bamhi Thermo Fisher Search Results


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  • 99
    Thermo Fisher bamhi
    A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With <t>BamHI</t> and <t>SalI</t> RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .
    Bamhi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13892 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pcdna3 1
    Zeb1 binds to Six2 promoter and up-regulates Six2 in mK3 cells. ( A ) The predicted binding motifs of Zeb1 to Six2 promoter, which was acquired from the JASPAR Database. The binding motifs across mammal species are conservatively shown in gray shadow representing the matched sequence among several mammal species, for instance, seven base pairs in eight were matched between mouse and human; ( B ) G401 cells were co-transfected with pGL-SV40 (renilla control), firefly luciferase reporter <t>pcDNA3.1-</t> Six2 promoter-luciferase, and either the m. Zeb1 expression plasmid pCDH-copGFP-m. Zeb1 or the respective control vector. Luciferase activity was assayed using dual luciferase reporter assay 48 h after transfection, normalized to renilla control. The result was analyzed by student’s t test and displayed with error bars representing mean ± SD ( n = 3), ** p
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 51485 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna3
    RGC-32 overexpression promotes EMT of BxPC-3 cells . BxPC-3 cells were transiently transfected with RGC-32 plasmid <t>(pcDNA3.1/</t> myc -His C-RGC32) or empty vector (pcDNA3.1/ myc -His C). 72 h after transfection, qPCR (A) and western blot (B and C) were performed to examine the expression of RGC-32, E-cadherin and vimentin at mRNA and protein levels respectively. β-actin was used as an internal control. * P
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    Thermo Fisher lipofectamine 2000
    LeTx causes caspase-1-dependent mΔψ hyperpolarization, and mitochondrial MLN64 and cholesterol accumulation. (A to F) RAW264.7 cells were transfected with scrambled siRNA or mouse caspase-1-specific siRNA (siCasp-1) using <t>Lipofectamine</t> RNAi MAX (Invitrogen) for 40 h. Procaspase-1 protein levels were analyzed by Western blotting (A) and cell death by MTT assays after treatment of cells with LeTx (500 ng/ml LF and 1 μg/ml PA) for 5 h (B). Data are expressed as means and SD ( n = 3). (C) RAW264.7 cells transfected with scrambled siRNA or siCasp-1 were incubated with LeTx (500 ng/ml LF and 1 μg/ml PA) for the indicated times, and total ROS generation was measured by flow cytometry using CM-H 2 DCFDA dye. Data are representative of two independent experiments. (D) Cells were treated with LeTx (500 ng/ml LF and 1 μg/ml PA) for 3 h, and mitochondrial membrane polarization was measured as described in Materials and Methods. Data are representative of three independent experiments. (E and F) Cells were treated with LeTx (500 ng/ml LF and 1 μg/ml PA) for 1 h. Mitochondrion- and late-endosome-rich fractions were isolated and cholesterol contents were measured as described in Materials and Methods. *, P
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ecori
    Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with <t>BamHI</t> and <t>EcoRI</t> (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).
    Ecori, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hindiii
    Schematic representation of the prototype recombinant virus M24-BAC and the promoterless shuttle vector pFLS-ICP4 . ( a ) M24-BAC recombinant virus has the BAC sequences inserted into the UL39 (ICP6) gene of mutant HSV d120. (upper) d120 has a 4.1 kb deletion in both copies of the diploid ICP4 gene. (middle) The ICP6 gene coding the large subunit of viral ribonucleotide reductase is located in the 9.4 kb <t>HindIII</t> restriction fragment of the HSV genome. (lower) The BAC vector sequences were inserted into the d120 genome between the StuI and the XhoI sites of UL39 via homologous recombination using the recombination plasmid pBAC-ICP6EF. The mini F plasmid sequences contain four regulatory genes ( oriS , repE , parA , and parB ) essential for replication and copy number control of the plasmid [19]. H, HindIII; S, StuI; X, XhoI; Open boxes, HSV inverted repeats. The numbers in rectangles at the top of the figure show the lengths in kb of HindIII restriction fragments of d120. The numbers in parentheses show the lengths in kb of terminal restriction fragments. ( b ) Schematic map of the promoterless shuttle vector pFLS-ICP4. MCS is situated upstream of the HSV ICP4 coding sequence so that an exogenous promoter can be inserted to drive ICP4 expression.
    Hindiii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9213 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher xhoi
    Southern blot analysis of plasmid <t>DNA</t> from Enterobacter sp. strains 1061 and 1434 and their transconjugants. Plasmid DNA was digested with the SmaI, <t>XhoI,</t> and BamHI endonucleases in lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12, respectively, and hybridized
    Xhoi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 dna ligase
    gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or <t>T4</t> DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25057 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bamh i
    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the <t>BamH</t> I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.
    Bamh I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher kpni
    Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by <t>KpnI</t> and <t>BamHI/Lane</t> 2: 100-bp DNA ladder marker
    Kpni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher buffer set
    Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by <t>KpnI</t> and <t>BamHI/Lane</t> 2: 100-bp DNA ladder marker
    Buffer Set, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher xbai
    Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the <t>BamHI</t> fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the <t>XbaI</t> fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.
    Xbai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher xhoi sites
    Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the <t>BamHI</t> fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the <t>XbaI</t> fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.
    Xhoi Sites, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2673 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher pcr2 1 topo
    Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the <t>BamHI</t> fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the <t>XbaI</t> fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.
    Pcr2 1 Topo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 11675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr2 1
    Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the <t>BamHI</t> fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the <t>XbaI</t> fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.
    Pcr2 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 12340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher topo ta cloning kit
    <t>PCR</t> results of screening of cloned plasmids after ligation into <t>pCR®-TOPO®</t> with M13 primers Left to right: M, molecular-weight standard 1kbp; lanes 3, 4,7,8 cloned plasmids for k39, respectively; lanes 2, 5,6 non-cloned plasmids; lane 9, Negative control
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 53970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr2 1 topo vector
    Schematic diagram of the construct used to measure expression of human AVPR2 in COS-7 cells. The entire coding region of the wild type AVPR2 cDNA was first subcloned into <t>pCR2.1-TOPO</t> vector, cut with EcoRI and KpnI and cloned into a compatible site of
    Pcr2 1 Topo Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 15098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion high fidelity dna polymerase
    Schematic diagram of the construct used to measure expression of human AVPR2 in COS-7 cells. The entire coding region of the wild type AVPR2 cDNA was first subcloned into <t>pCR2.1-TOPO</t> vector, cut with EcoRI and KpnI and cloned into a compatible site of
    Phusion High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18947 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ndei
    Assembling of the gp053 oligomers. ( A ) A schematic representation of the gp053 oligomeric constructs. The amino acids of the wild-type gp053 within the construct are shown in black, and the insertions with cloning sites are shown in orange. The constructs are named according to the number of copies of the gene 053 . ( B ) The agarose gel electrophoresis analysis of plasmids harbouring the fused 053 genes. The arrows indicate DNA fragments encoding gp053 after hydrolysis with <t>NdeI</t> and <t>XhoI.</t> Lanes: M—molecular mass marker, GeneRuler™ DNA Ladder Mix (Thermo Fisher Scientific, Vilnius, Lithuania); 1—gp053_N_C_mon; 2—gp053_N_C_dim; 3—gp053_N_C_trim; 4—gp053_N_C_tetr; 5—gp053_N_C_pent; and 6—gp053_N_C_hex. ( C ) The SDS-PAGE analysis of the cell-free extracts of the E. coli BL21 (DE3) cells producing the recombinant gp053 oligomers. Lanes: M—molecular mass marker, Page Ruler TM prestained Protein Ladder Plus (Thermo Fisher Scientific, Vilnius, Lithuania); K—pET21a (plasmid vector, control); 1—gp053_N_C_mon; 2—gp053_N_C_dim; 3—gp053_N_C_trim; 4—gp053_N_C_tetr; 5—gp053_N_C_pent; and 6—gp053_N_C_hex. ( D ) The TEM analysis of the structures formed by the recombinant gp053 oligomers. The cell-free extracts were analysed immediately after cell disruption and sample preparation or after incubation with periodical shaking at 22 °C. The incubation time is indicated on the left.
    Ndei, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With BamHI and SalI RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .

