a ). Southern hybridization with a bont /E gene probe showing that the gene probe hybridized to single restriction bands, as indicated by the black arrows ( 6b ).
    Figure Legend Snippet: PFGE of Xho I-digested DNA of strains ISS-145/1 (lane 1); ISS-20 (lane 2); ISS-21 (lane 3); ISS-109 (lane 4); ISS-86 (lane 5); ISS-190 (lane 6); KZ-1886 (lane 7); KZ-1890 (lane 8); LCL-063 (lane 9); LCL-155 (lane 10). S.e., XbaI -digested genomic DNA fragments of Salmonella enterica serotype Braenderup strain H9812 (ref. 34). PFGE conditions: 4–40 s pulse at 6 V/cm for 18 h ( 6a ). Southern hybridization with a bont /E gene probe showing that the gene probe hybridized to single restriction bands, as indicated by the black arrows ( 6b ).

    Techniques Used: Hybridization

    25) Product Images from "Escherichia coli outer membrane protein F (OmpF): an immunogenic protein induces cross-reactive antibodies against Escherichia coli and Shigella"

    Article Title: Escherichia coli outer membrane protein F (OmpF): an immunogenic protein induces cross-reactive antibodies against Escherichia coli and Shigella

    Journal: AMB Express

    doi: 10.1186/s13568-017-0452-8

    Cloning, expression, purification and Western blotting analysis of OmpF. aA PCR products of the ompF gene. Lane M1 Trans DNA Marker II (1500, 900, 700, 500, 400, 200 and 100 bp); lane 1 ompF PCR products. Lane 2 the recombinant pET-28a(+)-ompF plasmid. Lane 3 the recombinant pET-28a(+)-ompF plasmid digested with BamH I and Xho I; lane M2 Trans 5K DNA Marker (5000, 3000, 2000, 1500, 1000, 800, 500 and 300 bp); B schematic representation of the pET-28a(+)-ompF plasmid; C SDS-PAGE analysis of the expression of rOmpF in auto-inducing media at different time. 1 mL of cultured cells were collected at 4, 6, 8, 10, 12, 14, and 24 h, respectively. Lane M protein marker (94.4–14.4 kDa); lane 1 4 h; lane 2 6 h; lane 3 8 h; lane 4 10 h; lane 5 12 h; lane 6 14 h; lane 7 24 h; D SDS-PAGE analysis of the purified rOmpF protein. Lane M 5 µL of protein marker (94.4–14.4 kDa); lane 1 total proteins after auto-induction of E. coli BL21 (DE3) containing pET-28a(+)-ompF (15 µL of cell lysis); lane 2 precipitation containing inclusion body after sonication and centrifugation (15 µL of renaturation solution); Lane 3 flowthrough (15 µL of elution solution); Lane 4 the eluent washed with 70% elution buffer (15 µL of the purified OmpF protein); E Western blotting analysis of the OmpF protein. Lane M , protein marker; lane 1 , the OmpF protein
    Figure Legend Snippet: Cloning, expression, purification and Western blotting analysis of OmpF. aA PCR products of the ompF gene. Lane M1 Trans DNA Marker II (1500, 900, 700, 500, 400, 200 and 100 bp); lane 1 ompF PCR products. Lane 2 the recombinant pET-28a(+)-ompF plasmid. Lane 3 the recombinant pET-28a(+)-ompF plasmid digested with BamH I and Xho I; lane M2 Trans 5K DNA Marker (5000, 3000, 2000, 1500, 1000, 800, 500 and 300 bp); B schematic representation of the pET-28a(+)-ompF plasmid; C SDS-PAGE analysis of the expression of rOmpF in auto-inducing media at different time. 1 mL of cultured cells were collected at 4, 6, 8, 10, 12, 14, and 24 h, respectively. Lane M protein marker (94.4–14.4 kDa); lane 1 4 h; lane 2 6 h; lane 3 8 h; lane 4 10 h; lane 5 12 h; lane 6 14 h; lane 7 24 h; D SDS-PAGE analysis of the purified rOmpF protein. Lane M 5 µL of protein marker (94.4–14.4 kDa); lane 1 total proteins after auto-induction of E. coli BL21 (DE3) containing pET-28a(+)-ompF (15 µL of cell lysis); lane 2 precipitation containing inclusion body after sonication and centrifugation (15 µL of renaturation solution); Lane 3 flowthrough (15 µL of elution solution); Lane 4 the eluent washed with 70% elution buffer (15 µL of the purified OmpF protein); E Western blotting analysis of the OmpF protein. Lane M , protein marker; lane 1 , the OmpF protein

