pcr clean up system  (Promega)

 
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    Name:
    Wizard SV Gel and PCR Clean Up System
    Description:
    Purifies DNA fragments from gel slices or PCR in 15 minutes
    Catalog Number:
    a9281
    Price:
    None
    Category:
    Nucleic Acid Extraction Analysis Nucleic Acid Extraction Clean up and Concentration
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    Structured Review

    Promega pcr clean up system
    Comparison of the sensitivities for bla L1 gene detection by LAMP and conventional <t>PCR</t> methods. Pure genomic <t>DNA</t> extracted from S. maltophilia- K279a was diluted tenfold (379.0 ng/μl to 0.00379 pg/μl) and the DNA assayed by LAMP (A,B) and PCR (C) . (A) Turbidity was monitored using the Loopamp real-time turbidimeter and the OD recorded at 650 nm, at 6 s intervals. (B) Visual inspection of the color change, post-LAMP assay, and in the presence of calcein/Mn 2+ complex. (C) PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. The DNA marker is D2000 DNA Marker (Tiangen Biotech Co., Ltd.) The size is about 179 bp.
    Purifies DNA fragments from gel slices or PCR in 15 minutes
    https://www.bioz.com/result/pcr clean up system/product/Promega
    Average 99 stars, based on 3060 article reviews
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    pcr clean up system - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-β-lactamase blaL1 in Beijing, China"

    Article Title: Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-β-lactamase blaL1 in Beijing, China

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2014.00692

    Comparison of the sensitivities for bla L1 gene detection by LAMP and conventional PCR methods. Pure genomic DNA extracted from S. maltophilia- K279a was diluted tenfold (379.0 ng/μl to 0.00379 pg/μl) and the DNA assayed by LAMP (A,B) and PCR (C) . (A) Turbidity was monitored using the Loopamp real-time turbidimeter and the OD recorded at 650 nm, at 6 s intervals. (B) Visual inspection of the color change, post-LAMP assay, and in the presence of calcein/Mn 2+ complex. (C) PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. The DNA marker is D2000 DNA Marker (Tiangen Biotech Co., Ltd.) The size is about 179 bp.
    Figure Legend Snippet: Comparison of the sensitivities for bla L1 gene detection by LAMP and conventional PCR methods. Pure genomic DNA extracted from S. maltophilia- K279a was diluted tenfold (379.0 ng/μl to 0.00379 pg/μl) and the DNA assayed by LAMP (A,B) and PCR (C) . (A) Turbidity was monitored using the Loopamp real-time turbidimeter and the OD recorded at 650 nm, at 6 s intervals. (B) Visual inspection of the color change, post-LAMP assay, and in the presence of calcein/Mn 2+ complex. (C) PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. The DNA marker is D2000 DNA Marker (Tiangen Biotech Co., Ltd.) The size is about 179 bp.

    Techniques Used: Polymerase Chain Reaction, Lamp Assay, Agarose Gel Electrophoresis, Staining, Marker

    2) Product Images from "Vertebrate Ssu72 Regulates and Coordinates 3′-End Formation of RNAs Transcribed by RNA Polymerase II"

    Article Title: Vertebrate Ssu72 Regulates and Coordinates 3′-End Formation of RNAs Transcribed by RNA Polymerase II

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106040

    Establishment of conditional Ssu72-knockout DT40 cell lines. (A) Schematic representations of the chicken Ssu72 genomic fragment, knockout constructs, and configuration of the targeted alleles. Exons are shown as black boxes (E1–5), and the location of the 5′ probe used for Southern blotting is shown as a grey box. The double-headed arrows above the genes indicate length (in nucleotides). The XbaI and EcoRI restriction sites are indicated by vertical lines labeled X and R, respectively. (B) Southern blot analysis of wild-type (WT), heterozygous mutant (B15), homozygous mutant (P1, P2, P3), and unanticipated rearranged mutant (P10) clones. Genomic DNA obtained from each clone was digested with Xba I and Eco RI, and then hybridized with the 5′ probe shown in panel A. (C) RT-PCR analysis of the wild-type and mutant clones using primer pairs specific for the indicated gene. (D) Immunoblotting analysis of DT40 P3 (−/−) whole-cell extracts treated with Dox for the indicated times, using the indicated antibodies. Western blotting of β-actin was used as to confirm equal protein loading.
    Figure Legend Snippet: Establishment of conditional Ssu72-knockout DT40 cell lines. (A) Schematic representations of the chicken Ssu72 genomic fragment, knockout constructs, and configuration of the targeted alleles. Exons are shown as black boxes (E1–5), and the location of the 5′ probe used for Southern blotting is shown as a grey box. The double-headed arrows above the genes indicate length (in nucleotides). The XbaI and EcoRI restriction sites are indicated by vertical lines labeled X and R, respectively. (B) Southern blot analysis of wild-type (WT), heterozygous mutant (B15), homozygous mutant (P1, P2, P3), and unanticipated rearranged mutant (P10) clones. Genomic DNA obtained from each clone was digested with Xba I and Eco RI, and then hybridized with the 5′ probe shown in panel A. (C) RT-PCR analysis of the wild-type and mutant clones using primer pairs specific for the indicated gene. (D) Immunoblotting analysis of DT40 P3 (−/−) whole-cell extracts treated with Dox for the indicated times, using the indicated antibodies. Western blotting of β-actin was used as to confirm equal protein loading.

    Techniques Used: Knock-Out, Construct, Southern Blot, Labeling, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Western Blot

    3) Product Images from "Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels"

    Article Title: Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels

    Journal: The EMBO Journal

    doi: 10.15252/embj.201489559

    Cay1 and Rap1 genetically interact to maintain telomere homeostasis A Telomere length analysis of Apa I-digested DNA from cay1 Δ trt1 Δ cells harvested at increasing generation doublings (gen). B Telomere length analysis of Apa I-digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control. C PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1-arrested by nitrogen starvation (G1). Genomic DNA was digested with Not I and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused). D Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains. E qRT–PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1 + mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t -test. ** P
    Figure Legend Snippet: Cay1 and Rap1 genetically interact to maintain telomere homeostasis A Telomere length analysis of Apa I-digested DNA from cay1 Δ trt1 Δ cells harvested at increasing generation doublings (gen). B Telomere length analysis of Apa I-digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control. C PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1-arrested by nitrogen starvation (G1). Genomic DNA was digested with Not I and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused). D Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains. E qRT–PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1 + mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t -test. ** P

    Techniques Used: Northern Blot, Quantitative RT-PCR, Two Tailed Test

    4) Product Images from "A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells"

    Article Title: A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells

    Journal: Antioxidants

    doi: 10.3390/antiox8110518

    H 2 O 2 production and release by cultured KGN cells and effects of exogenous H 2 O 2 . ( A ) NOX4 RT-PCR analysis and Western blot of cultured KGN cells show single bands of 160 bp and 68 kDa, respectively. Controls using RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Hydrogen peroxide assay of untreated KGN cells showed increasing H 2 O 2 levels in the supernatant over a time period of 2 h ( n = 6). Signal intensities were normalized to start point values. Bars indicate SEM. ( C ) Exogenously added H 2 O 2 reduced cell viability in a dose dependent manner. Cell counts after treatment of KGN cells with different concentrations of H 2 O 2 for 24 h ( n = 2–5 for each concentration) are shown with an interpolated sigmoidal standard curve ( r 2 = 0.9361). Bars indicate SEM. ( D ) Images of KGN cells treated with H 2 O 2 (1 mM) for 24 h compared to untreated control cells. Scale bars indicate 200 µm.
    Figure Legend Snippet: H 2 O 2 production and release by cultured KGN cells and effects of exogenous H 2 O 2 . ( A ) NOX4 RT-PCR analysis and Western blot of cultured KGN cells show single bands of 160 bp and 68 kDa, respectively. Controls using RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Hydrogen peroxide assay of untreated KGN cells showed increasing H 2 O 2 levels in the supernatant over a time period of 2 h ( n = 6). Signal intensities were normalized to start point values. Bars indicate SEM. ( C ) Exogenously added H 2 O 2 reduced cell viability in a dose dependent manner. Cell counts after treatment of KGN cells with different concentrations of H 2 O 2 for 24 h ( n = 2–5 for each concentration) are shown with an interpolated sigmoidal standard curve ( r 2 = 0.9361). Bars indicate SEM. ( D ) Images of KGN cells treated with H 2 O 2 (1 mM) for 24 h compared to untreated control cells. Scale bars indicate 200 µm.

    Techniques Used: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, H2O2 Assay, Concentration Assay

    KGN cells express functional TRPM2. ( A ) TRPM2 RT-PCR shows a band at 103 bp. Controls with RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Addition of H 2 O 2 (1 mM) increased the fluorescence signal of the four individual KGN cells shown, which were loaded with the Ca 2+ -sensitive dye Fluoforte. Background signals were subtracted and fluorescence is shown relative to the respective start value of each region of interest (ROI). ( C ) Fluorescence images, taken before ( a ) and after ( b ) the first stimulation with H 2 O 2 . ( D ) Treatment with the inhibitor (ACA; 20 µM), 4 h prior to and during the measurement, blocked the Ca 2+ increase upon stimulation with H 2 O 2 , but not with 0.05‰ trypsin (T). Images ( c – f ) represent the indicated time points. ( E ) The H 2 O 2 -derived Ca 2+ increase was obtained in the DMSO control and thus ruled out solvent effects. Images ( g – j ) represent the indicated time points. The pseudo-color scale shown in ( c ) applies for all live cell images. Colored frames mark the cells represented in the corresponding graphs. Scale bars indicate 50 µm.
    Figure Legend Snippet: KGN cells express functional TRPM2. ( A ) TRPM2 RT-PCR shows a band at 103 bp. Controls with RNA (-RT) or H 2 O instead of cDNA (H 2 O) were negative. ( B ) Addition of H 2 O 2 (1 mM) increased the fluorescence signal of the four individual KGN cells shown, which were loaded with the Ca 2+ -sensitive dye Fluoforte. Background signals were subtracted and fluorescence is shown relative to the respective start value of each region of interest (ROI). ( C ) Fluorescence images, taken before ( a ) and after ( b ) the first stimulation with H 2 O 2 . ( D ) Treatment with the inhibitor (ACA; 20 µM), 4 h prior to and during the measurement, blocked the Ca 2+ increase upon stimulation with H 2 O 2 , but not with 0.05‰ trypsin (T). Images ( c – f ) represent the indicated time points. ( E ) The H 2 O 2 -derived Ca 2+ increase was obtained in the DMSO control and thus ruled out solvent effects. Images ( g – j ) represent the indicated time points. The pseudo-color scale shown in ( c ) applies for all live cell images. Colored frames mark the cells represented in the corresponding graphs. Scale bars indicate 50 µm.