    Journal: Jundishapur Journal of Microbiology

    Article Title: Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

    doi: 10.5812/jjm.40303

    Figure Lengend Snippet: A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With BamHI and SalI RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .

    Article Snippet: The pBlue Script II containing fusion truncated ORF2-NSP4 was transformed in E.coli DH5α, cultured in LB agar containing ampicillin (50 mg/L), then plasmid was extracted and double digested by BamHI and SalI restriction enzymes (Thermo Scientific, USA).

    Techniques: Clone Assay

    Characterization of part of the mouse Rad54B genomic locus and generation of mouse ES cells carrying a disrupted mRad54B allele. (A) Part of the mRad54B genomic locus and structure of the targeting construct. Exons 12 to 15 are indicated by black boxes. Shown are the locations of selected restriction sites, EcoRI (E), BamHI (B), BglII (Bg), HindIII (H), and XbaI (X). The positions of two different probes, named A and B, are indicated. (B) DNA blot analysis of G418-resistant ES clones with probe A and EcoRI digested DNA. The wild-type (wt) allele yields a 3.0-kb band, while the disrupted allele results in a 3.6-kb band. Lane 1, wild-type ES cell; lane 2, clone with a randomly integrated targeting construct; lane 3, clone with a homologously integrated targeting construct. (C) RNA blot analysis of mRad54B transcripts in mice carrying the disrupted allele. Total RNA (15 μg) isolated from testes of wild-type, mRad54B +/ − , and mRad54B −/− males was probed with 5′ and 3′ mRad54B cDNA probes. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe served as a loading control.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Contributions of Mammalian Rad54 Paralogs to Recombination, DNA Damage Repair, and Meiosis †

    doi: 10.1128/MCB.26.3.976-989.2006

    Figure Lengend Snippet: Characterization of part of the mouse Rad54B genomic locus and generation of mouse ES cells carrying a disrupted mRad54B allele. (A) Part of the mRad54B genomic locus and structure of the targeting construct. Exons 12 to 15 are indicated by black boxes. Shown are the locations of selected restriction sites, EcoRI (E), BamHI (B), BglII (Bg), HindIII (H), and XbaI (X). The positions of two different probes, named A and B, are indicated. (B) DNA blot analysis of G418-resistant ES clones with probe A and EcoRI digested DNA. The wild-type (wt) allele yields a 3.0-kb band, while the disrupted allele results in a 3.6-kb band. Lane 1, wild-type ES cell; lane 2, clone with a randomly integrated targeting construct; lane 3, clone with a homologously integrated targeting construct. (C) RNA blot analysis of mRad54B transcripts in mice carrying the disrupted allele. Total RNA (15 μg) isolated from testes of wild-type, mRad54B +/ − , and mRad54B −/− males was probed with 5′ and 3′ mRad54B cDNA probes. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe served as a loading control.

    Article Snippet: The hRAD54B cDNA was subcloned from pUC18 into the BamHI site of pFastBac (Invitrogen).

    Techniques: Construct, Clone Assay, Northern blot, Mouse Assay, Isolation

    Zeb1 binds to Six2 promoter and up-regulates Six2 in mK3 cells. ( A ) The predicted binding motifs of Zeb1 to Six2 promoter, which was acquired from the JASPAR Database. The binding motifs across mammal species are conservatively shown in gray shadow representing the matched sequence among several mammal species, for instance, seven base pairs in eight were matched between mouse and human; ( B ) G401 cells were co-transfected with pGL-SV40 (renilla control), firefly luciferase reporter pcDNA3.1- Six2 promoter-luciferase, and either the m. Zeb1 expression plasmid pCDH-copGFP-m. Zeb1 or the respective control vector. Luciferase activity was assayed using dual luciferase reporter assay 48 h after transfection, normalized to renilla control. The result was analyzed by student’s t test and displayed with error bars representing mean ± SD ( n = 3), ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Zeb1 Is a Potential Regulator of Six2 in the Proliferation, Apoptosis and Migration of Metanephric Mesenchyme Cells

    doi: 10.3390/ijms17081283

    Figure Lengend Snippet: Zeb1 binds to Six2 promoter and up-regulates Six2 in mK3 cells. ( A ) The predicted binding motifs of Zeb1 to Six2 promoter, which was acquired from the JASPAR Database. The binding motifs across mammal species are conservatively shown in gray shadow representing the matched sequence among several mammal species, for instance, seven base pairs in eight were matched between mouse and human; ( B ) G401 cells were co-transfected with pGL-SV40 (renilla control), firefly luciferase reporter pcDNA3.1- Six2 promoter-luciferase, and either the m. Zeb1 expression plasmid pCDH-copGFP-m. Zeb1 or the respective control vector. Luciferase activity was assayed using dual luciferase reporter assay 48 h after transfection, normalized to renilla control. The result was analyzed by student’s t test and displayed with error bars representing mean ± SD ( n = 3), ** p

    Article Snippet: The amplification was inserted into the HindIII/BamHI site of pCDNA3.1 (+) (Invitrogen, Carlsbad, CA, USA) using the ligation-independent cloning (LIC) [ , ].