    Techniques Used: Clone Assay, Expressing, Purification, Western Blot, Polymerase Chain Reaction, Marker, Recombinant, Positron Emission Tomography, Plasmid Preparation, SDS Page, Cell Culture, Lysis, Sonication, Centrifugation

    26) Product Images from "mRNA Vaccines Encoding the HA Protein of Influenza A H1N1 Virus Delivered by Cationic Lipid Nanoparticles Induce Protective Immune Responses in Mice"

    Article Title: mRNA Vaccines Encoding the HA Protein of Influenza A H1N1 Virus Delivered by Cationic Lipid Nanoparticles Induce Protective Immune Responses in Mice

    Journal: Vaccines

    doi: 10.3390/vaccines8010123

    Construction and verification of mRNA vaccine encoding the H1N1-HA protein. ( a ) Agarose gel electrophoresis of Xho I enzyme digestion products. 1 represents the intact plasmids of pGEM-H1N1-HA-n3 and 2 represents its linearized product of 5355 bp. M1: DL 5,000 DNA Marker (TaKaRa, Tokyo, Japan); M2: DL 2000 DNA Marker (TaKaRa, Tokyo, Japan). ( b,c ) Western blot and indirect immunofluorescence analyses. A549 cells were harvested 12 h and 48 h after transfection. The H1N1-HA protein was detected using rabbit anti-influenza A virus HA Mab. The H1N1-virus group was used as the positive control. Mock represents the negative control. DAPI was used to dye the nuclei.
    Figure Legend Snippet: Construction and verification of mRNA vaccine encoding the H1N1-HA protein. ( a ) Agarose gel electrophoresis of Xho I enzyme digestion products. 1 represents the intact plasmids of pGEM-H1N1-HA-n3 and 2 represents its linearized product of 5355 bp. M1: DL 5,000 DNA Marker (TaKaRa, Tokyo, Japan); M2: DL 2000 DNA Marker (TaKaRa, Tokyo, Japan). ( b,c ) Western blot and indirect immunofluorescence analyses. A549 cells were harvested 12 h and 48 h after transfection. The H1N1-HA protein was detected using rabbit anti-influenza A virus HA Mab. The H1N1-virus group was used as the positive control. Mock represents the negative control. DAPI was used to dye the nuclei.

    Techniques Used: Agarose Gel Electrophoresis, Marker, Western Blot, Immunofluorescence, Transfection, Positive Control, Negative Control

    27) Product Images from "Buffalo early pregnancy biomarker coding sequence cloning and partial length expression in E. coli after codon optimization"

    Article Title: Buffalo early pregnancy biomarker coding sequence cloning and partial length expression in E. coli after codon optimization