    Techniques Used: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Derivative Assay

    5) Product Images from "Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici"

    Article Title: Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici

    Journal: Fungal Genetics and Biology

    doi: 10.1016/j.fgb.2015.03.023

    Agarose gels showing the outcome of control PCR experiments. (A) DNA fragments of 5′ end of the myosin chitin synthase 1 ( mcs1 ) were amplified using primers CC-125 and CC-117 (see Table 1 ). In all three preparations, no PCR fragment was found in the absence of template (control), whereas strong bands of 707 bp appeared after PCR on total RNA preparations (lanes 3 and 4). These bands were not present when RNA which had been pre-treated with DNase I to remove contaminating genomic DNA (lanes 5 and 6). After transcribing this purified RNA into cDNA, PCR product of 585 bp appeared confirming the splicing of 122 bp predicted intron. The absence of 122 bp intron on the cDNA product was further confirmed by cDNA sequencing. Note that (1/10) and (1/50) indicate dilutions (1/10: 1 part RNA, 9 parts water; 1/50: 1 part RNA, 49 parts water). (B) Random amplification of yeast colonies with match maker PCR mix generated products with maximum sizes of 2000 bp in all three cDNA libraries (only IPO323_Yeasts shown). This suggests that entire open reading frames of proteins, up to ∼600–700 aa long, are represented in the library. Note that PCRs designed to amplify shorter fragments (585 bp and 1544 bp) of the chitin synthase gene mcs1 (5568 bp without introns) still produced positive bands (see main text). This suggests that fragments of larger genes are also represented in the libraries. (C) Primers were designed to amplify the entire open reading frame of the small GTPases rab7 (815 bp) and rab11 (807 bp) (see Table 1 , rab7 : primers SK-Sep-63 and SK-Sep-64; rab11 : primers SK-Sep-65 and SK-Sep-66). Both open reading frames were amplified from genomic DNA of IPO323. Smaller fragments (615 bp and 633 bp) were found after PCR reactions using cDNA from all three preparations (IPO323_Yeasts, IPO323_Hyphae, K4418_mixed). This corresponds with the predicted presence of introns in both genes ( rab7 : 815 bp; rab11 : 807 bp; see main text for more details) and further confirmed by DNA sequencing.
    Figure Legend Snippet: Agarose gels showing the outcome of control PCR experiments. (A) DNA fragments of 5′ end of the myosin chitin synthase 1 ( mcs1 ) were amplified using primers CC-125 and CC-117 (see Table 1 ). In all three preparations, no PCR fragment was found in the absence of template (control), whereas strong bands of 707 bp appeared after PCR on total RNA preparations (lanes 3 and 4). These bands were not present when RNA which had been pre-treated with DNase I to remove contaminating genomic DNA (lanes 5 and 6). After transcribing this purified RNA into cDNA, PCR product of 585 bp appeared confirming the splicing of 122 bp predicted intron. The absence of 122 bp intron on the cDNA product was further confirmed by cDNA sequencing. Note that (1/10) and (1/50) indicate dilutions (1/10: 1 part RNA, 9 parts water; 1/50: 1 part RNA, 49 parts water). (B) Random amplification of yeast colonies with match maker PCR mix generated products with maximum sizes of 2000 bp in all three cDNA libraries (only IPO323_Yeasts shown). This suggests that entire open reading frames of proteins, up to ∼600–700 aa long, are represented in the library. Note that PCRs designed to amplify shorter fragments (585 bp and 1544 bp) of the chitin synthase gene mcs1 (5568 bp without introns) still produced positive bands (see main text). This suggests that fragments of larger genes are also represented in the libraries. (C) Primers were designed to amplify the entire open reading frame of the small GTPases rab7 (815 bp) and rab11 (807 bp) (see Table 1 , rab7 : primers SK-Sep-63 and SK-Sep-64; rab11 : primers SK-Sep-65 and SK-Sep-66). Both open reading frames were amplified from genomic DNA of IPO323. Smaller fragments (615 bp and 633 bp) were found after PCR reactions using cDNA from all three preparations (IPO323_Yeasts, IPO323_Hyphae, K4418_mixed). This corresponds with the predicted presence of introns in both genes ( rab7 : 815 bp; rab11 : 807 bp; see main text for more details) and further confirmed by DNA sequencing.

    Techniques Used: Polymerase Chain Reaction, Amplification, Purification, Sequencing, Generated, Produced, DNA Sequencing

    6) Product Images from "Assessing product adulteration of Eurycoma longifolia (Tongkat Ali) herbal medicinal product using DNA barcoding and HPLC analysis"

    Article Title: Assessing product adulteration of Eurycoma longifolia (Tongkat Ali) herbal medicinal product using DNA barcoding and HPLC analysis

    Journal: Pharmaceutical Biology

    doi: 10.1080/13880209.2018.1479869

    Agarose gel electrophoresis of several rbc L PCR (A) and ITS2 fragment (B). Lane C shows negative control. The successful recombinant plasmid of PEasy- rbc L (C) and PEasy-ITS2 recombinant plasmid (D) is shown. 1 kb DNA ladder (Promega, Madison, WI).
    Figure Legend Snippet: Agarose gel electrophoresis of several rbc L PCR (A) and ITS2 fragment (B). Lane C shows negative control. The successful recombinant plasmid of PEasy- rbc L (C) and PEasy-ITS2 recombinant plasmid (D) is shown. 1 kb DNA ladder (Promega, Madison, WI).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Recombinant, Plasmid Preparation

    7) Product Images from "Switching secretory pathway direction for organelle acquisition in plants"

    Article Title: Switching secretory pathway direction for organelle acquisition in plants

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.956961

    Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from T-DNA-insertion lines. (B) A maximum likelihood phylogenetic tree of proteins with one ERF/AP2 domain. The color code is shown in Fig S1A . The branch lengths are proportional to the estimated number of substitutions per site. Bootstrap probability is indicated as a percentage on each branch with at least 50% support. A more detailed tree was presented previously ( 20 ). (C) Schematic representation of the Mp ERF13 gene structure and mutations generated in this study. Gray and black boxes indicate the UTR and coding sequences, respectively. Asterisks with numbers indicate sites of designed gRNA to generate Mp erf13-1 ge (*1 and *4) and Mp erf13-2 ge (*2 and *3). (D) PCR-based genotyping of Tak-1, Mp erf13 GOF , Mp erf13-1 ge , and Mp erf13-2 ge . The combinations and annealing sites of primers (a to d) are shown in (C). (E) The genomic and predicted amino acid sequences of the Mp ERF13 locus in Tak-1 and Mp erf13 mutants. Light blue, dark blue, and magenta letters indicate UTR, coding, and predicted amino acid sequences, respectively. The PAM sequences for gRNAs are underlined. The caret indicates the indel site. (F) Fluorescent and bright-field images of BODIPY-stained three-week-old thalli of Tak-1, Mp erf13-1 ge , and Mp erf13-2 ge . Bars = 0.5 mm. (G) The number of oil bodies visualized with BODIPY in a unit area (2.0 mm × 2.0 mm). Bars indicate means ± SD. Statistical analyses between Tak-1 and each genotype were conducted using a two-tailed Welch’s t -test. Sample numbers were 23 thalli for Tak-1, 24 for Mp erf13-1 ge , and 26 for Mp erf13-2 ge . p -values are 5.14×10 −11 for Mp erf13-1 ge and 5.14×10 −11 for Mp erf13-2 ge .
    Figure Legend Snippet: Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from T-DNA-insertion lines. (B) A maximum likelihood phylogenetic tree of proteins with one ERF/AP2 domain. The color code is shown in Fig S1A . The branch lengths are proportional to the estimated number of substitutions per site. Bootstrap probability is indicated as a percentage on each branch with at least 50% support. A more detailed tree was presented previously ( 20 ). (C) Schematic representation of the Mp ERF13 gene structure and mutations generated in this study. Gray and black boxes indicate the UTR and coding sequences, respectively. Asterisks with numbers indicate sites of designed gRNA to generate Mp erf13-1 ge (*1 and *4) and Mp erf13-2 ge (*2 and *3). (D) PCR-based genotyping of Tak-1, Mp erf13 GOF , Mp erf13-1 ge , and Mp erf13-2 ge . The combinations and annealing sites of primers (a to d) are shown in (C). (E) The genomic and predicted amino acid sequences of the Mp ERF13 locus in Tak-1 and Mp erf13 mutants. Light blue, dark blue, and magenta letters indicate UTR, coding, and predicted amino acid sequences, respectively. The PAM sequences for gRNAs are underlined. The caret indicates the indel site. (F) Fluorescent and bright-field images of BODIPY-stained three-week-old thalli of Tak-1, Mp erf13-1 ge , and Mp erf13-2 ge . Bars = 0.5 mm. (G) The number of oil bodies visualized with BODIPY in a unit area (2.0 mm × 2.0 mm). Bars indicate means ± SD. Statistical analyses between Tak-1 and each genotype were conducted using a two-tailed Welch’s t -test. Sample numbers were 23 thalli for Tak-1, 24 for Mp erf13-1 ge , and 26 for Mp erf13-2 ge . p -values are 5.14×10 −11 for Mp erf13-1 ge and 5.14×10 −11 for Mp erf13-2 ge .

    Techniques Used: Generated, Polymerase Chain Reaction, Staining, Two Tailed Test

    8) Product Images from "Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants"

    Article Title: Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190526

    Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Multigene cloning with Plant X-tender expression vectors. Two expression cassettes were cloned into pCAMBIA_ASX and introduced into N . benthamiana . (A-F) Scheme of cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extension homologies in the case of p35S::H2BRFP_tNOS expression cassette. PCR amplification of subunits (pNOS, ECFP, t35S) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid in the case of pNOS::ECFP_t35S expression cassette. (B) Assembly of subunits into Hin dIII digested Level 0 vectors by NEBuilder HiFi assembly method. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions were released from the backbone using Pme I. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by TAR or NEBuilder HiFi. (E) Release of the multigene construct from Level 1 vector using I- Sce I homing endonuclease, cutting outside the homology regions A0 and B0. (F) Assembly of two expression cassettes and yeast selection marker ( URA3 ) into Hin dIII digested Plant X-tender expression vectors with SLiCE of NEBuilder HiFi. (G–J) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_multigene (upper panel) or with empty A . tumefaciens (bottom panel). (G) Nuclear localisation of RFP. Fluorescence is represented as a maximum projection of z-stacks. (H) ECFP is localised in the cytoplasm. Fluorescence is represented as maximum projections of z-stacks. (I) Bright field. (J) Overlay of G, H and I. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, pNOS: nopaline synthase promoter, ECFP: cyan fluorescent protein, t35S: cauliflower mosaic virus CaMV 35S terminator, A0, A1 AR, B0: homology regions, Rp: selection marker conferring hygromycin resistance in plants, Re: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method. TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Techniques Used: Clone Assay, Expressing, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Construct, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay

    Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Functional evaluation of constructed vectors by cloning expression cassette p35S::H2BRFP_tNOS into Plant X-tender expression vectors. (A-F) Scheme of the cloning procedure. (A) Amplification of expression cassette from template plasmid using primers with appropriate 5’ and 3’ extensions to add A0 and AR homology regions. (B) Expression cassette assembly in Hin dIII restricted pL0A_0-R Level 0 vector by NEBuilder HiFi assembly method. (C) Release of expression cassette with flanking homology regions A0 and AR from Level 0 vector by Pme I digestion. (D) Assembly of expression cassette with flanking homology regions A0 and AR into Pac I digested pL1A-hc / pL1A-lc (A0/AR) Level 1 vector by TAR or NEBuilder HiFi. (E) Release of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 from Level 1 vector by I- Sce I digestion. (F) Assembly of expression cassette flanked by URA3 yeast selection marker and homology regions A0 and B0 into Plant X-tender expression vectors by SLiCE or NEBuilder HiFi. (G-I) Images of agroinfiltrated N . benthamiana leaves obtained by laser scanning confocal microscopy. Leaves were agroinfiltrated with agrobacteria containing pCAMBIA_ASX_cassette, pK7WG_ASX_cassette, pH7WG_ASX_cassette, pB7WG_ASX_cassette or empty agrobacteria (top to bottom). (G) Nuclear localisation of RFP. Fluorescence is represented as maximum projections of z-stacks. (H) Bright field. (I) Overlay of G with H. Scale bars are 100 μm. p35S: cauliflower mosaic virus CaMV 35S promoter, H2BRFP: histon sequence fused to red fluorescence protein (mRFP1), tNOS: nopaline synthase terminator, A0, AR, B0: homology regions, Rp: selection marker conferring resistance in plants (hygromycin in the case of pCAMBIA_ASX and pH7WG_ASX, kanamycin in the case of pK7WG_ASX, glufosinate-ammonium in the case of pB7WG_ASX), Re: selection marker conferring resistance in E . coli and A . tumefaciens (kanamycin in the case of pCAMBIA_ASX, spectinomycinin in the case of pK7WG_ASX, pH7WG_ASX and pB7WG_ASX), Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , LB: left border of T-DNA, RB: right border of T-DNA, Hin dIII, I- Sce I, Pac I, Pme I: restriction enzyme recognition sites, URA3 : yeast selection marker, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: NEBuilder HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, ASX: Plant X-tender expression vector.