    Techniques: Binding Assay, Sequencing, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Reporter Assay

    Generation of prM-E cell line for VLP production. ZIKV prM-E was PCR amplified by use of specific primers, using the codon-optimized C-prM-E construct as the template (A), and cloned into the pCDNA3.1 expression vector (B). (C) E protein expression was determined by fluorescence microscopy after staining with the MAB10216 antibody. (D) Culture supernatants were harvested from ZIKV prM-E-expressing cells and ultracentrifuged. Cell and virus pellets were lysed, and E protein expression was determined by Western blotting. (E) ZIKV prM-E was PCR amplified and cloned into the lentiviral vector pLenti6/5-D-Topo. 293T cells were then transfected with the pLenti-prM-E construct along with the helper plasmid and VSV-G envelope, and ZIKV prM-E lentiviral particles were harvested at 48 h posttransfection. 293T cells were then transduced with the lentiviral particles, and cells were either bulk selected or selected as single-cell clones by culture in the presence of blasticidin. Selected cells were confirmed to have E protein expression via immunofluorescence assay after staining with the MAB10216 antibody (F) and by flow cytometry (G). The mean fluorescence intensity (MFI) of E protein expression for each prM-E clone is indicated on the right. (H) The indicated pLenti-ZIKV-prM-E cell clones were seeded in equal cell numbers, and culture supernatants were harvested and ultracentrifuged. VLP pellets were lysed and resolved by SDS-PAGE, and E protein expression was determined by Western blotting.

    Journal: Journal of Virology

    Article Title: Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

    doi: 10.1128/JVI.00834-17

    Figure Lengend Snippet: Generation of prM-E cell line for VLP production. ZIKV prM-E was PCR amplified by use of specific primers, using the codon-optimized C-prM-E construct as the template (A), and cloned into the pCDNA3.1 expression vector (B). (C) E protein expression was determined by fluorescence microscopy after staining with the MAB10216 antibody. (D) Culture supernatants were harvested from ZIKV prM-E-expressing cells and ultracentrifuged. Cell and virus pellets were lysed, and E protein expression was determined by Western blotting. (E) ZIKV prM-E was PCR amplified and cloned into the lentiviral vector pLenti6/5-D-Topo. 293T cells were then transfected with the pLenti-prM-E construct along with the helper plasmid and VSV-G envelope, and ZIKV prM-E lentiviral particles were harvested at 48 h posttransfection. 293T cells were then transduced with the lentiviral particles, and cells were either bulk selected or selected as single-cell clones by culture in the presence of blasticidin. Selected cells were confirmed to have E protein expression via immunofluorescence assay after staining with the MAB10216 antibody (F) and by flow cytometry (G). The mean fluorescence intensity (MFI) of E protein expression for each prM-E clone is indicated on the right. (H) The indicated pLenti-ZIKV-prM-E cell clones were seeded in equal cell numbers, and culture supernatants were harvested and ultracentrifuged. VLP pellets were lysed and resolved by SDS-PAGE, and E protein expression was determined by Western blotting.

    Article Snippet: The prM-E construct was generated by PCR amplification of the prM-E region spanning amino acids 105 to 795, using a Phusion high-fidelity PCR kit (New England BioLabs), and was cloned into the pcDNA3.1+ vector (Invitrogen).

    Techniques: Polymerase Chain Reaction, Amplification, Construct, Clone Assay, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Staining, Western Blot, Transfection, Transduction, Immunofluorescence, Flow Cytometry, Cytometry, SDS Page

    Expression of ZIKV C-prM-E by use of a codon-optimized synthetic construct. (A) A codon-optimized ZIKV C-prM-E gene was synthesized via gene synthesis technology, using the sequence available from the current outbreak in the Americas, and cloned into the pcDNA3.1 vector. (B) 293T cells were transfected with the ZIKV C-prM-E or WNV C-prM-E construct. Cells were fixed, stained with the MAB10216 and MAB8150 antibodies, and analyzed for E protein expression by fluorescence microscopy. (C) 293T cells were transfected as described above. At 48 h posttransfection, cells were radiolabeled with [ 35 S]Met/Cys. Cell lysates were immunoprecipitated with protein A beads coated with MAB10216 or anti-WNV serum, resolved by SDS-PAGE, and visualized by PhosphorImager analysis.

    Journal: Journal of Virology

    Article Title: Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

    doi: 10.1128/JVI.00834-17

    Figure Lengend Snippet: Expression of ZIKV C-prM-E by use of a codon-optimized synthetic construct. (A) A codon-optimized ZIKV C-prM-E gene was synthesized via gene synthesis technology, using the sequence available from the current outbreak in the Americas, and cloned into the pcDNA3.1 vector. (B) 293T cells were transfected with the ZIKV C-prM-E or WNV C-prM-E construct. Cells were fixed, stained with the MAB10216 and MAB8150 antibodies, and analyzed for E protein expression by fluorescence microscopy. (C) 293T cells were transfected as described above. At 48 h posttransfection, cells were radiolabeled with [ 35 S]Met/Cys. Cell lysates were immunoprecipitated with protein A beads coated with MAB10216 or anti-WNV serum, resolved by SDS-PAGE, and visualized by PhosphorImager analysis.

    Article Snippet: The prM-E construct was generated by PCR amplification of the prM-E region spanning amino acids 105 to 795, using a Phusion high-fidelity PCR kit (New England BioLabs), and was cloned into the pcDNA3.1+ vector (Invitrogen).

    Techniques: Expressing, Construct, Synthesized, Sequencing, Clone Assay, Plasmid Preparation, Transfection, Staining, Fluorescence, Microscopy, Immunoprecipitation, SDS Page

    Expression of ZIKV prM-E alone releases VLPs into the supernatant, while C-prM-E requires the NS2B-3 protease for efficient VLP release. (A) 293T cells were transfected with the pCDNA3.1 vector expressing ZIKV prM-E or C-prM-E along with the indicated expression vectors. Cells were radiolabeled with [ 35 S]Met/Cys, and culture supernatants were harvested and ultracentrifuged. Cell and virion samples were lysed and immunoprecipitated with MAB10216-coated protein A beads, resolved by SDS-PAGE, and visualized by PhosphorImager analysis. (B) Culture supernatants were harvested from cells expressing ZIKV C-prM-E or prM-E as indicated in Materials and Methods. Twenty-five to 30 ml of supernatant was transferred to ultracentrifuge tubes and carefully underlaid with 5 ml of 25% glycerol in TNE buffer. VLPs were pelleted by centrifugation at 110,500 × g for 3 h at 4°C. Thereafter, the supernatant was carefully removed and the VLP pellet resuspended in TNE buffer. An aliquot of concentrated VLPs was subjected to lysis with 10× RIPA buffer, and E protein in the preparations was detected by Western blotting. (C) VLPs were concentrated as described above, and images were acquired after negative staining, using a JEOL1010 transmission electron microscope with a Hamamatsu digital camera. Bars, ∼30 nm. (D and E) Immunization studies in mice. (D) BALB/c mice were divided into groups of six mice each. Mice received the primary immunization on day 0, followed by 2 boosters, at days 14 and 28, and were finally sacrificed at day 63 post-primary immunization. (E) Mice were divided into 5 groups and received immunizations with either C-prM-E/prM-E DNA or VLPs. For DNA immunization, a total of 50 μg of DNA in a volume of 100 μl PBS was injected intramuscularly (i.m.). For VLPs, the first immunization consisted of a VLP preparation mixed with TiterMax Gold adjuvant in a total volume of 100 μl injected intramuscularly. For subsequent boosters, mice received VLPs alone, without adjuvant. Control mice were injected with PBS.