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2019.e02863

    (A) Amplification of PAG in buffalo cotyledon and non-pregnant endometrium samples using single L and four right (R1-R4) primer pairs. Lanes: 1-RNA, 2-L-R1, 3-L-R2, 4-L-R3, 5-L-R4, L- DNA ladder (Gene Ruler™ 1kb DNA Ladder Plus cat no N3232S, NEB, USA); (B) Cloning of 1163 and 603bp PCR products in pJET1.2 vector; (C) Amplification of isolated plasmids by gene-specific primers; (D) restriction enzymes digestion results of positive plasmids (L: DNA ladder, lane 1: uncut cloned vector, 2: BamH I digest (single cut), 3: Sal I digest (single cut), 4: Xho I digest (3 cut sites:1164bp, 990bp and 170bp products), 5: Bgl II digest (3 cut sites-1194bp, 1069bp and 113bp products), 6: Eag I digest (single cut) and 7: Pme I digest (single cut) indicating the authenticity of sequence reads.
    Figure Legend Snippet: (A) Amplification of PAG in buffalo cotyledon and non-pregnant endometrium samples using single L and four right (R1-R4) primer pairs. Lanes: 1-RNA, 2-L-R1, 3-L-R2, 4-L-R3, 5-L-R4, L- DNA ladder (Gene Ruler™ 1kb DNA Ladder Plus cat no N3232S, NEB, USA); (B) Cloning of 1163 and 603bp PCR products in pJET1.2 vector; (C) Amplification of isolated plasmids by gene-specific primers; (D) restriction enzymes digestion results of positive plasmids (L: DNA ladder, lane 1: uncut cloned vector, 2: BamH I digest (single cut), 3: Sal I digest (single cut), 4: Xho I digest (3 cut sites:1164bp, 990bp and 170bp products), 5: Bgl II digest (3 cut sites-1194bp, 1069bp and 113bp products), 6: Eag I digest (single cut) and 7: Pme I digest (single cut) indicating the authenticity of sequence reads.

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

    28) Product Images from "Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection"

    Article Title: Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2008.0792

    Strategy to generate insertion vector for rapid selection of rVVs. ( A ) The sequence of the mutated HIV clade C env gene, termed envC , was obtained by PCR (blue box) and cloned into the Xho I and Bam HI sites of the vector pIRES2-GFP. In the second step, the sequence of the expression cassette envC –IRES–GFP was PCR amplified and cloned into vector pCS59 (sites Xho I– Eco RI) under the control of strong synthetic early/late VV promoter (red box). Gray boxes, flanking sequences of the tk gene used for targeting the insertion vector and thereby disrupt the normal VV tk gene. As a consequence, rVVs containing the desired insert are no longer able to express thymidine kinase, and thus cells infected with rVVs become resistant to the toxic effects of BrdU. ( B ) Experimental protocol to accelerate the generation and purification of rVVs. First, 293T cells are exposed to wt VV, followed by transfection of the insertion vector encoding the gene of interest ( envC in our example). After 12 h, the GFP-positive cells are selected using flow cytometry. As an alternative to FACS sorting, rVVs can also be isolated by picking of green 293T cell plaques under a fluorescent microscope. After freezing and thawing, the GFP-positive 293T cell lysates are used to infect 143B Tk − cells in the presence of BrdU (the latter prevents replication of wt VV). To obtain pure clonal rVV stocks, several rounds of plaque purification can be performed. Finally, the amplified rVVs are collected and frozen in aliquots.
    Figure Legend Snippet: Strategy to generate insertion vector for rapid selection of rVVs. ( A ) The sequence of the mutated HIV clade C env gene, termed envC , was obtained by PCR (blue box) and cloned into the Xho I and Bam HI sites of the vector pIRES2-GFP. In the second step, the sequence of the expression cassette envC –IRES–GFP was PCR amplified and cloned into vector pCS59 (sites Xho I– Eco RI) under the control of strong synthetic early/late VV promoter (red box). Gray boxes, flanking sequences of the tk gene used for targeting the insertion vector and thereby disrupt the normal VV tk gene. As a consequence, rVVs containing the desired insert are no longer able to express thymidine kinase, and thus cells infected with rVVs become resistant to the toxic effects of BrdU. ( B ) Experimental protocol to accelerate the generation and purification of rVVs. First, 293T cells are exposed to wt VV, followed by transfection of the insertion vector encoding the gene of interest ( envC in our example). After 12 h, the GFP-positive cells are selected using flow cytometry. As an alternative to FACS sorting, rVVs can also be isolated by picking of green 293T cell plaques under a fluorescent microscope. After freezing and thawing, the GFP-positive 293T cell lysates are used to infect 143B Tk − cells in the presence of BrdU (the latter prevents replication of wt VV). To obtain pure clonal rVV stocks, several rounds of plaque purification can be performed. Finally, the amplified rVVs are collected and frozen in aliquots.