    Techniques Used: Functional Assay, Construct, Clone Assay, Expressing, Amplification, Plasmid Preparation, Selection, Marker, Confocal Microscopy, Fluorescence, Sequencing, Ligation, Transformation Assay, Polymerase Chain Reaction

    Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.
    Figure Legend Snippet: Plant X-tender cloning strategy. Diagram showing example of assembly of two expression cassettes into a plant expression vector using Plant X-tender. Definition of parts and design of Level 0 units is done using GenoCAD. Design of multigene cassettes and computation of primers is performed using the AssemblX webtool. (A-D) Assembly of two expression cassettes into a Level 1 vector. (A) PCR amplification of subunits (e.g. promoter, CDS, terminator) using custom-designed primers with appropriate 5’ extensions to add overlaps between the individual subunits and chosen Level 0 plasmid. (B) Assembly of subunits into Hin dIII digested Level 0 vectors via overlap-based assembly methods. Only the restriction of Level 0 vector with A0/A1 homology regions is shown. (C) Assembled cassettes flanked by homology regions are released from the backbone using one of five rare 8-base cutter recognition sites ( Asc I, Sbf I, Swa I, Fsa I, Pme I) flanking the homology regions. (D) Assembly of expression cassettes into Pac I digested Level 1 vector by of the preferred overlap-based assembly method. (E-G) Multigene assembly into Plant X-tender expression vector. (E) Digestion with I- Sce I allows the release of a multigene construct flanked by homology regions A0 and B0 from the Level 1 AssemblX vector. (F) Hin dIII digestion enables the linearization of Plant X-tender expression vector and the release of ccd B cassette prior the assembly. (G) Assembly of a multigene construct and a yeast selection marker ( URA3 ) flanked by homology regions into Plant X-tender expression vector by overlap-based methods exploiting homologous recombination between the homology regions A0 and B0 of the Plant X-tender expression vector and the homology regions A0 and B0 of the insert. A0, A1, AR, B0: homology regions, Hin dIII, I- Sce I, Pac I, Asc I, Sbf I, Swa I, Fse I, Pme I: restriction enzyme recognition sites, Rp: selection marker conferring resistance in plants, Re: selection marker conferring resistance in E . coli and A . tumefaciens , Amp: selection marker conferring ampicillin resistance in E . coli and A . tumefaciens , Kan: selection marker conferring kanamycin resistance in E . coli and A . tumefaciens , URA3 : yeast selection marker, LB: left border of T-DNA, RB: right border of T-DNA, ccd B: bacterial suicide gene, SLiCE: Seamless ligation cloning extract cloning method, HiFi: HiFi DNA assembly method, Gibson: Gibson DNA assembly method, TAR: cloning based on transformation-associated recombination, PCR: Polymerase chain reaction, CDS: coding sequence, ASX: Plant X-tender expression vector.

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Construct, Selection, Marker, Homologous Recombination, Ligation, Transformation Assay, Sequencing

    9) Product Images from "Premature Sperm Activation and Defective Spermatogenesis Caused by Loss of spe-46 Function in Caenorhabditis elegans"

    Article Title: Premature Sperm Activation and Defective Spermatogenesis Caused by Loss of spe-46 Function in Caenorhabditis elegans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057266

    Products of RT-PCR reactions using RNA from wild-type (N2) worms, hermaphrodites that produce only sperm [ fem-3(q23) ], hermaphrodties that produce only oocytes [ fem-1(hc17) ], and spe-46(hc197) mutant hermaphrodites. The reactions were multiplexed with primers for both the spe-46 transcript and the transcript for the C. elegans β-Actin homolog act-2 . The molecular weight marker (MW) is lambda phage cut with PstI. While non-specific products were amplified, the specific amplicons for both spe-46 and act-2 were present. In particular, the spe-46 product is present in all lanes but fem-1 , a strain that makes no sperm. The size of each specific amplicon is indicated.
    Figure Legend Snippet: Products of RT-PCR reactions using RNA from wild-type (N2) worms, hermaphrodites that produce only sperm [ fem-3(q23) ], hermaphrodties that produce only oocytes [ fem-1(hc17) ], and spe-46(hc197) mutant hermaphrodites. The reactions were multiplexed with primers for both the spe-46 transcript and the transcript for the C. elegans β-Actin homolog act-2 . The molecular weight marker (MW) is lambda phage cut with PstI. While non-specific products were amplified, the specific amplicons for both spe-46 and act-2 were present. In particular, the spe-46 product is present in all lanes but fem-1 , a strain that makes no sperm. The size of each specific amplicon is indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Activated Clotting Time Assay, Molecular Weight, Marker, Amplification

    10) Product Images from "Identification and characterisation of short chain rhamnolipid production in a previously uninvestigated, non-pathogenic marine pseudomonad"

    Article Title: Identification and characterisation of short chain rhamnolipid production in a previously uninvestigated, non-pathogenic marine pseudomonad

    Journal: Applied Microbiology and Biotechnology

    doi: 10.1007/s00253-018-9202-3

    DNA fragments resulting from PCR amplification of rhamnolipid synthesis genes rhlA ( a ) and rhlB ( b ). PCR products were separated by molecular weight on a 1.5% ( w / v ) agarose gel, imaged under UV light using SybrSafe DNA strains ( Thermo Fisher Scientific ). Samples from left to right on each gel; 1 kb Plus DNA marker ( Thermo Fisher Scientific ), amplification product from P. aeruginosa PAO1, amplification product from Pseudomonas sp. MCTG214(3b1)
    Figure Legend Snippet: DNA fragments resulting from PCR amplification of rhamnolipid synthesis genes rhlA ( a ) and rhlB ( b ). PCR products were separated by molecular weight on a 1.5% ( w / v ) agarose gel, imaged under UV light using SybrSafe DNA strains ( Thermo Fisher Scientific ). Samples from left to right on each gel; 1 kb Plus DNA marker ( Thermo Fisher Scientific ), amplification product from P. aeruginosa PAO1, amplification product from Pseudomonas sp. MCTG214(3b1)

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Agarose Gel Electrophoresis, Marker

    11) Product Images from "SIMON: Simple methods for analyzing DNA methylation by targeted bisulfite next-generation sequencing"

    Article Title: SIMON: Simple methods for analyzing DNA methylation by targeted bisulfite next-generation sequencing

    Journal: Plant Biotechnology

    doi: 10.5511/plantbiotechnology.19.0822a

    Figure 2. Experimental workflow and data processing of the SIMON method. A) Schematics of the steps for the preparation of DNA samples from plant material and bisulfite treatment, B) schematics of the steps for PCR amplification of loci of interest by using barcode-extended primers and a mixture of the amplicons into one solution, C) schematics of the sample preparation steps required for the NGS run and NGS, and D) schematics of the steps constituting the primary data analysis of the NGS raw data. Each experimental procedure and each analytical step is indicated by an arrow. The details of each step are illustrated or described. The method explains how sample materials are prepared for NGS, and then how the raw data from NGS are processed to provide methylation counts at each cytosine position.
    Figure Legend Snippet: Figure 2. Experimental workflow and data processing of the SIMON method. A) Schematics of the steps for the preparation of DNA samples from plant material and bisulfite treatment, B) schematics of the steps for PCR amplification of loci of interest by using barcode-extended primers and a mixture of the amplicons into one solution, C) schematics of the sample preparation steps required for the NGS run and NGS, and D) schematics of the steps constituting the primary data analysis of the NGS raw data. Each experimental procedure and each analytical step is indicated by an arrow. The details of each step are illustrated or described. The method explains how sample materials are prepared for NGS, and then how the raw data from NGS are processed to provide methylation counts at each cytosine position.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sample Prep, Next-Generation Sequencing, Methylation

    12) Product Images from "Water Transport by Aquaporins in the Extant Plant Physcomitrella patens 1 1 [W]"

    Article Title: Water Transport by Aquaporins in the Extant Plant Physcomitrella patens 1 1 [W]

    Journal: Plant Physiology

    doi: 10.1104/pp.107.111351

    Expression pattern of PIP2;1 , PIP2;2 , and PIP2;3 transcripts in P. patens wild type. For each gene, amplification with 20-mer specific primers on genomic DNA was included as a control (A, lane G). RT-PCR analyses were performed on RNA from 4-d-old protonema
    Figure Legend Snippet: Expression pattern of PIP2;1 , PIP2;2 , and PIP2;3 transcripts in P. patens wild type. For each gene, amplification with 20-mer specific primers on genomic DNA was included as a control (A, lane G). RT-PCR analyses were performed on RNA from 4-d-old protonema

    Techniques Used: Expressing, Amplification, Reverse Transcription Polymerase Chain Reaction

    13) Product Images from "Anti-inflammatory and Intestinal Barrier-protective Activities of Commensal Lactobacilli and Bifidobacteria in Thoroughbreds: Role of Probiotics in Diarrhea Prevention in Neonatal Thoroughbreds"

    Article Title: Anti-inflammatory and Intestinal Barrier-protective Activities of Commensal Lactobacilli and Bifidobacteria in Thoroughbreds: Role of Probiotics in Diarrhea Prevention in Neonatal Thoroughbreds

    Journal: Journal of Equine Science

    doi: 10.1294/jes.25.37

    DGGE analysis of the PCR products of lactic acid bacteria present in the neonatal Thoroughbred feces by lactic acid bacteria-specific primers [ 25 ]. Approximately 200 bp 16S rDNA of E. coli No. 341–534 were amplified by PCR. Lane 1: meconium, lane 2: feces obtained on the 7th day after birth, lane 3: feces obtained on the 14th day after birth, lane 4: feces obtained on the 21st day after birth. Band a: L. johnsonii (100% similarity), band b: L. equi (100% similarity), band c: L. ruminis (99.9% similarity), and band d: L. reuteri (99.8% similarity).
    Figure Legend Snippet: DGGE analysis of the PCR products of lactic acid bacteria present in the neonatal Thoroughbred feces by lactic acid bacteria-specific primers [ 25 ]. Approximately 200 bp 16S rDNA of E. coli No. 341–534 were amplified by PCR. Lane 1: meconium, lane 2: feces obtained on the 7th day after birth, lane 3: feces obtained on the 14th day after birth, lane 4: feces obtained on the 21st day after birth. Band a: L. johnsonii (100% similarity), band b: L. equi (100% similarity), band c: L. ruminis (99.9% similarity), and band d: L. reuteri (99.8% similarity).

    Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction, Amplification

    14) Product Images from "Hepatocyte nuclear factor (HNF) 4α transactivation of cytochrome P450 (Cyp) 2d40 promoter is enhanced during pregnancy in mice"

    Article Title: Hepatocyte nuclear factor (HNF) 4α transactivation of cytochrome P450 (Cyp) 2d40 promoter is enhanced during pregnancy in mice

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2015.01.001

    HNF4α recruitment to Cyp2d40 promoter increases at term pregnancy Liver tissues were collected from Tg-CYP2D6 mice at pre-pregnancy (P0), 17 days of pregnancy (P17), and 7 days post-partum (PP7). ChIP assays were performed using HNF4α antibody (or IgG as a control), and the pulled-down DNA was quantified by qRT-PCR using a set of primers that bind −171/−67 of Cyp2d40 (n=7, mean ± S.D.; *, p
    Figure Legend Snippet: HNF4α recruitment to Cyp2d40 promoter increases at term pregnancy Liver tissues were collected from Tg-CYP2D6 mice at pre-pregnancy (P0), 17 days of pregnancy (P17), and 7 days post-partum (PP7). ChIP assays were performed using HNF4α antibody (or IgG as a control), and the pulled-down DNA was quantified by qRT-PCR using a set of primers that bind −171/−67 of Cyp2d40 (n=7, mean ± S.D.; *, p

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR

    HNF4α is critical for Cyp2d40 basal expression and induction during pregnancy Liver tissues were collected from Hnf4α(wt/wt) and Hnf4α( −/ wt) mice at pre-pregnancy (P0), 17 days of pregnancy (P17), and 7 days post-partum (PP7). mRNA levels of Cyp2d40 were determined by qRT-PCR and normalized by that of Hnf4α(wt/wt) mice at P0 (n=4, mean ± S.D.; **, p
    Figure Legend Snippet: HNF4α is critical for Cyp2d40 basal expression and induction during pregnancy Liver tissues were collected from Hnf4α(wt/wt) and Hnf4α( −/ wt) mice at pre-pregnancy (P0), 17 days of pregnancy (P17), and 7 days post-partum (PP7). mRNA levels of Cyp2d40 were determined by qRT-PCR and normalized by that of Hnf4α(wt/wt) mice at P0 (n=4, mean ± S.D.; **, p

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR

    Hepatic Cyp2d40 is induced in both wild-type and Tg-CYP2D6 mice during pregnancy (A) Liver tissues of wild-type nonpregnant female mice were collected (n=4). mRNA expression levels of Cyp2ds were determined by qRT-PCR and normalized by Cyp2d40 expression. (B) and (C) Liver tissues of wild-type (B) and Tg-CYP2D6 (C) mice were collected at different gestational time points: pre-pregnancy (P0), 7, 14, or 21 days of pregnancy (P7, P14, P21, respectively), 7 days postpartum (PP7). mRNA levels of Cyp2d40 were determined by qRT-PCR and normalized by that in the pre-pregnancy group (n=4, mean ± S.D.; **, p
    Figure Legend Snippet: Hepatic Cyp2d40 is induced in both wild-type and Tg-CYP2D6 mice during pregnancy (A) Liver tissues of wild-type nonpregnant female mice were collected (n=4). mRNA expression levels of Cyp2ds were determined by qRT-PCR and normalized by Cyp2d40 expression. (B) and (C) Liver tissues of wild-type (B) and Tg-CYP2D6 (C) mice were collected at different gestational time points: pre-pregnancy (P0), 7, 14, or 21 days of pregnancy (P7, P14, P21, respectively), 7 days postpartum (PP7). mRNA levels of Cyp2d40 were determined by qRT-PCR and normalized by that in the pre-pregnancy group (n=4, mean ± S.D.; **, p

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR

    15) Product Images from "Fibronectin-binding protein B variation in Staphylococcus aureus"

    Article Title: Fibronectin-binding protein B variation in Staphylococcus aureus

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-10-160

    FnBPB A domain typing of S.aureus strains by dot blot hybridisation . DNA fragments coding for the entire A domain of fnbB were amplified by PCR from clinical S.aureus isolates. PCR products were spotted onto nitrocellulose membranes and probed with DIG-labelled probes specific for fnbB isotype I (A), II (B), III (C) and IV (D). fnbB DNA from strains 8325-4, N315, MSSA476 and P1 was used as control.
    Figure Legend Snippet: FnBPB A domain typing of S.aureus strains by dot blot hybridisation . DNA fragments coding for the entire A domain of fnbB were amplified by PCR from clinical S.aureus isolates. PCR products were spotted onto nitrocellulose membranes and probed with DIG-labelled probes specific for fnbB isotype I (A), II (B), III (C) and IV (D). fnbB DNA from strains 8325-4, N315, MSSA476 and P1 was used as control.

    Techniques Used: Dot Blot, Hybridization, Amplification, Polymerase Chain Reaction

    16) Product Images from "Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri ▿"

    Article Title: Requirement of the galU Gene for Polysaccharide Production by and Pathogenicity and Growth In Planta of Xanthomonas citri subsp. citri ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02897-09

    Sequence analysis of EZ-Tn5 insertion in the galU mutants. (A) Genomic location of galU on the X. citri subsp. citri chromosome and transposon insertion sites in the galU mutants. kefB encodes a transport protein, galU encodes a UTP-glucose-1-phosphate uridylyltransferase, and XCC2293 encodes a dehydratase protein. Bg, BglII restriction site. (B) PCR analysis confirming insertion of EZ-Tn5 into the galU gene: agarose gel electrophoresis of DNA amplified using primers galU -F1 and galU -R1 targeting the interior region of the galU gene from the X. citri subsp. citri wild-type 306, D12, and F6 strains. Lane 1, Invitrogen 1 Kb Plus DNA size marker; lane 2, D12, lane 3, F6, lane 4, X. citri subsp. citri 306. (C) Southern blot of DNA of X. citri subsp. citri wild-type strain 306 and galU mutants F6 and D12 digested with BglII. The membrane was probed with a 675-bp kan-2 gene fragment that was amplified using PCR with primers Kan-F1 and Kan-R1. Wt, wild type.
    Figure Legend Snippet: Sequence analysis of EZ-Tn5 insertion in the galU mutants. (A) Genomic location of galU on the X. citri subsp. citri chromosome and transposon insertion sites in the galU mutants. kefB encodes a transport protein, galU encodes a UTP-glucose-1-phosphate uridylyltransferase, and XCC2293 encodes a dehydratase protein. Bg, BglII restriction site. (B) PCR analysis confirming insertion of EZ-Tn5 into the galU gene: agarose gel electrophoresis of DNA amplified using primers galU -F1 and galU -R1 targeting the interior region of the galU gene from the X. citri subsp. citri wild-type 306, D12, and F6 strains. Lane 1, Invitrogen 1 Kb Plus DNA size marker; lane 2, D12, lane 3, F6, lane 4, X. citri subsp. citri 306. (C) Southern blot of DNA of X. citri subsp. citri wild-type strain 306 and galU mutants F6 and D12 digested with BglII. The membrane was probed with a 675-bp kan-2 gene fragment that was amplified using PCR with primers Kan-F1 and Kan-R1. Wt, wild type.

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

    17) Product Images from "Functional Analysis of a Novel FOXL2 Indel Mutation in Chinese Families with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome Type I"

    Article Title: Functional Analysis of a Novel FOXL2 Indel Mutation in Chinese Families with Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome Type I

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.19532

    StAR promoter activity, as determined using reporter gene assays, RT-PCR and EMSA. (A) As reflected by the luciferase activity, WT FOXL2 represses StAR promoter activity in a dose-dependent manner. However, there is no such change in luciferase activity in the presence of mutant FOXL2 or empty vector. Statistically significant differences are indicated by * P
    Figure Legend Snippet: StAR promoter activity, as determined using reporter gene assays, RT-PCR and EMSA. (A) As reflected by the luciferase activity, WT FOXL2 represses StAR promoter activity in a dose-dependent manner. However, there is no such change in luciferase activity in the presence of mutant FOXL2 or empty vector. Statistically significant differences are indicated by * P

    Techniques Used: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Luciferase, Mutagenesis, Plasmid Preparation

    18) Product Images from "Distribution of genes related to Type 6 secretion system and lipooligosaccharide that induced ganglioside mimicry among Campylobacter jejuni isolated from human diarrhea in Thailand"

    Article Title: Distribution of genes related to Type 6 secretion system and lipooligosaccharide that induced ganglioside mimicry among Campylobacter jejuni isolated from human diarrhea in Thailand

    Journal: Gut Pathogens

    doi: 10.1186/s13099-020-00357-6

    Gel electrophoresis of amplified products of GBS-associated LOS genes from selected C. jejuni isolates from travelers and children. PCR products representing cgtA (527 bp), cgtB (502 bp), cstII HS2 (417 bp), cstII HS19 (400 bp), and lpxA genes from C. jejuni isolates in this study. Lane M, 100 bp DNA maker; Lane 1, ATCC 43432 (LOS class A1); Lane 2, ATCC 43438 (LOS class A2); Lane 3, ATCC 43449 (LOS class B2); Lane 4, ATCC 43456 (LOS class B1); Lane 5, 6: NCTC 11168 and ATCC 43429 (LOS class C); Lane 7, LIO87 (LOS class D); Lane 8, ATCC 43434 (LOS class E); Lane 9, negative control (nuclease-free water); Lane 10, 11, and 13, amplified product of selected C. jejuni isolates from travelers CG-01-09510 (HS2), CG-03-4049 (HS23/36), and CG-99-8135 (HS23/36), respectively; Lane 12, 14, and 15, amplified products of selected C. jejuni isolates from children, SR-08-150 (HS2), BH-01-193 (HS15), and KR-08-207 (HS4 cpxAB ), respectively
    Figure Legend Snippet: Gel electrophoresis of amplified products of GBS-associated LOS genes from selected C. jejuni isolates from travelers and children. PCR products representing cgtA (527 bp), cgtB (502 bp), cstII HS2 (417 bp), cstII HS19 (400 bp), and lpxA genes from C. jejuni isolates in this study. Lane M, 100 bp DNA maker; Lane 1, ATCC 43432 (LOS class A1); Lane 2, ATCC 43438 (LOS class A2); Lane 3, ATCC 43449 (LOS class B2); Lane 4, ATCC 43456 (LOS class B1); Lane 5, 6: NCTC 11168 and ATCC 43429 (LOS class C); Lane 7, LIO87 (LOS class D); Lane 8, ATCC 43434 (LOS class E); Lane 9, negative control (nuclease-free water); Lane 10, 11, and 13, amplified product of selected C. jejuni isolates from travelers CG-01-09510 (HS2), CG-03-4049 (HS23/36), and CG-99-8135 (HS23/36), respectively; Lane 12, 14, and 15, amplified products of selected C. jejuni isolates from children, SR-08-150 (HS2), BH-01-193 (HS15), and KR-08-207 (HS4 cpxAB ), respectively

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Polymerase Chain Reaction, Negative Control

    19) Product Images from "Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load"

    Article Title: Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

    Journal: Biotechnology Research International

    doi: 10.4061/2011/964831

    Correlation between the NucliSens EasyQ HIV-1 v1.1 test (bioMerieux, Boxtel, The Netherlands) and the in-house qRT-PCR assay, on 14 plasma specimens from HIV-1 infected patients. The solid line represents the identity line, where all determinations should fall if a perfect correlation between the two assays was achieved. r 2 , determination coefficient; r , correlation coefficient.
    Figure Legend Snippet: Correlation between the NucliSens EasyQ HIV-1 v1.1 test (bioMerieux, Boxtel, The Netherlands) and the in-house qRT-PCR assay, on 14 plasma specimens from HIV-1 infected patients. The solid line represents the identity line, where all determinations should fall if a perfect correlation between the two assays was achieved. r 2 , determination coefficient; r , correlation coefficient.

    Techniques Used: Quantitative RT-PCR, Infection

    20) Product Images from "Allele frequency of antiretroviral host factor TRIMCyp in wild-caught cynomolgus macaques (Macaca fascicularis)"

    Article Title: Allele frequency of antiretroviral host factor TRIMCyp in wild-caught cynomolgus macaques (Macaca fascicularis)

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2012.00314

    Determination of CypA insertion. (A) Diagram indicating splicing of TRIM5α or TRIMCyp. Noncoding and coding exons and CypA sequences are shown in white, black, and shaded white, respectively. F and R denote forward and reverse primers used in this study, respectively. (B) The genomic DNA was extracted from whole blood. To test for CypA insertion, the 3′ region of the TRIM5 gene was amplified by PCR with primers spanning the 3′ UTR and the putative CypA insertion. M and DW denote DNA molecular weight standard marker and water control, respectively.
    Figure Legend Snippet: Determination of CypA insertion. (A) Diagram indicating splicing of TRIM5α or TRIMCyp. Noncoding and coding exons and CypA sequences are shown in white, black, and shaded white, respectively. F and R denote forward and reverse primers used in this study, respectively. (B) The genomic DNA was extracted from whole blood. To test for CypA insertion, the 3′ region of the TRIM5 gene was amplified by PCR with primers spanning the 3′ UTR and the putative CypA insertion. M and DW denote DNA molecular weight standard marker and water control, respectively.