    Journal: Journal of Virology

    Article Title: Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

    doi: 10.1128/JVI.00834-17

    Figure Lengend Snippet: Expression of ZIKV prM-E alone releases VLPs into the supernatant, while C-prM-E requires the NS2B-3 protease for efficient VLP release. (A) 293T cells were transfected with the pCDNA3.1 vector expressing ZIKV prM-E or C-prM-E along with the indicated expression vectors. Cells were radiolabeled with [ 35 S]Met/Cys, and culture supernatants were harvested and ultracentrifuged. Cell and virion samples were lysed and immunoprecipitated with MAB10216-coated protein A beads, resolved by SDS-PAGE, and visualized by PhosphorImager analysis. (B) Culture supernatants were harvested from cells expressing ZIKV C-prM-E or prM-E as indicated in Materials and Methods. Twenty-five to 30 ml of supernatant was transferred to ultracentrifuge tubes and carefully underlaid with 5 ml of 25% glycerol in TNE buffer. VLPs were pelleted by centrifugation at 110,500 × g for 3 h at 4°C. Thereafter, the supernatant was carefully removed and the VLP pellet resuspended in TNE buffer. An aliquot of concentrated VLPs was subjected to lysis with 10× RIPA buffer, and E protein in the preparations was detected by Western blotting. (C) VLPs were concentrated as described above, and images were acquired after negative staining, using a JEOL1010 transmission electron microscope with a Hamamatsu digital camera. Bars, ∼30 nm. (D and E) Immunization studies in mice. (D) BALB/c mice were divided into groups of six mice each. Mice received the primary immunization on day 0, followed by 2 boosters, at days 14 and 28, and were finally sacrificed at day 63 post-primary immunization. (E) Mice were divided into 5 groups and received immunizations with either C-prM-E/prM-E DNA or VLPs. For DNA immunization, a total of 50 μg of DNA in a volume of 100 μl PBS was injected intramuscularly (i.m.). For VLPs, the first immunization consisted of a VLP preparation mixed with TiterMax Gold adjuvant in a total volume of 100 μl injected intramuscularly. For subsequent boosters, mice received VLPs alone, without adjuvant. Control mice were injected with PBS.

    Article Snippet: The prM-E construct was generated by PCR amplification of the prM-E region spanning amino acids 105 to 795, using a Phusion high-fidelity PCR kit (New England BioLabs), and was cloned into the pcDNA3.1+ vector (Invitrogen).

    Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Centrifugation, Lysis, Western Blot, Negative Staining, Transmission Assay, Microscopy, Mouse Assay, Injection

    In vivo tumor protection experiment to demonstrate the antitumor effect generated by pcDNA3-CRT/E6 against E6-expressing TC-1 tumors and the effects of lymphocyte subsets on tumor protection. (A) Mice were immunized with pcDNA3-CRT, pcDNA3-E6, pcDNA3-CRT/E6, or pcDNA3-CRT/mtE6. One week after vaccination, mice were challenged subcutaneously with 5 × 10 4 TC-1 cells/mouse; they were then monitored for evidence of tumor growth by palpation and inspection twice a week. (B) In vivo antibody depletion experiments to determine the effects of lymphocyte subsets on the tumor protection of the CRT/E6 DNA vaccine. CD4, CD8, and NK1.1 depletions were initiated 1 week before tumor challenge. Data shown in this figure are from one experiment representative of two performed.

    Journal: Journal of Virology

    Article Title: Development of a DNA Vaccine Targeting Human Papillomavirus Type 16 Oncoprotein E6

    doi: 10.1128/JVI.78.16.8468-8476.2004

    Figure Lengend Snippet: In vivo tumor protection experiment to demonstrate the antitumor effect generated by pcDNA3-CRT/E6 against E6-expressing TC-1 tumors and the effects of lymphocyte subsets on tumor protection. (A) Mice were immunized with pcDNA3-CRT, pcDNA3-E6, pcDNA3-CRT/E6, or pcDNA3-CRT/mtE6. One week after vaccination, mice were challenged subcutaneously with 5 × 10 4 TC-1 cells/mouse; they were then monitored for evidence of tumor growth by palpation and inspection twice a week. (B) In vivo antibody depletion experiments to determine the effects of lymphocyte subsets on the tumor protection of the CRT/E6 DNA vaccine. CD4, CD8, and NK1.1 depletions were initiated 1 week before tumor challenge. Data shown in this figure are from one experiment representative of two performed.

    Article Snippet: The amplified E6 DNA was then cloned into the EcoRI/BamHI site of the pcDNA3 vector (Invitrogen, Carlsbad, Calif.).

    Techniques: In Vivo, Generated, Expressing, Mouse Assay

    Intracellular cytokine staining with flow cytometry analysis and chromium release assay to demonstrate that TC-1 cells naturally process the E6 epitope containing aa 50 to 57. (A) Mice were immunized with pcDNA3-CRT/E6 or pcDNA3-CRT/mtE6. Flow cytometry data indicate the number of IFN-γ-expressing CD8 + T-cell precursors generated from splenocytes harvested from vaccinated mice and pulsed with either E6 aa48-57 or E6-expressing TC-1 cells. (B) Bar graph shows the number of E6-specific IFN-γ + CD8 + T cells/3 × 10 5 splenocytes (mean ± SD). (C) The lytic activity of T cells was assessed by using a standard chromium release assay. TC-1 and EL-4 cells were used as target cells. EL-4 cells pulsed with the E6 peptide aa48-57 were used as a positive control. Splenocytes stimulated in vitro with E6 peptide served as effector cells. Percent cytotoxicity (specific lysis) was calculated. Data were collected from cultures measured at E:T ratios of 1:10, 1:1, 10:1, and 25:1. Results are expressed as percent cytotoxicity ± SD.

    Journal: Journal of Virology

    Article Title: Development of a DNA Vaccine Targeting Human Papillomavirus Type 16 Oncoprotein E6

    doi: 10.1128/JVI.78.16.8468-8476.2004

    Figure Lengend Snippet: Intracellular cytokine staining with flow cytometry analysis and chromium release assay to demonstrate that TC-1 cells naturally process the E6 epitope containing aa 50 to 57. (A) Mice were immunized with pcDNA3-CRT/E6 or pcDNA3-CRT/mtE6. Flow cytometry data indicate the number of IFN-γ-expressing CD8 + T-cell precursors generated from splenocytes harvested from vaccinated mice and pulsed with either E6 aa48-57 or E6-expressing TC-1 cells. (B) Bar graph shows the number of E6-specific IFN-γ + CD8 + T cells/3 × 10 5 splenocytes (mean ± SD). (C) The lytic activity of T cells was assessed by using a standard chromium release assay. TC-1 and EL-4 cells were used as target cells. EL-4 cells pulsed with the E6 peptide aa48-57 were used as a positive control. Splenocytes stimulated in vitro with E6 peptide served as effector cells. Percent cytotoxicity (specific lysis) was calculated. Data were collected from cultures measured at E:T ratios of 1:10, 1:1, 10:1, and 25:1. Results are expressed as percent cytotoxicity ± SD.