    Techniques Used: Plasmid Preparation, Selection, Sequencing, Polymerase Chain Reaction, Clone Assay, Expressing, Amplification, Infection, Purification, Transfection, Flow Cytometry, Cytometry, FACS, Isolation, Microscopy

    Strategy to generate insertion vector for rapid selection of rVVs. ( A ) The sequence of the mutated HIV clade C env gene, termed envC , was obtained by PCR (blue box) and cloned into the Xho I and Bam HI sites of the vector pIRES2-GFP. In the second step, the sequence of the expression cassette envC –IRES–GFP was PCR amplified and cloned into vector pCS59 (sites Xho I– Eco RI) under the control of strong synthetic early/late VV promoter (red box). Gray boxes, flanking sequences of the tk gene used for targeting the insertion vector and thereby disrupt the normal VV tk gene. As a consequence, rVVs containing the desired insert are no longer able to express thymidine kinase, and thus cells infected with rVVs become resistant to the toxic effects of BrdU. ( B ) Experimental protocol to accelerate the generation and purification of rVVs. First, 293T cells are exposed to wt VV, followed by transfection of the insertion vector encoding the gene of interest ( envC in our example). After 12 h, the GFP-positive cells are selected using flow cytometry. As an alternative to FACS sorting, rVVs can also be isolated by picking of green 293T cell plaques under a fluorescent microscope. After freezing and thawing, the GFP-positive 293T cell lysates are used to infect 143B Tk − cells in the presence of BrdU (the latter prevents replication of wt VV). To obtain pure clonal rVV stocks, several rounds of plaque purification can be performed. Finally, the amplified rVVs are collected and frozen in aliquots.
    Figure Legend Snippet: Strategy to generate insertion vector for rapid selection of rVVs. ( A ) The sequence of the mutated HIV clade C env gene, termed envC , was obtained by PCR (blue box) and cloned into the Xho I and Bam HI sites of the vector pIRES2-GFP. In the second step, the sequence of the expression cassette envC –IRES–GFP was PCR amplified and cloned into vector pCS59 (sites Xho I– Eco RI) under the control of strong synthetic early/late VV promoter (red box). Gray boxes, flanking sequences of the tk gene used for targeting the insertion vector and thereby disrupt the normal VV tk gene. As a consequence, rVVs containing the desired insert are no longer able to express thymidine kinase, and thus cells infected with rVVs become resistant to the toxic effects of BrdU. ( B ) Experimental protocol to accelerate the generation and purification of rVVs. First, 293T cells are exposed to wt VV, followed by transfection of the insertion vector encoding the gene of interest ( envC in our example). After 12 h, the GFP-positive cells are selected using flow cytometry. As an alternative to FACS sorting, rVVs can also be isolated by picking of green 293T cell plaques under a fluorescent microscope. After freezing and thawing, the GFP-positive 293T cell lysates are used to infect 143B Tk − cells in the presence of BrdU (the latter prevents replication of wt VV). To obtain pure clonal rVV stocks, several rounds of plaque purification can be performed. Finally, the amplified rVVs are collected and frozen in aliquots.

    Techniques Used: Plasmid Preparation, Selection, Sequencing, Polymerase Chain Reaction, Clone Assay, Expressing, Amplification, Infection, Purification, Transfection, Flow Cytometry, Cytometry, FACS, Isolation, Microscopy

    Related Articles

    Polymerase Chain Reaction:

    Article Title: A partial form of recessive STAT1 deficiency in humans
    Article Snippet: .. PCR products and empty pSPL3 plasmids (provided by Ralph Burkhardt, The Rockefeller University) were digested with Xho I and BamH 1 (New England BioLabs). .. Plasmids were then dephosphorylated, and 50 ng of purified plasmids and 250 ng of purified PCR products were ligated with T4 ligase (New England BioLabs).

    Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies
    Article Snippet: .. The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions. .. The pCL6-2AEGwo lentiviral backbone vector, a kind gift from Dr. Zanxian Xia, School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China, contains both a “ribosome-skip” fragment encoding the 2A-like peptide APVKQTLNFDLLKLAGDVESNPGP and an in-frame eGFP fluorescent coding sequence downstream of a multiple cloning site.

    Clone Assay:

    Article Title: Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection
    Article Snippet: .. Next, the fragment was digested with Xho I and Bam HI, purified in a 1% agarose gel, and cloned into vector pIRES2-EGFP, which had been linearized with Xho I and Bam HI (New England Biolabs, Ipswich, MA). .. The resulting construct was used as a template to obtain the expression cassette GFP–IRES–envC by PCR.

    Agarose Gel Electrophoresis:

    Article Title: Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection
    Article Snippet: .. Next, the fragment was digested with Xho I and Bam HI, purified in a 1% agarose gel, and cloned into vector pIRES2-EGFP, which had been linearized with Xho I and Bam HI (New England Biolabs, Ipswich, MA). .. The resulting construct was used as a template to obtain the expression cassette GFP–IRES–envC by PCR.

    Article Title: A Germination-Specific Endo-?-Mannanase Gene Is Expressed in the Micropylar Endosperm Cap of Tomato Seeds 1
    Article Snippet: .. Genomic DNA (10 μg) was digested with the restriction enzymes Bam HI, Xba I, and Xho I (New England Biolabs), separated on a 1.0% (w/v) agarose gel, and transferred to positively charged membranes (Hybond-N+ , Amersham Pharmacia Biotech). .. Prehybridization, hybridization, washing, and detection were performed as described for cDNA library screening.

    Article Title: Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site
    Article Snippet: .. The DNA samples were digested with 10 U of Xho I for 2 h at 37 °C (New England BioLabs) and electrophoresed in a 0.8% agarose gel in 0.5X TBE. .. Results Since the BAC cloning vector pBeloBAc11 was commercially available from New England BioLabs, I decided to modify this plasmid to increase its utility for genomics studies.

    Plasmid Preparation:

    Article Title: Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection
    Article Snippet: .. Next, the fragment was digested with Xho I and Bam HI, purified in a 1% agarose gel, and cloned into vector pIRES2-EGFP, which had been linearized with Xho I and Bam HI (New England Biolabs, Ipswich, MA). .. The resulting construct was used as a template to obtain the expression cassette GFP–IRES–envC by PCR.

    Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies
    Article Snippet: .. The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions. .. The pCL6-2AEGwo lentiviral backbone vector, a kind gift from Dr. Zanxian Xia, School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China, contains both a “ribosome-skip” fragment encoding the 2A-like peptide APVKQTLNFDLLKLAGDVESNPGP and an in-frame eGFP fluorescent coding sequence downstream of a multiple cloning site.

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta
    Article Snippet: .. Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs). .. The ExoIII-digested DNAs were treated with S1 nuclease (Sigma) followed by a fill-in reaction with the Klenow fragment of DNA polymerase I, ligation, and transformation into DH5αMCR cells (Gibco BRL).

    Purification:

    Article Title: Generation of Recombinant Vaccinia Viruses via Green Fluorescent Protein Selection
    Article Snippet: .. Next, the fragment was digested with Xho I and Bam HI, purified in a 1% agarose gel, and cloned into vector pIRES2-EGFP, which had been linearized with Xho I and Bam HI (New England Biolabs, Ipswich, MA). .. The resulting construct was used as a template to obtain the expression cassette GFP–IRES–envC by PCR.