    Techniques Used: Amplification, Polymerase Chain Reaction, Molecular Weight, Marker

    21) Product Images from "Prevalence of virulence genes in strains of Campylobacter jejuni isolated from human, bovine and broiler"

    Article Title: Prevalence of virulence genes in strains of Campylobacter jejuni isolated from human, bovine and broiler

    Journal: Brazilian Journal of Microbiology

    doi:

    Purified polymerase chain reaction products from the cccj , cdtB , csrA, cst-II, ggt and virB11 genes. Strain: C. jejuni 81176. M, DNA Molecular Weight Marker.
    Figure Legend Snippet: Purified polymerase chain reaction products from the cccj , cdtB , csrA, cst-II, ggt and virB11 genes. Strain: C. jejuni 81176. M, DNA Molecular Weight Marker.

    Techniques Used: Purification, Polymerase Chain Reaction, Molecular Weight, Marker

    22) Product Images from "Alteration of Light-Dependent Gene Regulation by the Absence of the RCO-1/RCM-1 Repressor Complex in the Fungus Neurospora crassa"

    Article Title: Alteration of Light-Dependent Gene Regulation by the Absence of the RCO-1/RCM-1 Repressor Complex in the Fungus Neurospora crassa

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095069

    The absence of RCO-1 modifies the kinetics of WCC binding to the promoters of light-regulated genes. A. WCC binding sites in the promoters of several light-regulated genes. The position of the WCC binding sites in the promoters of frq (proximal site, frq-p ; distal site, frq-d ), wc-1 , vvd , al-1 and al-3 are shown relative to the initiator ATG. B. Kinetics of WCC binding to the promoters measured by chromatin immunoprecipitation with an antibody against WC-2. Mycelia were grown for 48 hours at 30°C in the dark and then exposed for different times to light. Nuclei were extracted prior to each ChIP experiment. Quantitative PCR were performed to measure the relative accumulation of each DNA segment in immunoprecipitated samples and inputs. Each plot shows the average and standard error of the mean of DNA accumulation in three independent experiments. Results from each PCR for each gene were normalized to the corresponding PCR for 28S DNA to correct for sampling errors, and plotted relative to the amount obtained in the dark.
    Figure Legend Snippet: The absence of RCO-1 modifies the kinetics of WCC binding to the promoters of light-regulated genes. A. WCC binding sites in the promoters of several light-regulated genes. The position of the WCC binding sites in the promoters of frq (proximal site, frq-p ; distal site, frq-d ), wc-1 , vvd , al-1 and al-3 are shown relative to the initiator ATG. B. Kinetics of WCC binding to the promoters measured by chromatin immunoprecipitation with an antibody against WC-2. Mycelia were grown for 48 hours at 30°C in the dark and then exposed for different times to light. Nuclei were extracted prior to each ChIP experiment. Quantitative PCR were performed to measure the relative accumulation of each DNA segment in immunoprecipitated samples and inputs. Each plot shows the average and standard error of the mean of DNA accumulation in three independent experiments. Results from each PCR for each gene were normalized to the corresponding PCR for 28S DNA to correct for sampling errors, and plotted relative to the amount obtained in the dark.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction, Sampling

    23) Product Images from "CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice"

    Article Title: CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

    Journal: Science Advances

    doi: 10.1126/sciadv.1602814

    DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated exon skipping. ( A ) Two gRNAs [either gRNA (g2 or g3), which target intron 50, and the other (g1), which targets exon 51] were used to direct Cpf1-mediated removal of the exon 51 splice acceptor site. ( B ) T7E1 assay using 293T cells transfected with LbCpf1 and gRNA2 (g2) or gRNA3 (g3) shows cleavage of the DMD locus at intron 50. Red arrowheads denote cleavage products. M, marker; Ctrl, control. ( C ) PCR products of genomic DNA isolated from DMD-iPSCs transfected with a plasmid expressing LbCpf1, g1 + g2, and GFP. The lower band (red arrowhead) indicates removal of the exon 51 splice acceptor site. ( D ) Sequence of the lower PCR band from (C) shows a 200-bp deletion, spanning from the 3′ end of intron 50 to the 5′ end of exon 51. This confirms removal of the “ag” splice acceptor of exon 51. The sequence of the uncorrected allele is shown above that of the LbCpf1-edited allele. ( E ) RT-PCR of iPSC-derived cardiomyocytes using primer sets described in Fig. 2B . The 700-bp band in the WT lane is the dystrophin transcript from exons 47 to 52; the 300-bp band in the uncorrected lane is the dystrophin transcript from exons 47 to 52 with exon 48 to 50 deletion; and the lower band in the g1 + g2 mixture lane (edited by LbCpf1) shows exon 51 skipping. ( F )Sequence of the lower band from (E) (g1 + g2 mixture lane) confirms skipping of exon 51, which reframed the DMD ORF. ( G ) Western blot analysis shows dystrophin protein expression in iPSC-derived cardiomyocyte mixtures after exon 51 skipping by LbCpf1 with g1 + g2. αMHC is loading control. ( H ) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte mixtures following Cpf1-mediated exon skipping with g1 + g2 gRNA compared to WT and uncorrected cardiomyocyte mixtures. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm.
    Figure Legend Snippet: DMD iPSC-derived cardiomyocytes express dystrophin after Cpf1-mediated exon skipping. ( A ) Two gRNAs [either gRNA (g2 or g3), which target intron 50, and the other (g1), which targets exon 51] were used to direct Cpf1-mediated removal of the exon 51 splice acceptor site. ( B ) T7E1 assay using 293T cells transfected with LbCpf1 and gRNA2 (g2) or gRNA3 (g3) shows cleavage of the DMD locus at intron 50. Red arrowheads denote cleavage products. M, marker; Ctrl, control. ( C ) PCR products of genomic DNA isolated from DMD-iPSCs transfected with a plasmid expressing LbCpf1, g1 + g2, and GFP. The lower band (red arrowhead) indicates removal of the exon 51 splice acceptor site. ( D ) Sequence of the lower PCR band from (C) shows a 200-bp deletion, spanning from the 3′ end of intron 50 to the 5′ end of exon 51. This confirms removal of the “ag” splice acceptor of exon 51. The sequence of the uncorrected allele is shown above that of the LbCpf1-edited allele. ( E ) RT-PCR of iPSC-derived cardiomyocytes using primer sets described in Fig. 2B . The 700-bp band in the WT lane is the dystrophin transcript from exons 47 to 52; the 300-bp band in the uncorrected lane is the dystrophin transcript from exons 47 to 52 with exon 48 to 50 deletion; and the lower band in the g1 + g2 mixture lane (edited by LbCpf1) shows exon 51 skipping. ( F )Sequence of the lower band from (E) (g1 + g2 mixture lane) confirms skipping of exon 51, which reframed the DMD ORF. ( G ) Western blot analysis shows dystrophin protein expression in iPSC-derived cardiomyocyte mixtures after exon 51 skipping by LbCpf1 with g1 + g2. αMHC is loading control. ( H ) Immunocytochemistry shows dystrophin expression in iPSC-derived cardiomyocyte mixtures following Cpf1-mediated exon skipping with g1 + g2 gRNA compared to WT and uncorrected cardiomyocyte mixtures. Red, dystrophin staining; green, troponin I staining. Scale bar, 100 μm.

    Techniques Used: Derivative Assay, Transfection, Marker, Polymerase Chain Reaction, Isolation, Plasmid Preparation, Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunocytochemistry, Staining

    24) Product Images from "The CCTL ( Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro"

    Article Title: The CCTL ( Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx018

    Cpf1-assisted substitution of actII-orf4 P with erm P in act cluster. ( A ) Schematic chart for substitution of the actII-orf4 promoter, employing Cpf1-assisted cleavage. A strong promoter was PCR amplified, containing the same flanking sequences as the actII-orf4 promoter. A pair of 17-nt spacer sequences were chosen nearby the promoter region, and both promoters were then cleaved by Cpf1 in vitro , followed by DNA ligation to form an engineered cluster. ( B and C ) The production of actinorhodin in both solid (B) and liquid (C) R2YE medium. HIW- erm P and HIW represented Streptomyces sp . 4F harboring pHIW- erm P and pHIW expression vector, while blank represented the wild-type Streptomyces sp . 4F. ( D ) Transcriptional levels of actII-orf4 and the target genes of actI and actIII in HIW- erm P and HIW strains, respectively.
    Figure Legend Snippet: Cpf1-assisted substitution of actII-orf4 P with erm P in act cluster. ( A ) Schematic chart for substitution of the actII-orf4 promoter, employing Cpf1-assisted cleavage. A strong promoter was PCR amplified, containing the same flanking sequences as the actII-orf4 promoter. A pair of 17-nt spacer sequences were chosen nearby the promoter region, and both promoters were then cleaved by Cpf1 in vitro , followed by DNA ligation to form an engineered cluster. ( B and C ) The production of actinorhodin in both solid (B) and liquid (C) R2YE medium. HIW- erm P and HIW represented Streptomyces sp . 4F harboring pHIW- erm P and pHIW expression vector, while blank represented the wild-type Streptomyces sp . 4F. ( D ) Transcriptional levels of actII-orf4 and the target genes of actI and actIII in HIW- erm P and HIW strains, respectively.

    Techniques Used: Activated Clotting Time Assay, Polymerase Chain Reaction, Amplification, In Vitro, DNA Ligation, Expressing, Plasmid Preparation

    25) Product Images from "Characterisation of a New Fungal Immunomodulatory Protein from Tiger Milk mushroom, Lignosus rhinocerotis"

    Article Title: Characterisation of a New Fungal Immunomodulatory Protein from Tiger Milk mushroom, Lignosus rhinocerotis

    Journal: Scientific Reports

    doi: 10.1038/srep30010

    RT-PCR and cloning of FIP-Lrh cDNA. ( a ) Total RNA extracted from sclerotia of L. rhinocerotis as analysed on a 1% agarose gel electrophoresis. Lane M 1 contained 1.5 μg of λ Hin dIII DNA marker (NEB, USA) whereas Lane 1 and 2 contained ~0.5 μg and 1.5 μg of total RNA. ( b ) An approximately 500 bp PCR product of FIP-Lrh cDNA was obtained through RT-PCR using FIPf and FIPr primers, as shown in Lane 3. M 2 contained 1.25 μg of 100 bp DNA marker (NEB, USA). ( c ) Four pGEMT clones containing FIP-Lrh cDNA were subjected to PCR using FIPf and FIPr. A total of 5 μL PCR product obtained using clone pGEM_FIP_Lrh_1, pGEM_FIP_Lrh_2, pGEM_FIP_Lrh_3 and pGEM_FIP_Lrh_4 as template was loaded in Lane 4–7 respectively. Arrow indicates the presence of insert of approximately 400–500 bp in size.
    Figure Legend Snippet: RT-PCR and cloning of FIP-Lrh cDNA. ( a ) Total RNA extracted from sclerotia of L. rhinocerotis as analysed on a 1% agarose gel electrophoresis. Lane M 1 contained 1.5 μg of λ Hin dIII DNA marker (NEB, USA) whereas Lane 1 and 2 contained ~0.5 μg and 1.5 μg of total RNA. ( b ) An approximately 500 bp PCR product of FIP-Lrh cDNA was obtained through RT-PCR using FIPf and FIPr primers, as shown in Lane 3. M 2 contained 1.25 μg of 100 bp DNA marker (NEB, USA). ( c ) Four pGEMT clones containing FIP-Lrh cDNA were subjected to PCR using FIPf and FIPr. A total of 5 μL PCR product obtained using clone pGEM_FIP_Lrh_1, pGEM_FIP_Lrh_2, pGEM_FIP_Lrh_3 and pGEM_FIP_Lrh_4 as template was loaded in Lane 4–7 respectively. Arrow indicates the presence of insert of approximately 400–500 bp in size.