    Article Snippet: The amplified E6 DNA was then cloned into the EcoRI/BamHI site of the pcDNA3 vector (Invitrogen, Carlsbad, Calif.).

    Techniques: Staining, Flow Cytometry, Cytometry, Release Assay, Mouse Assay, Expressing, Generated, Activity Assay, Positive Control, In Vitro, Lysis

    Intracellular cytokine staining with flow cytometry analysis of IFN-γ-expressing, E6-specific CD8 + T-cell precursors generated by splenocytes stimulated with various E6 peptides. Mice were immunized with pcDNA3-CRT/E6, and splenocytes were collected and cultured. (A) Representative flow cytometry data for splenocytes harvested from mice and either left unstimulated or stimulated overnight with the E6 peptide aa36-80, aa48-57, or aa50-57. (B) Bar graph showing the number of E6-specific IFN-γ-expressing CD8 + T-cell precursors per 3 × 10 5 splenocytes (mean ± SD) generated by in vitro stimulation. Data presented in this figure are from one experiment representative of two performed.

    Journal: Journal of Virology

    Article Title: Development of a DNA Vaccine Targeting Human Papillomavirus Type 16 Oncoprotein E6

    doi: 10.1128/JVI.78.16.8468-8476.2004

    Figure Lengend Snippet: Intracellular cytokine staining with flow cytometry analysis of IFN-γ-expressing, E6-specific CD8 + T-cell precursors generated by splenocytes stimulated with various E6 peptides. Mice were immunized with pcDNA3-CRT/E6, and splenocytes were collected and cultured. (A) Representative flow cytometry data for splenocytes harvested from mice and either left unstimulated or stimulated overnight with the E6 peptide aa36-80, aa48-57, or aa50-57. (B) Bar graph showing the number of E6-specific IFN-γ-expressing CD8 + T-cell precursors per 3 × 10 5 splenocytes (mean ± SD) generated by in vitro stimulation. Data presented in this figure are from one experiment representative of two performed.

    Article Snippet: The amplified E6 DNA was then cloned into the EcoRI/BamHI site of the pcDNA3 vector (Invitrogen, Carlsbad, Calif.).

    Techniques: Staining, Flow Cytometry, Cytometry, Expressing, Generated, Mouse Assay, Cell Culture, In Vitro

    RGC-32 overexpression promotes EMT of BxPC-3 cells . BxPC-3 cells were transiently transfected with RGC-32 plasmid (pcDNA3.1/ myc -His C-RGC32) or empty vector (pcDNA3.1/ myc -His C). 72 h after transfection, qPCR (A) and western blot (B and C) were performed to examine the expression of RGC-32, E-cadherin and vimentin at mRNA and protein levels respectively. β-actin was used as an internal control. * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Response gene to complement-32 enhances metastatic phenotype by mediating transforming growth factor beta-induced epithelial-mesenchymal transition in human pancreatic cancer cell line BxPC-3

    doi: 10.1186/1756-9966-31-29

    Figure Lengend Snippet: RGC-32 overexpression promotes EMT of BxPC-3 cells . BxPC-3 cells were transiently transfected with RGC-32 plasmid (pcDNA3.1/ myc -His C-RGC32) or empty vector (pcDNA3.1/ myc -His C). 72 h after transfection, qPCR (A) and western blot (B and C) were performed to examine the expression of RGC-32, E-cadherin and vimentin at mRNA and protein levels respectively. β-actin was used as an internal control. * P

    Article Snippet: Construction of RGC-32 expression plasmid and RGC-32 short interfering RNA (siRNA) RGC-32 cDNA was amplified from mRNA extracted from BxPC-3 cells and then cloned into pcDNA3.1/myc -His C expression vector(Invitrogen, USA)between Hind III and BamH I restriction sites.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Expressing

    LeTx causes caspase-1-dependent mΔψ hyperpolarization, and mitochondrial MLN64 and cholesterol accumulation. (A to F) RAW264.7 cells were transfected with scrambled siRNA or mouse caspase-1-specific siRNA (siCasp-1) using Lipofectamine RNAi MAX (Invitrogen) for 40 h. Procaspase-1 protein levels were analyzed by Western blotting (A) and cell death by MTT assays after treatment of cells with LeTx (500 ng/ml LF and 1 μg/ml PA) for 5 h (B). Data are expressed as means and SD ( n = 3). (C) RAW264.7 cells transfected with scrambled siRNA or siCasp-1 were incubated with LeTx (500 ng/ml LF and 1 μg/ml PA) for the indicated times, and total ROS generation was measured by flow cytometry using CM-H 2 DCFDA dye. Data are representative of two independent experiments. (D) Cells were treated with LeTx (500 ng/ml LF and 1 μg/ml PA) for 3 h, and mitochondrial membrane polarization was measured as described in Materials and Methods. Data are representative of three independent experiments. (E and F) Cells were treated with LeTx (500 ng/ml LF and 1 μg/ml PA) for 1 h. Mitochondrion- and late-endosome-rich fractions were isolated and cholesterol contents were measured as described in Materials and Methods. *, P

    Journal: Molecular and Cellular Biology

    Article Title: Cellular Adaptation to Anthrax Lethal Toxin-Induced Mitochondrial Cholesterol Enrichment, Hyperpolarization, and Reactive Oxygen Species Generation through Downregulating MLN64 in Macrophages

    doi: 10.1128/MCB.00494-12

    Figure Lengend Snippet: LeTx causes caspase-1-dependent mΔψ hyperpolarization, and mitochondrial MLN64 and cholesterol accumulation. (A to F) RAW264.7 cells were transfected with scrambled siRNA or mouse caspase-1-specific siRNA (siCasp-1) using Lipofectamine RNAi MAX (Invitrogen) for 40 h. Procaspase-1 protein levels were analyzed by Western blotting (A) and cell death by MTT assays after treatment of cells with LeTx (500 ng/ml LF and 1 μg/ml PA) for 5 h (B). Data are expressed as means and SD ( n = 3). (C) RAW264.7 cells transfected with scrambled siRNA or siCasp-1 were incubated with LeTx (500 ng/ml LF and 1 μg/ml PA) for the indicated times, and total ROS generation was measured by flow cytometry using CM-H 2 DCFDA dye. Data are representative of two independent experiments. (D) Cells were treated with LeTx (500 ng/ml LF and 1 μg/ml PA) for 3 h, and mitochondrial membrane polarization was measured as described in Materials and Methods. Data are representative of three independent experiments. (E and F) Cells were treated with LeTx (500 ng/ml LF and 1 μg/ml PA) for 1 h. Mitochondrion- and late-endosome-rich fractions were isolated and cholesterol contents were measured as described in Materials and Methods. *, P

    Article Snippet: Lipofectamine 2000 (Invitrogen) was used for plasmid transfections into RAW 264.7 cells or TIR cells.