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    New England Biolabs xho i
    Structure of the <t>DNA</t> molecules used in this study. A . Structure of the parent c-DNA. B . l-DNA formed by restriction digest of c-DNA with the enzyme Xho I. The size of the linearized plasmid was 4272 bp, C . pcr-DNA (2209 bp) generated by site-specific primers that amplified the region of the c-DNA containing the HTLV promoter, the hOPG open reading frame, and the SV40 Poly-A site. D . Structure of the parent c-DNA used in transfection. E . l-DNA formed by digest of pEGFP-N2 with the enzyme Cla I, which cuts in the vector backbone. F . pcr-DNA (1713 bp) generated by primers that are complementary to the sequence upstream of the CMV promoter and downstream of the polyA site.
    Xho I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs xho i site
    Expression constructs with various LTSM - TSS distances . (A) Schema of the vector construct pRPL18: the promoter region of RPL18 containing an internal start codon (ATG) was cloned into the gene trap vector pT1β-geo in frame with the β-geo gene lacking its start codon. An <t>Xho</t> I restriction site was added to facilitate the insertion of linker sequences of different lengths. (B, C, D) The cells were transfected with a GFP control construct (pGFP) and the five β-geo constructs, one with the endogenous RPL18 promoter including the Xho I site (pRPL18- Xho ) and four with additional linkers of the lengths 4 bp, 29 bp, 53 bp and 117 bp. (B) Northern and Western blotting analyses of β-geo mRNA and protein using a β-geo specific DNA probe and an anti-β-Galactosidase antibody in the respective experiment. (C) One representative X-Gal staining of cells transfected with the different constructs. (D) Overlay of the results of three measures of expression of the construct: mRNA, protein, and enzymatic activity (see B, C). The images were scanned and the signals quantified using ImageQuant. Northern blotting signals were normalized to the rRNA fluorescence intensities of the agarose gels (not shown) and Western blotting signals by reprobing the membranes with an anti-β-actin antibody (not shown). For each experiment the signal of pRPL18- Xho was assigned 100% and the other signals were quantified relative to it.
    Xho I Site, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Structure of the DNA molecules used in this study. A . Structure of the parent c-DNA. B . l-DNA formed by restriction digest of c-DNA with the enzyme Xho I. The size of the linearized plasmid was 4272 bp, C . pcr-DNA (2209 bp) generated by site-specific primers that amplified the region of the c-DNA containing the HTLV promoter, the hOPG open reading frame, and the SV40 Poly-A site. D . Structure of the parent c-DNA used in transfection. E . l-DNA formed by digest of pEGFP-N2 with the enzyme Cla I, which cuts in the vector backbone. F . pcr-DNA (1713 bp) generated by primers that are complementary to the sequence upstream of the CMV promoter and downstream of the polyA site.

    Journal: BMC Biotechnology

    Article Title: Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers

    doi: 10.1186/1472-6750-8-23

    Figure Lengend Snippet: Structure of the DNA molecules used in this study. A . Structure of the parent c-DNA. B . l-DNA formed by restriction digest of c-DNA with the enzyme Xho I. The size of the linearized plasmid was 4272 bp, C . pcr-DNA (2209 bp) generated by site-specific primers that amplified the region of the c-DNA containing the HTLV promoter, the hOPG open reading frame, and the SV40 Poly-A site. D . Structure of the parent c-DNA used in transfection. E . l-DNA formed by digest of pEGFP-N2 with the enzyme Cla I, which cuts in the vector backbone. F . pcr-DNA (1713 bp) generated by primers that are complementary to the sequence upstream of the CMV promoter and downstream of the polyA site.