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

    26) Product Images from "Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae"

    Article Title: Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq583

    CPA-sRNA validation using 3′-RACE. ( A ) Total RNA from M. oryzae was used to purify 5′ methylguanosine-capped RNAs using recombinant eIF4E K119A bound to beads ( 21 ). 5′ methylguanosine-capped RNA was treated with DNase I and single-stranded cDNA synthesized using an oligo (dT) 20 VN primer. PCR amplification was performed using a forward primer to the 5′-end of specific CPA-sRNAs and reverse primer specific to the oligo (dT) 20 VN linker. PCR products were analyzed on 3% agarose gels, bands eluted, cloned into pGEM-T vectors and Sanger sequenced. PCR products were resolved on a 3% agarose gels for ( B ) protein-coding mRNA (MGG_0383.6, MGG_6594.6, MGG_0469.6, MGG_0592.6, MGG_02597.6, MGG_07928.6, MGG_10680.6, MGG_14279.6 and MGG_01210.6); ( C ) tRNAs (Ala: MGG_20297.6, Cys: MGG_20209.6, Gln: MGG_20266.6 and Leu: MGG_20218.6); ( D ) rRNAs (18S and 28S); (E ) snRNAs (U6 and U2) and ( F ) retroelements (MAGGY-LTR). A DNA ladder is shown on the left of each panel. Arrows indicate PCR products that were sequenced.
    Figure Legend Snippet: CPA-sRNA validation using 3′-RACE. ( A ) Total RNA from M. oryzae was used to purify 5′ methylguanosine-capped RNAs using recombinant eIF4E K119A bound to beads ( 21 ). 5′ methylguanosine-capped RNA was treated with DNase I and single-stranded cDNA synthesized using an oligo (dT) 20 VN primer. PCR amplification was performed using a forward primer to the 5′-end of specific CPA-sRNAs and reverse primer specific to the oligo (dT) 20 VN linker. PCR products were analyzed on 3% agarose gels, bands eluted, cloned into pGEM-T vectors and Sanger sequenced. PCR products were resolved on a 3% agarose gels for ( B ) protein-coding mRNA (MGG_0383.6, MGG_6594.6, MGG_0469.6, MGG_0592.6, MGG_02597.6, MGG_07928.6, MGG_10680.6, MGG_14279.6 and MGG_01210.6); ( C ) tRNAs (Ala: MGG_20297.6, Cys: MGG_20209.6, Gln: MGG_20266.6 and Leu: MGG_20218.6); ( D ) rRNAs (18S and 28S); (E ) snRNAs (U6 and U2) and ( F ) retroelements (MAGGY-LTR). A DNA ladder is shown on the left of each panel. Arrows indicate PCR products that were sequenced.

    Techniques Used: Recombinant, Synthesized, Polymerase Chain Reaction, Amplification, Clone Assay

    CPA-sRNA isolation and size distribution. ( A ) Strategy for CPA-sRNA preparation from mycelial total RNA. The protocol ensures capture of RNA species that possess both a 5′-cap and a 3′-polyadenylated tail. The first treatment with BAP prevents RNA containing a 5′-free phosphate from being able to ligate to the 5′-linker. The use of (dT) 20 VN oligo for single-strand cDNA priming allows cDNA to be synthesized exclusively from RNA containing polyA. Following amplification by PCR, small cDNAs (
    Figure Legend Snippet: CPA-sRNA isolation and size distribution. ( A ) Strategy for CPA-sRNA preparation from mycelial total RNA. The protocol ensures capture of RNA species that possess both a 5′-cap and a 3′-polyadenylated tail. The first treatment with BAP prevents RNA containing a 5′-free phosphate from being able to ligate to the 5′-linker. The use of (dT) 20 VN oligo for single-strand cDNA priming allows cDNA to be synthesized exclusively from RNA containing polyA. Following amplification by PCR, small cDNAs (

    Techniques Used: Isolation, Synthesized, Amplification, Polymerase Chain Reaction

    27) Product Images from "Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases"

    Article Title: Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

    Journal: Genetics

    doi: 10.1534/genetics.112.147645

    Dose-dependent mutagenesis by DJ1-TALENs. (A) MultiNA gel images of Hae III digestion. A PCR fragment containing the TALEN target site was digested with Hae III. Gel images from two representative embryos injected with 0–300 ng/µl RNA for
    Figure Legend Snippet: Dose-dependent mutagenesis by DJ1-TALENs. (A) MultiNA gel images of Hae III digestion. A PCR fragment containing the TALEN target site was digested with Hae III. Gel images from two representative embryos injected with 0–300 ng/µl RNA for

    Techniques Used: Mutagenesis, TALENs, Polymerase Chain Reaction, Injection

    28) Product Images from "Type VI Secretion System Transports Zn2+ to Combat Multiple Stresses and Host Immunity"

    Article Title: Type VI Secretion System Transports Zn2+ to Combat Multiple Stresses and Host Immunity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005020

    OxyR directly activates T6SS-4 expression. A. OxyR binds the T6SS-4 promoter. Biotin-labeled probe or its mutant was incubated with OxyR. The protein-DNA complexes were detected by streptavidin-conjugated HRP and chemiluminescent substrate. Unlabeled promoter was added to determine the binding specificity of OxyR. Bio-T6SS-4p: biotin-labeled T6SS-4 promoter. Bio-T6SS-4pM: biotin-labeled T6SS-4 promoter mutant. B. Identification of the OxyR protected region in T6SS-4 promoter region. Complexes formed between FAM dye-labeled probes and His 6 -OxyR were subjected to DNase I digestion. DNA was sequenced and the 4 nucleotides marked in different colors were merged. The electropherograms were aligned using GeneScan-LIZ500. C-D. OxyR activates the expression of T6SS-4. β -galactosidase activity ( C ) or relative expression measured by quantitative RT-PCR in indicated bacterial strains was determined. Relative levels of transcripts were presented as the mean values ± SD calculated from three sets of independent experiments ( D ). E. The protein level of Hcp4 in relevant Yptb strains. Lysates from bacteria were resolved by SDS-PAGE, and Hcp4 was detected by immunoblotting. The metabolic protein phosphoglucose isomerase (Pgi) was probed as a loading control. F . OxyR does not activate T6SS1-3. β -galactosidase activity from chromosomal lacZ fusions in relevant Yptb was measured. Data shown were the average of three independent experiments; error bars indicate SD from three independent experiments. ***, p
    Figure Legend Snippet: OxyR directly activates T6SS-4 expression. A. OxyR binds the T6SS-4 promoter. Biotin-labeled probe or its mutant was incubated with OxyR. The protein-DNA complexes were detected by streptavidin-conjugated HRP and chemiluminescent substrate. Unlabeled promoter was added to determine the binding specificity of OxyR. Bio-T6SS-4p: biotin-labeled T6SS-4 promoter. Bio-T6SS-4pM: biotin-labeled T6SS-4 promoter mutant. B. Identification of the OxyR protected region in T6SS-4 promoter region. Complexes formed between FAM dye-labeled probes and His 6 -OxyR were subjected to DNase I digestion. DNA was sequenced and the 4 nucleotides marked in different colors were merged. The electropherograms were aligned using GeneScan-LIZ500. C-D. OxyR activates the expression of T6SS-4. β -galactosidase activity ( C ) or relative expression measured by quantitative RT-PCR in indicated bacterial strains was determined. Relative levels of transcripts were presented as the mean values ± SD calculated from three sets of independent experiments ( D ). E. The protein level of Hcp4 in relevant Yptb strains. Lysates from bacteria were resolved by SDS-PAGE, and Hcp4 was detected by immunoblotting. The metabolic protein phosphoglucose isomerase (Pgi) was probed as a loading control. F . OxyR does not activate T6SS1-3. β -galactosidase activity from chromosomal lacZ fusions in relevant Yptb was measured. Data shown were the average of three independent experiments; error bars indicate SD from three independent experiments. ***, p

    Techniques Used: Expressing, Labeling, Mutagenesis, Incubation, Binding Assay, Activity Assay, Quantitative RT-PCR, SDS Page

    29) Product Images from "A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays"

    Article Title: A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-13-34

    Agarose gel with PCR–RFLP products for analysis of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C . Lane 1 contains Fermentas O’ GeneRuler® Ultra Low Range DNA Ladder (25 bp - 700 bp); Lanes 2, 3 and 4 contain multiplex PCR-RFLP products digested with 1 U of NEB® BanII restriction enzyme (NEB®, England), 1 U Fermentas Fast Digest® HinfI restriction enzyme (Fermentas, Lithuania) and 1 U Fermentas Fast Digest® MspI restriction enzyme (Fermentas, Lithuania), respectively. The band sizes were 371 bp, 248 bp, 178 bp, 130 bp and 118 bp for all lanes. This indicates that the sample is a TT genotype for MTHFR 677 C > T, GG genotype for eNOS +894 G > T , and TT genotype for eNOS −786 T > C.
    Figure Legend Snippet: Agarose gel with PCR–RFLP products for analysis of MTHFR 677 C > T and eNOS +894 G > T and eNOS −786 T > C . Lane 1 contains Fermentas O’ GeneRuler® Ultra Low Range DNA Ladder (25 bp - 700 bp); Lanes 2, 3 and 4 contain multiplex PCR-RFLP products digested with 1 U of NEB® BanII restriction enzyme (NEB®, England), 1 U Fermentas Fast Digest® HinfI restriction enzyme (Fermentas, Lithuania) and 1 U Fermentas Fast Digest® MspI restriction enzyme (Fermentas, Lithuania), respectively. The band sizes were 371 bp, 248 bp, 178 bp, 130 bp and 118 bp for all lanes. This indicates that the sample is a TT genotype for MTHFR 677 C > T, GG genotype for eNOS +894 G > T , and TT genotype for eNOS −786 T > C.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Multiplex Assay

    Agarose gel with PCR products from multiplex PCR for MTHFR 677 C > T , eNOS +894 G > T and eNOS −786 T > C . Lane 1 contains Fermentas O’ GeneRuler® Ultra Low Range DNA Ladder (25 bp - 700 bp); Lane 2 contains multiplex PCR products with band sizes 178 bp, 248 bp and 371 bp; Lane 3 contains a positive control for eNOS +894 G > T with band size 371 bp; Lane 4 contains a positive control for MTHFR 677 C > T with band size 248 bp; Lane 5 contains a positive control for eNOS −786 T > C with band size 178; and Lane 6 is a negative control.
    Figure Legend Snippet: Agarose gel with PCR products from multiplex PCR for MTHFR 677 C > T , eNOS +894 G > T and eNOS −786 T > C . Lane 1 contains Fermentas O’ GeneRuler® Ultra Low Range DNA Ladder (25 bp - 700 bp); Lane 2 contains multiplex PCR products with band sizes 178 bp, 248 bp and 371 bp; Lane 3 contains a positive control for eNOS +894 G > T with band size 371 bp; Lane 4 contains a positive control for MTHFR 677 C > T with band size 248 bp; Lane 5 contains a positive control for eNOS −786 T > C with band size 178; and Lane 6 is a negative control.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Multiplex Assay, Positive Control, Negative Control

    30) Product Images from "Hepatitis C Virus Deletion Mutants Are Found in Individuals Chronically Infected with Genotype 1 Hepatitis C Virus in Association with Age, High Viral Load and Liver Inflammatory Activity"

    Article Title: Hepatitis C Virus Deletion Mutants Are Found in Individuals Chronically Infected with Genotype 1 Hepatitis C Virus in Association with Age, High Viral Load and Liver Inflammatory Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0138546

    Viral genome architecture of the defective forms identified in the serum of chronic hepatitis C patients (genotype 1 HCV). (A) Pictures of agarose-gel showing amplicons obtained after the second round of nested PCR for all the patients in which defective forms were identified (lanes 1–25) and a subset of patients negative for the defective form (lanes 26–36). Lanes M: molecular size markers. (B) Schematic representation of the architecture of the 25 defective variants identified in the sera of subjects chronically infected with genotype 1 HCV. (C) Picture of agarose gel showing amplicons obtained after nested PCR performed on synthetic HCV RNA mixtures assessing full-length/defective ratios from 1 to 1000 (lanes 3–12). FL: full-length RNA only (lane 1); D: deleted RNA only (lane 2). Lane M: molecular size markers.
    Figure Legend Snippet: Viral genome architecture of the defective forms identified in the serum of chronic hepatitis C patients (genotype 1 HCV). (A) Pictures of agarose-gel showing amplicons obtained after the second round of nested PCR for all the patients in which defective forms were identified (lanes 1–25) and a subset of patients negative for the defective form (lanes 26–36). Lanes M: molecular size markers. (B) Schematic representation of the architecture of the 25 defective variants identified in the sera of subjects chronically infected with genotype 1 HCV. (C) Picture of agarose gel showing amplicons obtained after nested PCR performed on synthetic HCV RNA mixtures assessing full-length/defective ratios from 1 to 1000 (lanes 3–12). FL: full-length RNA only (lane 1); D: deleted RNA only (lane 2). Lane M: molecular size markers.