    Techniques: Transfection, Western Blot, MTT Assay, Incubation, Flow Cytometry, Cytometry, Isolation

    Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).

    Journal: Genome Research

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences

    doi: 10.1101/gr.094953.109

    Figure Lengend Snippet: Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).

    Article Snippet: The pellets containing ∼5 × 108 nuclei were then resuspended in BamHI buffer and digested with 2000 U each of EcoRI and BamHI (Invitrogen) for 90 min at 37°C with gentle agitation, allowing digested DNA to leach out of the nuclei.

    Techniques:

    Schematic representation of the prototype recombinant virus M24-BAC and the promoterless shuttle vector pFLS-ICP4 . ( a ) M24-BAC recombinant virus has the BAC sequences inserted into the UL39 (ICP6) gene of mutant HSV d120. (upper) d120 has a 4.1 kb deletion in both copies of the diploid ICP4 gene. (middle) The ICP6 gene coding the large subunit of viral ribonucleotide reductase is located in the 9.4 kb HindIII restriction fragment of the HSV genome. (lower) The BAC vector sequences were inserted into the d120 genome between the StuI and the XhoI sites of UL39 via homologous recombination using the recombination plasmid pBAC-ICP6EF. The mini F plasmid sequences contain four regulatory genes ( oriS , repE , parA , and parB ) essential for replication and copy number control of the plasmid [19]. H, HindIII; S, StuI; X, XhoI; Open boxes, HSV inverted repeats. The numbers in rectangles at the top of the figure show the lengths in kb of HindIII restriction fragments of d120. The numbers in parentheses show the lengths in kb of terminal restriction fragments. ( b ) Schematic map of the promoterless shuttle vector pFLS-ICP4. MCS is situated upstream of the HSV ICP4 coding sequence so that an exogenous promoter can be inserted to drive ICP4 expression.

    Journal: BMC Biotechnology

    Article Title: Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

    doi: 10.1186/1472-6750-6-40

    Figure Lengend Snippet: Schematic representation of the prototype recombinant virus M24-BAC and the promoterless shuttle vector pFLS-ICP4 . ( a ) M24-BAC recombinant virus has the BAC sequences inserted into the UL39 (ICP6) gene of mutant HSV d120. (upper) d120 has a 4.1 kb deletion in both copies of the diploid ICP4 gene. (middle) The ICP6 gene coding the large subunit of viral ribonucleotide reductase is located in the 9.4 kb HindIII restriction fragment of the HSV genome. (lower) The BAC vector sequences were inserted into the d120 genome between the StuI and the XhoI sites of UL39 via homologous recombination using the recombination plasmid pBAC-ICP6EF. The mini F plasmid sequences contain four regulatory genes ( oriS , repE , parA , and parB ) essential for replication and copy number control of the plasmid [19]. H, HindIII; S, StuI; X, XhoI; Open boxes, HSV inverted repeats. The numbers in rectangles at the top of the figure show the lengths in kb of HindIII restriction fragments of d120. The numbers in parentheses show the lengths in kb of terminal restriction fragments. ( b ) Schematic map of the promoterless shuttle vector pFLS-ICP4. MCS is situated upstream of the HSV ICP4 coding sequence so that an exogenous promoter can be inserted to drive ICP4 expression.

    Article Snippet: Then the FRT sequence oligomers described above were subcloned into the BamHI/EcoRI sites of pUC-loxP to create pUC-LF, which was subsequently digested with HindIII and NdeI, filled-in and self-ligated to create pUC-LF2. pCMV-LacZV was constructed from pcDNA6-E/Uni-lacZ (Invitrogen) by AgeI and PmlI digestion followed by self-ligation.

    Techniques: Recombinant, BAC Assay, Plasmid Preparation, Mutagenesis, Homologous Recombination, Sequencing, Expressing

    Genomic structure of M24-BAC virus and pM24-BAC plasmid DNA . HindIII restriction analysis of M24-BAC virus DNA (left panel) and pM24-BAC plasmid DNA (right panel) is shown. Closed arrowhead: 9.4 kb HindIII fragment of d120 (containing ICP6 region), Open arrowheads: 15 kb and 2.2 kb HindIII fragments generated by insertion of the BAC sequences. A band present in M24-BAC digest just above the closed triangle is the submolar terminal restriction fragment (9.8 kb) derived from the short terminal repeat. This band is overlapped by the 9.4 kb restriction fragment of d120 and absent from pM24-BAC. The restriction pattern of pM24-BAC indicates that this clone contains both the U L and U S sequences of HSV genome in the forward orientation. Other bands present in the M24-BAC digest but not in pM24-BAC are submolar terminal fragments or L-S junction fragments derived from other isomeric forms of virus genome.

    Journal: BMC Biotechnology

    Article Title: Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

    doi: 10.1186/1472-6750-6-40

    Figure Lengend Snippet: Genomic structure of M24-BAC virus and pM24-BAC plasmid DNA . HindIII restriction analysis of M24-BAC virus DNA (left panel) and pM24-BAC plasmid DNA (right panel) is shown. Closed arrowhead: 9.4 kb HindIII fragment of d120 (containing ICP6 region), Open arrowheads: 15 kb and 2.2 kb HindIII fragments generated by insertion of the BAC sequences. A band present in M24-BAC digest just above the closed triangle is the submolar terminal restriction fragment (9.8 kb) derived from the short terminal repeat. This band is overlapped by the 9.4 kb restriction fragment of d120 and absent from pM24-BAC. The restriction pattern of pM24-BAC indicates that this clone contains both the U L and U S sequences of HSV genome in the forward orientation. Other bands present in the M24-BAC digest but not in pM24-BAC are submolar terminal fragments or L-S junction fragments derived from other isomeric forms of virus genome.

    Article Snippet: Then the FRT sequence oligomers described above were subcloned into the BamHI/EcoRI sites of pUC-loxP to create pUC-LF, which was subsequently digested with HindIII and NdeI, filled-in and self-ligated to create pUC-LF2. pCMV-LacZV was constructed from pcDNA6-E/Uni-lacZ (Invitrogen) by AgeI and PmlI digestion followed by self-ligation.