    Article Snippet: Linear DNA (l-DNA) Purified c-DNA was linearized using the restriction enzyme, Xho I (New England Biolabs; Pickering, ON) for pORF9-hTNFRS11b or Cla I (Invitrogen; Burlington, ON) for pEGFP-N2, Restriction digestion were set up with 5 μg of DNA per 50 μL of reaction volume containing 3 units of enzyme and incubated at 37°C for 16 hours.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Generated, Amplification, Transfection, Sequencing

    Xho I digests of miniprep DNA. Each DNA sample was digested with Xho I and electrophoresed on a 0.8% agarose gel. Lanes 1–7 represent various transformants. Lane 8 is a 1 kb ladder

    Journal: BMC Research Notes

    Article Title: Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site

    doi: 10.1186/s13104-017-2631-8

    Figure Lengend Snippet: Xho I digests of miniprep DNA. Each DNA sample was digested with Xho I and electrophoresed on a 0.8% agarose gel. Lanes 1–7 represent various transformants. Lane 8 is a 1 kb ladder

    Article Snippet: The DNA samples were digested with 10 U of Xho I for 2 h at 37 °C (New England BioLabs) and electrophoresed in a 0.8% agarose gel in 0.5X TBE.

    Techniques: Agarose Gel Electrophoresis

    Design of the mutant loxP oligonucleotide. The 13 bp inverted repeats are underlined . The mutated bases in the 8 bp central spacer are shown in shadowed grey font . The Xho I site is also indicated

    Journal: BMC Research Notes

    Article Title: Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site

    doi: 10.1186/s13104-017-2631-8

    Figure Lengend Snippet: Design of the mutant loxP oligonucleotide. The 13 bp inverted repeats are underlined . The mutated bases in the 8 bp central spacer are shown in shadowed grey font . The Xho I site is also indicated

    Article Snippet: The DNA samples were digested with 10 U of Xho I for 2 h at 37 °C (New England BioLabs) and electrophoresed in a 0.8% agarose gel in 0.5X TBE.

    Techniques: Mutagenesis

    STAT1 P696S would be associated with an ESE defect. ( A ). Nucleotides from exon 23 are shown, with the C2086 nucleotide in the WT sequence and the C2086T substitution in the P696S sequence shown in red. The horizontal blue and green bars show the significance threshold homology score for the binding of SC35 and SRp40 proteins, respectively. The predicted binding sites of these proteins are shown as rectangles along the length of the nucleotide sequence at the height of the homology score. ( B ) The genomic STAT1 region from nucleotide 36989 to 38523 (NC_000002) was inserted into an exon-trapping vector using Xho I and BamH 1, with or without the C2086T (P696S) nucleotide substitution. The exons are numbered in Roman numerals and shown in gray boxes, with the introns between them in white boxes, with the exception of exon 23, which is shown in a red box. The vector is shown as black boxes. HEK293 and COS-7 cells were transfected with the various constructs, the exon-trapping pSPL3 mock vector (pmock-p), or no vector (–). RNA was isolated, and the various spliced products were amplified and are shown on an agarose gel with GADPH amplification. The various products were isolated and sequenced, and the resulting sequences are also shown with corresponding exons and MW. These results are representative of 2 independent experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: A partial form of recessive STAT1 deficiency in humans

    doi: 10.1172/JCI37083

    Figure Lengend Snippet: STAT1 P696S would be associated with an ESE defect. ( A ). Nucleotides from exon 23 are shown, with the C2086 nucleotide in the WT sequence and the C2086T substitution in the P696S sequence shown in red. The horizontal blue and green bars show the significance threshold homology score for the binding of SC35 and SRp40 proteins, respectively. The predicted binding sites of these proteins are shown as rectangles along the length of the nucleotide sequence at the height of the homology score. ( B ) The genomic STAT1 region from nucleotide 36989 to 38523 (NC_000002) was inserted into an exon-trapping vector using Xho I and BamH 1, with or without the C2086T (P696S) nucleotide substitution. The exons are numbered in Roman numerals and shown in gray boxes, with the introns between them in white boxes, with the exception of exon 23, which is shown in a red box. The vector is shown as black boxes. HEK293 and COS-7 cells were transfected with the various constructs, the exon-trapping pSPL3 mock vector (pmock-p), or no vector (–). RNA was isolated, and the various spliced products were amplified and are shown on an agarose gel with GADPH amplification. The various products were isolated and sequenced, and the resulting sequences are also shown with corresponding exons and MW. These results are representative of 2 independent experiments.