    Techniques Used: Agarose Gel Electrophoresis, Nested PCR, Infection

    31) Product Images from "SiteFinding-PCR: a simple and efficient PCR method for chromosome walking"

    Article Title: SiteFinding-PCR: a simple and efficient PCR method for chromosome walking

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni124

    Chromosome walking for the Cyanophage P4 and an Arabidopsis mutant using the SiteFinding-PCR method. SiteFinder-1 was used to initialize the SiteFinding reaction. ( A ) Products of the secondary round of PCR (lanes 1–2 for P4 Cyanophage and 3–4 for Arabidopsis mutant). Lanes 1–4 contained PCR products obtained with primer couples SFP2/P4-2, SFP2/P4-3, SFP2/DL2 and SFP2/DL3, respectively. ( B ) Cloned and sequenced PCR products. Lanes 1 and 2 both showed three specific products (A), and the largest one in lane 1 was cloned and sequenced as indicated in (I). There were another two GCCT sites on the 4617 bp fragment of the Cyanophage P4 sequence, which are indicated with the black arrowheads in (I) (first site and second site). These findings were consistent with the gel electrophoresis results (white arrows in lane 1). Lanes 3 and 4 both showed two specific products (A), and the larger one in lane 3 was cloned and sequenced as indicated in (II). There was another GCCT site on the 2270 bp fragment of the Arabidopsis DNA as indicated with the black arrowheads in (II) (first site), which was also consistent with the gel electrophoresis (white arrow in lane 3).
    Figure Legend Snippet: Chromosome walking for the Cyanophage P4 and an Arabidopsis mutant using the SiteFinding-PCR method. SiteFinder-1 was used to initialize the SiteFinding reaction. ( A ) Products of the secondary round of PCR (lanes 1–2 for P4 Cyanophage and 3–4 for Arabidopsis mutant). Lanes 1–4 contained PCR products obtained with primer couples SFP2/P4-2, SFP2/P4-3, SFP2/DL2 and SFP2/DL3, respectively. ( B ) Cloned and sequenced PCR products. Lanes 1 and 2 both showed three specific products (A), and the largest one in lane 1 was cloned and sequenced as indicated in (I). There were another two GCCT sites on the 4617 bp fragment of the Cyanophage P4 sequence, which are indicated with the black arrowheads in (I) (first site and second site). These findings were consistent with the gel electrophoresis results (white arrows in lane 1). Lanes 3 and 4 both showed two specific products (A), and the larger one in lane 3 was cloned and sequenced as indicated in (II). There was another GCCT site on the 2270 bp fragment of the Arabidopsis DNA as indicated with the black arrowheads in (II) (first site), which was also consistent with the gel electrophoresis (white arrow in lane 3).

    Techniques Used: Chromosome Walking, Mutagenesis, Polymerase Chain Reaction, Clone Assay, Sequencing, Nucleic Acid Electrophoresis

    Identification of T-DNA insertion sites of 14 Arabidopsis mutants with SiteFinding-PCR. Lanes II and III: the products generated by SFP2/DL2 and SFP2/DL3, respectively. Samples are labeled as S1–S14. ( A ) The products of S1–S7, initially primed by SiteFinder-1. White arrows in lane II indicated the cloned products. Digitals in boxed areas show different insertion sites. As for S3, 1-1 and 1-2 show two different products of the 1st insertion site; 2-1, 2-2 and 2-3 in S3 refer to three products of the 2nd insertion site. ( B ) The products of samples 8–14, initially primed by SiteFinder-2. White arrows in lane II indicated the cloned products. ( C ) Comparison of the 3′ end of the SiteFinder with the largest or larger fragment. Bold black line segments and grey line segments represent the T-DNA and Arabidopsis DNAs, respectively. The arrows represent SFP2. The sizes of the largest or larger specific fragment of the inserted site are indicated on the right, and the smaller ones are marked in the middle with blue bars. NNNNNN with 0–3 mismatch nt (courier) helped the 3′ ends (underlined with blue bars) of SiteFinder-1 and SiteFinder-2 to anneal accurately with 3′-CGGA-5′ (GCCT site) and 3′-CGCG-5′ (GCGC site), respectively.
    Figure Legend Snippet: Identification of T-DNA insertion sites of 14 Arabidopsis mutants with SiteFinding-PCR. Lanes II and III: the products generated by SFP2/DL2 and SFP2/DL3, respectively. Samples are labeled as S1–S14. ( A ) The products of S1–S7, initially primed by SiteFinder-1. White arrows in lane II indicated the cloned products. Digitals in boxed areas show different insertion sites. As for S3, 1-1 and 1-2 show two different products of the 1st insertion site; 2-1, 2-2 and 2-3 in S3 refer to three products of the 2nd insertion site. ( B ) The products of samples 8–14, initially primed by SiteFinder-2. White arrows in lane II indicated the cloned products. ( C ) Comparison of the 3′ end of the SiteFinder with the largest or larger fragment. Bold black line segments and grey line segments represent the T-DNA and Arabidopsis DNAs, respectively. The arrows represent SFP2. The sizes of the largest or larger specific fragment of the inserted site are indicated on the right, and the smaller ones are marked in the middle with blue bars. NNNNNN with 0–3 mismatch nt (courier) helped the 3′ ends (underlined with blue bars) of SiteFinder-1 and SiteFinder-2 to anneal accurately with 3′-CGGA-5′ (GCCT site) and 3′-CGCG-5′ (GCGC site), respectively.

    Techniques Used: Polymerase Chain Reaction, Generated, Labeling, Clone Assay

    Schematic outline of SiteFinding-PCR method for chromosome walking. Known and unknown sequences are depicted with thick and thin lines, respectively. Blue segments show the expected SiteFinder targets. Gene-specific primers (GSPs) 1–3 can anneal with known sequences (white arrows). (1) Original genomic double-strand templates, showing target molecule and non-target molecules. (2) SiteFinding reaction: after low temperature priming by a SiteFinder, one strand of the target gene was replaced by long Taq DNA polymerase, which generated double-stranded target molecules of different lengths. (3) Nested PCR: the target DNA was exponentially amplified by nested PCR with GSPs and SiteFinder primers (SFPs) 1 and 2, while non-target gene amplification was suppressed by the stem–loop structure of the DNA. (4) Cloning target molecules: after being cleaved with NotI, the PCR products (generated by GSP2 and SFP2) were purified by agarose gel electrophoresis, and then the purified DNA was cloned into a pBluescript SK(+) vector linearized by NotI and EcoRV. (5) Screening and sequencing: the clones were screened by colony-PCR with the third gene-specific primer (GSP3) and a vector primer (M13 reverse primer or T3 primer), and the target molecules were screened out and sequenced subsequently.
    Figure Legend Snippet: Schematic outline of SiteFinding-PCR method for chromosome walking. Known and unknown sequences are depicted with thick and thin lines, respectively. Blue segments show the expected SiteFinder targets. Gene-specific primers (GSPs) 1–3 can anneal with known sequences (white arrows). (1) Original genomic double-strand templates, showing target molecule and non-target molecules. (2) SiteFinding reaction: after low temperature priming by a SiteFinder, one strand of the target gene was replaced by long Taq DNA polymerase, which generated double-stranded target molecules of different lengths. (3) Nested PCR: the target DNA was exponentially amplified by nested PCR with GSPs and SiteFinder primers (SFPs) 1 and 2, while non-target gene amplification was suppressed by the stem–loop structure of the DNA. (4) Cloning target molecules: after being cleaved with NotI, the PCR products (generated by GSP2 and SFP2) were purified by agarose gel electrophoresis, and then the purified DNA was cloned into a pBluescript SK(+) vector linearized by NotI and EcoRV. (5) Screening and sequencing: the clones were screened by colony-PCR with the third gene-specific primer (GSP3) and a vector primer (M13 reverse primer or T3 primer), and the target molecules were screened out and sequenced subsequently.

    Techniques Used: Polymerase Chain Reaction, Chromosome Walking, Generated, Nested PCR, Amplification, Clone Assay, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Sequencing

    ( A ) Sequences of two SiteFinders and their primers (SFP1 and SFP2). SiteFinder-1 and 2 differed only at their 3′ ends, and contained a rare restriction enzyme site for NotI, which facilitates cloning with commonly used vectors, such as pBluescript SK(+). The four specific nucleotides underlined with blue bar at the 3′ ends of the SiteFinders, with the help of NNNNNN, were used to anneal with the complimentary sites on genomic DNAs at low temperature and initiate SiteFinding-PCR. SFP1 and SFP2 were used in the primary and secondary reactions, respectively. ( B ) Three gene-specific primers (GSPs) for Cyanophage P4 (P4-1, P4-2 and P4-3) are indicated by black arrows. The distance from P4-2 to P4-3 was 31 bp. ( C ) Three GSPs for Arabidopsis T-DNA insertion mutants (DL1, DL2 and DL3) designed based on the T-DNA sequence of pSki015 are indicated by black arrows. The distance from DL2 to DL3 was 59 bp.
    Figure Legend Snippet: ( A ) Sequences of two SiteFinders and their primers (SFP1 and SFP2). SiteFinder-1 and 2 differed only at their 3′ ends, and contained a rare restriction enzyme site for NotI, which facilitates cloning with commonly used vectors, such as pBluescript SK(+). The four specific nucleotides underlined with blue bar at the 3′ ends of the SiteFinders, with the help of NNNNNN, were used to anneal with the complimentary sites on genomic DNAs at low temperature and initiate SiteFinding-PCR. SFP1 and SFP2 were used in the primary and secondary reactions, respectively. ( B ) Three gene-specific primers (GSPs) for Cyanophage P4 (P4-1, P4-2 and P4-3) are indicated by black arrows. The distance from P4-2 to P4-3 was 31 bp. ( C ) Three GSPs for Arabidopsis T-DNA insertion mutants (DL1, DL2 and DL3) designed based on the T-DNA sequence of pSki015 are indicated by black arrows. The distance from DL2 to DL3 was 59 bp.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Sequencing

    32) Product Images from "Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification"

    Article Title: Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026217

    IGFBP1 promoter sequence and FoxO1 binding assay. (A) The 180 bp mouse IGFBP1 promoter sequence (−204 to −25) contains three FoxO1 binding sites, two within the IRE (insulin response element) and one new binding site designated FNBS. This promoter fragment also contains a binding site for the transcription factor HNF-1 (hepatocyte nuclear factor 1) and two binding sites for GR (glucocorticoid receptor). (B) Electrophoretic mobility shift assay (EMSA) confirms specific binding of recombinant FoxO1 protein to the PCR amplicon. The band in lane one corresponds to free IGFBP1 DNA. Two new bands, indicated by the arrows, appear in lane two when FoxO1 protein is mixed with the IGFBP1 DNA at a molar ratio of 1.5∶1.0 corresponding to the formation of the 1∶1 and 2∶1 FoxO1-IGFBP1 protein-DNA complex. Increasing the molar ratio of FoxO1 protein to IGFBP1 DNA to 3.0∶1.0 results in a nearly complete depletion of free DNA and an increase in the intensity of the band assigned to the 2∶1 complex.
    Figure Legend Snippet: IGFBP1 promoter sequence and FoxO1 binding assay. (A) The 180 bp mouse IGFBP1 promoter sequence (−204 to −25) contains three FoxO1 binding sites, two within the IRE (insulin response element) and one new binding site designated FNBS. This promoter fragment also contains a binding site for the transcription factor HNF-1 (hepatocyte nuclear factor 1) and two binding sites for GR (glucocorticoid receptor). (B) Electrophoretic mobility shift assay (EMSA) confirms specific binding of recombinant FoxO1 protein to the PCR amplicon. The band in lane one corresponds to free IGFBP1 DNA. Two new bands, indicated by the arrows, appear in lane two when FoxO1 protein is mixed with the IGFBP1 DNA at a molar ratio of 1.5∶1.0 corresponding to the formation of the 1∶1 and 2∶1 FoxO1-IGFBP1 protein-DNA complex. Increasing the molar ratio of FoxO1 protein to IGFBP1 DNA to 3.0∶1.0 results in a nearly complete depletion of free DNA and an increase in the intensity of the band assigned to the 2∶1 complex.