    Techniques: BAC Assay, Plasmid Preparation, Generated, Derivative Assay

    Analysis of BAC clones after site-specific recombination . (a) HindIII restriction analysis of chloramphenicol/kanamycin double-resistant clones (pM24-BAC-CMV) after Cre-mediated integration between pM24-BAC and pFLS-CMV shuttle vector. Clones #3, 6, and 7 (marked with asterisks) show the expected restriction pattern. Clones #1, 2, 4, 5, and 8 contain an additional 7.8 kb fragment ( closed arrowhead ) and a greater amount of the 6.2 kb fragment ( open arrowhead ) which is consistent with the expected digestion pattern of pM24-BAC-CMV with doubly inserted shuttle vector [see Additional file 3 ]. Clone #10 also contains a greater amount of the 7.8 kb fragment, suggesting insertion of three or more copies of the shuttle vector. The remaining clone (#9) did not undergo recombination and shows a pattern identical to that of pM24-BAC. (b) HindIII restriction analysis of pM24-BAC-null clones obtained after Cre-mediated integration between pM24-BAC and pFLS-XICP4 shuttle vector. Clones #1, 2, 5, 9, 10 and 11 (marked with asterisks) show a restriction pattern consistent with singly integrated pM24-BAC-null. The other seven clones contain a partially deleted, incomplete HSV-BAC genome. The 8.2 kb HindIII fragment containing the BAC backbone ( arrow ) and the two neighboring fragments (20.1 and 2.2 kb; closed arrowheads ) are preserved in all clones, and the 8.6 kb fragment ( open arrowhead ) adjacent to the 2.2 kb fragment is preserved in all clones except clone #13. Other fragments are lost in the deletion clones and new fragments of varying lengths are observed, suggesting that these deletions occurred randomly at different locations. For the HindIII restriction map, see Fig 2a and Additional file 3 .

    Journal: BMC Biotechnology

    Article Title: Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

    doi: 10.1186/1472-6750-6-40

    Figure Lengend Snippet: Analysis of BAC clones after site-specific recombination . (a) HindIII restriction analysis of chloramphenicol/kanamycin double-resistant clones (pM24-BAC-CMV) after Cre-mediated integration between pM24-BAC and pFLS-CMV shuttle vector. Clones #3, 6, and 7 (marked with asterisks) show the expected restriction pattern. Clones #1, 2, 4, 5, and 8 contain an additional 7.8 kb fragment ( closed arrowhead ) and a greater amount of the 6.2 kb fragment ( open arrowhead ) which is consistent with the expected digestion pattern of pM24-BAC-CMV with doubly inserted shuttle vector [see Additional file 3 ]. Clone #10 also contains a greater amount of the 7.8 kb fragment, suggesting insertion of three or more copies of the shuttle vector. The remaining clone (#9) did not undergo recombination and shows a pattern identical to that of pM24-BAC. (b) HindIII restriction analysis of pM24-BAC-null clones obtained after Cre-mediated integration between pM24-BAC and pFLS-XICP4 shuttle vector. Clones #1, 2, 5, 9, 10 and 11 (marked with asterisks) show a restriction pattern consistent with singly integrated pM24-BAC-null. The other seven clones contain a partially deleted, incomplete HSV-BAC genome. The 8.2 kb HindIII fragment containing the BAC backbone ( arrow ) and the two neighboring fragments (20.1 and 2.2 kb; closed arrowheads ) are preserved in all clones, and the 8.6 kb fragment ( open arrowhead ) adjacent to the 2.2 kb fragment is preserved in all clones except clone #13. Other fragments are lost in the deletion clones and new fragments of varying lengths are observed, suggesting that these deletions occurred randomly at different locations. For the HindIII restriction map, see Fig 2a and Additional file 3 .

    Article Snippet: Then the FRT sequence oligomers described above were subcloned into the BamHI/EcoRI sites of pUC-loxP to create pUC-LF, which was subsequently digested with HindIII and NdeI, filled-in and self-ligated to create pUC-LF2. pCMV-LacZV was constructed from pcDNA6-E/Uni-lacZ (Invitrogen) by AgeI and PmlI digestion followed by self-ligation.

    Techniques: BAC Assay, Clone Assay, Plasmid Preparation

    Analysis of genomic DNA from recombinant viruses generated using the Flip-Flop HSV-BAC system . DNA from two independent isolates was purified, digested with HindIII and electrophoresed on an agarose gel. The DNAs of parental viruses bM24-BAC and d120 illustrate the common fragments, while the asterisks denote fragments containing inserts derived from the shuttle vector. [see Additional file 3 ].

    Journal: BMC Biotechnology

    Article Title: Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

    doi: 10.1186/1472-6750-6-40

    Figure Lengend Snippet: Analysis of genomic DNA from recombinant viruses generated using the Flip-Flop HSV-BAC system . DNA from two independent isolates was purified, digested with HindIII and electrophoresed on an agarose gel. The DNAs of parental viruses bM24-BAC and d120 illustrate the common fragments, while the asterisks denote fragments containing inserts derived from the shuttle vector. [see Additional file 3 ].

    Article Snippet: Then the FRT sequence oligomers described above were subcloned into the BamHI/EcoRI sites of pUC-loxP to create pUC-LF, which was subsequently digested with HindIII and NdeI, filled-in and self-ligated to create pUC-LF2. pCMV-LacZV was constructed from pcDNA6-E/Uni-lacZ (Invitrogen) by AgeI and PmlI digestion followed by self-ligation.

    Techniques: Recombinant, Generated, BAC Assay, Purification, Agarose Gel Electrophoresis, Derivative Assay, Plasmid Preparation

    Southern blot analysis of plasmid DNA from Enterobacter sp. strains 1061 and 1434 and their transconjugants. Plasmid DNA was digested with the SmaI, XhoI, and BamHI endonucleases in lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12, respectively, and hybridized

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel

    doi: 10.1128/AAC.49.3.1150-1156.2005

    Figure Lengend Snippet: Southern blot analysis of plasmid DNA from Enterobacter sp. strains 1061 and 1434 and their transconjugants. Plasmid DNA was digested with the SmaI, XhoI, and BamHI endonucleases in lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12, respectively, and hybridized

    Article Snippet: Plasmid DNA was digested with SmaI, XhoI, and BamHI endonucleases (MBI Fermentas); and the resulting restriction pattern was visualized in a 1% agarose gel by ethidium bromide staining.

    Techniques: Southern Blot, Plasmid Preparation

    gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Journal: Nucleic Acids Research

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    doi: 10.1093/nar/gkq1191

    Figure Lengend Snippet: gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Article Snippet: The digested DNA was precipitated by 8 M ammonium acetate and resuspended in water, followed by the addition of T4 DNA ligase (Fermentas) or E. coli DNA ligase (New England Biolabs) in a 25 µl reaction mixture.

    Techniques: Incubation, Plasmid Preparation, Labeling

    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Luciferase, Activity Assay, Generated, Mutagenesis, Construct, Sequencing

    ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Construct, Luciferase, Sequencing

    Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Journal: Iranian Journal of Parasitology

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    doi:

    Figure Lengend Snippet: Electrophoresis of digested pBSC-ptr using enzyme/ Lane 1: Digested pBSC-ptr by KpnI and BamHI/Lane 2: 100-bp DNA ladder marker

    Article Snippet: Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI.