    Article Snippet: PCR products and empty pSPL3 plasmids (provided by Ralph Burkhardt, The Rockefeller University) were digested with Xho I and BamH 1 (New England BioLabs).

    Techniques: Sequencing, Binding Assay, Plasmid Preparation, Transfection, Construct, Isolation, Amplification, Agarose Gel Electrophoresis

    Expression constructs with various LTSM - TSS distances . (A) Schema of the vector construct pRPL18: the promoter region of RPL18 containing an internal start codon (ATG) was cloned into the gene trap vector pT1β-geo in frame with the β-geo gene lacking its start codon. An Xho I restriction site was added to facilitate the insertion of linker sequences of different lengths. (B, C, D) The cells were transfected with a GFP control construct (pGFP) and the five β-geo constructs, one with the endogenous RPL18 promoter including the Xho I site (pRPL18- Xho ) and four with additional linkers of the lengths 4 bp, 29 bp, 53 bp and 117 bp. (B) Northern and Western blotting analyses of β-geo mRNA and protein using a β-geo specific DNA probe and an anti-β-Galactosidase antibody in the respective experiment. (C) One representative X-Gal staining of cells transfected with the different constructs. (D) Overlay of the results of three measures of expression of the construct: mRNA, protein, and enzymatic activity (see B, C). The images were scanned and the signals quantified using ImageQuant. Northern blotting signals were normalized to the rRNA fluorescence intensities of the agarose gels (not shown) and Western blotting signals by reprobing the membranes with an anti-β-actin antibody (not shown). For each experiment the signal of pRPL18- Xho was assigned 100% and the other signals were quantified relative to it.

    Journal: BMC Genomics

    Article Title: A tandem sequence motif acts as a distance-dependent enhancer in a set of genes involved in translation by binding the proteins NonO and SFPQ

    doi: 10.1186/1471-2164-12-624

    Figure Lengend Snippet: Expression constructs with various LTSM - TSS distances . (A) Schema of the vector construct pRPL18: the promoter region of RPL18 containing an internal start codon (ATG) was cloned into the gene trap vector pT1β-geo in frame with the β-geo gene lacking its start codon. An Xho I restriction site was added to facilitate the insertion of linker sequences of different lengths. (B, C, D) The cells were transfected with a GFP control construct (pGFP) and the five β-geo constructs, one with the endogenous RPL18 promoter including the Xho I site (pRPL18- Xho ) and four with additional linkers of the lengths 4 bp, 29 bp, 53 bp and 117 bp. (B) Northern and Western blotting analyses of β-geo mRNA and protein using a β-geo specific DNA probe and an anti-β-Galactosidase antibody in the respective experiment. (C) One representative X-Gal staining of cells transfected with the different constructs. (D) Overlay of the results of three measures of expression of the construct: mRNA, protein, and enzymatic activity (see B, C). The images were scanned and the signals quantified using ImageQuant. Northern blotting signals were normalized to the rRNA fluorescence intensities of the agarose gels (not shown) and Western blotting signals by reprobing the membranes with an anti-β-actin antibody (not shown). For each experiment the signal of pRPL18- Xho was assigned 100% and the other signals were quantified relative to it.

    Article Snippet: Three oligonucleotides linkers were generated with using the primer pairs Geo-link 25-fw/rev, Geo-link 50-fw/rev and Geo-link 75-fw/rev (Additional file ) and a fill-in of the Xho I site took place (T4 DNA polymerase, NEB) resulting in a relocation of the LTSM to position +4, +29, +53 and +117 related to the original position of +68 of the RPL18 LTSM.

    Techniques: Expressing, Construct, Plasmid Preparation, Clone Assay, Transfection, Northern Blot, Western Blot, Staining, Activity Assay, Fluorescence