    Techniques: Electrophoresis, Marker

    Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Journal: Iranian Journal of Parasitology

    Article Title: Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct

    doi:

    Figure Lengend Snippet: Identification of recombinant plasmid pcDNA-rPTR using enzyme digestion. Lane 1:100-bp DNA ladder marker. Lane 2: pcDNA-rPTR digested with KpnI and BamHI

    Article Snippet: Construction of the antisense ptr1 gene Recombinant pBluescript containing L. major ptr1 gene (accession no. EF113119) was digested with KpnI and BamHI enzymes and purified using a Fermentas DNA purification kit (Cat. No. k0513) and then subcloned into pcDNA3 digested with KpnI and BamHI.

    Techniques: Recombinant, Plasmid Preparation, Marker

    Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the BamHI fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the XbaI fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.

    Journal: Nucleic Acids Research

    Article Title: Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum

    doi: 10.1093/nar/gni037

    Figure Lengend Snippet: Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the BamHI fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the XbaI fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.

    Article Snippet: The DNA was digested and end-labeled using a five-enzyme, four-color labeling kit consisting of (i) four 6 bp restriction endonucleases, BamHI, Hind III, XbaI and XhoI, producing four different 3′ recessed ends to be labeled, (ii) one 4 bp restriction endonuclease, HaeIII, producing blunted ends and (iii) the SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems).

    Techniques: Generated, Labeling, Multiplex Assay

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

    Journal: Iranian Journal of Parasitology

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

    doi:

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

    Article Snippet: Cloning of PCR products and confirmation of pCR®-TOPO® -k39 and pET-32(+)-k39 Cloning The k39 band, of a predicted length of approximately 843 bp, was ligated into pCR®-TOPO® vector by TOPO®TA Cloning® Kit (Invitrogen, USA) and transformed into E. coli (DH5-alpha competent cells).

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

    Schematic diagram of the construct used to measure expression of human AVPR2 in COS-7 cells. The entire coding region of the wild type AVPR2 cDNA was first subcloned into pCR2.1-TOPO vector, cut with EcoRI and KpnI and cloned into a compatible site of

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Analysis of a novel AVPR2 mutation in a family with nephrogenic diabetes insipidus

    doi:

    Figure Lengend Snippet: Schematic diagram of the construct used to measure expression of human AVPR2 in COS-7 cells. The entire coding region of the wild type AVPR2 cDNA was first subcloned into pCR2.1-TOPO vector, cut with EcoRI and KpnI and cloned into a compatible site of

    Article Snippet: The resulting PCR product was first subcloned into pCR2.1-TOPO vector (Invitrogen) and then cloned between the EcoRI and KpnI sites of pcDNA3 vector with a C-terminal Flag tag [ ] (kindly donated by Dr. Hyang-Sook Rhim [Research Institute of Molecular Genetics, Catholic Research Institute of Medical Science, The Catholic University of Korea, Seoul, Korea]) ( ).

    Techniques: Construct, Expressing, Plasmid Preparation, Clone Assay

    Assembling of the gp053 oligomers. ( A ) A schematic representation of the gp053 oligomeric constructs. The amino acids of the wild-type gp053 within the construct are shown in black, and the insertions with cloning sites are shown in orange. The constructs are named according to the number of copies of the gene 053 . ( B ) The agarose gel electrophoresis analysis of plasmids harbouring the fused 053 genes. The arrows indicate DNA fragments encoding gp053 after hydrolysis with NdeI and XhoI. Lanes: M—molecular mass marker, GeneRuler™ DNA Ladder Mix (Thermo Fisher Scientific, Vilnius, Lithuania); 1—gp053_N_C_mon; 2—gp053_N_C_dim; 3—gp053_N_C_trim; 4—gp053_N_C_tetr; 5—gp053_N_C_pent; and 6—gp053_N_C_hex. ( C ) The SDS-PAGE analysis of the cell-free extracts of the E. coli BL21 (DE3) cells producing the recombinant gp053 oligomers. Lanes: M—molecular mass marker, Page Ruler TM prestained Protein Ladder Plus (Thermo Fisher Scientific, Vilnius, Lithuania); K—pET21a (plasmid vector, control); 1—gp053_N_C_mon; 2—gp053_N_C_dim; 3—gp053_N_C_trim; 4—gp053_N_C_tetr; 5—gp053_N_C_pent; and 6—gp053_N_C_hex. ( D ) The TEM analysis of the structures formed by the recombinant gp053 oligomers. The cell-free extracts were analysed immediately after cell disruption and sample preparation or after incubation with periodical shaking at 22 °C. The incubation time is indicated on the left.

    Journal: Viruses

    Article Title: The Robust Self-Assembling Tubular Nanostructures Formed by gp053 from Phage vB_EcoM_FV3

    doi: 10.3390/v11010050

    Figure Lengend Snippet: Assembling of the gp053 oligomers. ( A ) A schematic representation of the gp053 oligomeric constructs. The amino acids of the wild-type gp053 within the construct are shown in black, and the insertions with cloning sites are shown in orange. The constructs are named according to the number of copies of the gene 053 . ( B ) The agarose gel electrophoresis analysis of plasmids harbouring the fused 053 genes. The arrows indicate DNA fragments encoding gp053 after hydrolysis with NdeI and XhoI. Lanes: M—molecular mass marker, GeneRuler™ DNA Ladder Mix (Thermo Fisher Scientific, Vilnius, Lithuania); 1—gp053_N_C_mon; 2—gp053_N_C_dim; 3—gp053_N_C_trim; 4—gp053_N_C_tetr; 5—gp053_N_C_pent; and 6—gp053_N_C_hex. ( C ) The SDS-PAGE analysis of the cell-free extracts of the E. coli BL21 (DE3) cells producing the recombinant gp053 oligomers. Lanes: M—molecular mass marker, Page Ruler TM prestained Protein Ladder Plus (Thermo Fisher Scientific, Vilnius, Lithuania); K—pET21a (plasmid vector, control); 1—gp053_N_C_mon; 2—gp053_N_C_dim; 3—gp053_N_C_trim; 4—gp053_N_C_tetr; 5—gp053_N_C_pent; and 6—gp053_N_C_hex. ( D ) The TEM analysis of the structures formed by the recombinant gp053 oligomers. The cell-free extracts were analysed immediately after cell disruption and sample preparation or after incubation with periodical shaking at 22 °C. The incubation time is indicated on the left.

    Article Snippet: The purified PCR products were cleaved with NdeI and BamHI/XhoI (Thermo Fisher Scientific, Vilnius, Lithuania) and then inserted into the pET-16b or pET-21a (Novagene, Madison, WI, USA) vectors, digested with the appropriate restriction endonucleases.

    Techniques: Construct, Clone Assay, Agarose Gel Electrophoresis, Marker, SDS Page, Recombinant, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Transmission Electron Microscopy, Sample Prep, Incubation