    Techniques Used: Sequencing, Binding Assay, Electrophoretic Mobility Shift Assay, Recombinant, Polymerase Chain Reaction, Amplification

    33) Product Images from "A Novel Fatty Acyl Desaturase from the Pheromone Glands of Ctenopseustis obliquana and C. herana with Specific Z5-Desaturase Activity on Myristic Acid"

    Article Title: A Novel Fatty Acyl Desaturase from the Pheromone Glands of Ctenopseustis obliquana and C. herana with Specific Z5-Desaturase Activity on Myristic Acid

    Journal: Journal of Chemical Ecology

    doi: 10.1007/s10886-013-0373-1

    Quantitative RT-PCR of desat7 in pheromone gland and abdominal tissues of Ctenopseustis obliquana and C. herana adult females. Co Ctenopseustis obliquana , Ch C. herana , PG pheromone gland, Ab abdominal tissue, BLD below limits of detection. Error bars are standard errors of the means of three biological replicates
    Figure Legend Snippet: Quantitative RT-PCR of desat7 in pheromone gland and abdominal tissues of Ctenopseustis obliquana and C. herana adult females. Co Ctenopseustis obliquana , Ch C. herana , PG pheromone gland, Ab abdominal tissue, BLD below limits of detection. Error bars are standard errors of the means of three biological replicates

    Techniques Used: Quantitative RT-PCR

    34) Product Images from "RNA Aptamers Rescue Mitochondrial Dysfunction in a Yeast Model of Huntington’s Disease"

    Article Title: RNA Aptamers Rescue Mitochondrial Dysfunction in a Yeast Model of Huntington’s Disease

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.04.010

    Estimation of mtDNA Abundance (A) PCR amplification of the Cob , Atp6 , Atp9 , and Cox2 genes was used to assess mtDNA loss using genomic DNA isolated from cells expressing 103Q-htt with intramers or non-inhibitors. Hsp31 served as the control for nuclear DNA. Yeast cells treated with EtBr (black bars) to induce mtDNA loss were used as a negative control. (B–E) Densitometric analysis for band intensities was carried out, and the ratio of mitochondrial to nuclear marker was plotted for (B) Cob , (C) Atp6 , (D) Atp9 , and (E) Cox2 . The ratio of intensities of mitochondrial to nuclear markers in cells expressing 103Q-htt in the presence of a pair of non-inhibitors (gray bars) was assigned an arbitrary value of 1 in each case. Values shown are mean ± SEM of three independent experiments. ***p
    Figure Legend Snippet: Estimation of mtDNA Abundance (A) PCR amplification of the Cob , Atp6 , Atp9 , and Cox2 genes was used to assess mtDNA loss using genomic DNA isolated from cells expressing 103Q-htt with intramers or non-inhibitors. Hsp31 served as the control for nuclear DNA. Yeast cells treated with EtBr (black bars) to induce mtDNA loss were used as a negative control. (B–E) Densitometric analysis for band intensities was carried out, and the ratio of mitochondrial to nuclear marker was plotted for (B) Cob , (C) Atp6 , (D) Atp9 , and (E) Cox2 . The ratio of intensities of mitochondrial to nuclear markers in cells expressing 103Q-htt in the presence of a pair of non-inhibitors (gray bars) was assigned an arbitrary value of 1 in each case. Values shown are mean ± SEM of three independent experiments. ***p

    Techniques Used: Polymerase Chain Reaction, Amplification, Isolation, Expressing, Negative Control, Marker

    35) Product Images from "Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing"

    Article Title: Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing

    Journal: Infection, Genetics and Evolution

    doi: 10.1016/j.meegid.2018.07.016

    Internal transcribed spacer 1 (ITS1)–PCR analysis of Sudanese Leishmania donovani strain SD9 (LEM3472). Panel A – Parasites were cloned ( Garin et al., 2002 ), and ITS-1–PCR RFLP carried out ( Schonian et al., 2003 ). Ethidium-bromide stained agarose gel showing the parent strain SD9 (MHOM/SD/97/LEM3472) with an extra band at 239 bp*, and another Sudanese strain SD13 (MHOM/SD/98/LEM3570) displaying typical HaeIII digestion patterns (189, 77 and 51 bp arrows). Panel B - Sequence alignment of ITS1–PCR region of the Sudanese PKDL strain MHOM/SD/97/LEM3472 (SD9). The PCR products were cloned into pGEM-T Easy and colonies picked for sequencing, and DNA sequences were compared using MultAlin ( http://sacs.ucsf.edu/cgi-bin/multalin.py ). Sequences examined: Genebank – SD9_database (accession number emb|AJ634370.1| ), Parental strain – SD9_parental (this study), and SD9 amplicons subcloned into pGEM –T Easy - colony_5, _2, _14 and _18 (this study). In colony 14 a single nucleotide point mutation (C189A) is present eliminating the HaeIII restriction site (GGCC) resulting in an extra 239 bp product. Additional single nucleotide point mutations, colony 18 (T169C) and colony 2 (G318A) were noted suggesting that different ITS-1 sequences exist in the tandem repeats ( Downing et al., 2011 ; Gelanew et al., 2010a ) of the ITS1 regions between the SSU rRNA and the 5.8S genes on chromosome 27.
    Figure Legend Snippet: Internal transcribed spacer 1 (ITS1)–PCR analysis of Sudanese Leishmania donovani strain SD9 (LEM3472). Panel A – Parasites were cloned ( Garin et al., 2002 ), and ITS-1–PCR RFLP carried out ( Schonian et al., 2003 ). Ethidium-bromide stained agarose gel showing the parent strain SD9 (MHOM/SD/97/LEM3472) with an extra band at 239 bp*, and another Sudanese strain SD13 (MHOM/SD/98/LEM3570) displaying typical HaeIII digestion patterns (189, 77 and 51 bp arrows). Panel B - Sequence alignment of ITS1–PCR region of the Sudanese PKDL strain MHOM/SD/97/LEM3472 (SD9). The PCR products were cloned into pGEM-T Easy and colonies picked for sequencing, and DNA sequences were compared using MultAlin ( http://sacs.ucsf.edu/cgi-bin/multalin.py ). Sequences examined: Genebank – SD9_database (accession number emb|AJ634370.1| ), Parental strain – SD9_parental (this study), and SD9 amplicons subcloned into pGEM –T Easy - colony_5, _2, _14 and _18 (this study). In colony 14 a single nucleotide point mutation (C189A) is present eliminating the HaeIII restriction site (GGCC) resulting in an extra 239 bp product. Additional single nucleotide point mutations, colony 18 (T169C) and colony 2 (G318A) were noted suggesting that different ITS-1 sequences exist in the tandem repeats ( Downing et al., 2011 ; Gelanew et al., 2010a ) of the ITS1 regions between the SSU rRNA and the 5.8S genes on chromosome 27.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Staining, Agarose Gel Electrophoresis, Sequencing, Mutagenesis

    36) Product Images from "Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing"

    Article Title: Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing

    Journal: Infection, Genetics and Evolution

    doi: 10.1016/j.meegid.2018.07.016

    Internal transcribed spacer 1 (ITS1)–PCR analysis of Sudanese Leishmania donovani strain SD9 (LEM3472). Panel A – Parasites were cloned ( Garin et al., 2002 ), and ITS-1–PCR RFLP carried out ( Schonian et al., 2003 ). Ethidium-bromide stained agarose gel showing the parent strain SD9 (MHOM/SD/97/LEM3472) with an extra band at 239 bp*, and another Sudanese strain SD13 (MHOM/SD/98/LEM3570) displaying typical HaeIII digestion patterns (189, 77 and 51 bp arrows). Panel B - Sequence alignment of ITS1–PCR region of the Sudanese PKDL strain MHOM/SD/97/LEM3472 (SD9). The PCR products were cloned into pGEM-T Easy and colonies picked for sequencing, and DNA sequences were compared using MultAlin ( http://sacs.ucsf.edu/cgi-bin/multalin.py ). Sequences examined: Genebank – SD9_database (accession number emb|AJ634370.1| ), Parental strain – SD9_parental (this study), and SD9 amplicons subcloned into pGEM –T Easy - colony_5, _2, _14 and _18 (this study). In colony 14 a single nucleotide point mutation (C189A) is present eliminating the HaeIII restriction site (GGCC) resulting in an extra 239 bp product. Additional single nucleotide point mutations, colony 18 (T169C) and colony 2 (G318A) were noted suggesting that different ITS-1 sequences exist in the tandem repeats ( Downing et al., 2011 ; Gelanew et al., 2010a ) of the ITS1 regions between the SSU rRNA and the 5.8S genes on chromosome 27.
    Figure Legend Snippet: Internal transcribed spacer 1 (ITS1)–PCR analysis of Sudanese Leishmania donovani strain SD9 (LEM3472). Panel A – Parasites were cloned ( Garin et al., 2002 ), and ITS-1–PCR RFLP carried out ( Schonian et al., 2003 ). Ethidium-bromide stained agarose gel showing the parent strain SD9 (MHOM/SD/97/LEM3472) with an extra band at 239 bp*, and another Sudanese strain SD13 (MHOM/SD/98/LEM3570) displaying typical HaeIII digestion patterns (189, 77 and 51 bp arrows). Panel B - Sequence alignment of ITS1–PCR region of the Sudanese PKDL strain MHOM/SD/97/LEM3472 (SD9). The PCR products were cloned into pGEM-T Easy and colonies picked for sequencing, and DNA sequences were compared using MultAlin ( http://sacs.ucsf.edu/cgi-bin/multalin.py ). Sequences examined: Genebank – SD9_database (accession number emb|AJ634370.1| ), Parental strain – SD9_parental (this study), and SD9 amplicons subcloned into pGEM –T Easy - colony_5, _2, _14 and _18 (this study). In colony 14 a single nucleotide point mutation (C189A) is present eliminating the HaeIII restriction site (GGCC) resulting in an extra 239 bp product. Additional single nucleotide point mutations, colony 18 (T169C) and colony 2 (G318A) were noted suggesting that different ITS-1 sequences exist in the tandem repeats ( Downing et al., 2011 ; Gelanew et al., 2010a ) of the ITS1 regions between the SSU rRNA and the 5.8S genes on chromosome 27.

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Staining, Agarose Gel Electrophoresis, Sequencing, Mutagenesis

    37) Product Images from "A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions"

    Article Title: A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions

    Journal: RNA

    doi: 10.1261/rna.2184010

    Mapping of the cleavage site of the I-LtrII LHEase in the mt rns gene. Shown is a representative sequencing ladder generated for the top ( A ) and bottom ( B ) DNA strands that flank the intron IS. The uncleaved product represents the 248-bp PCR amplicon
    Figure Legend Snippet: Mapping of the cleavage site of the I-LtrII LHEase in the mt rns gene. Shown is a representative sequencing ladder generated for the top ( A ) and bottom ( B ) DNA strands that flank the intron IS. The uncleaved product represents the 248-bp PCR amplicon

    Techniques Used: Sequencing, Generated, Polymerase Chain Reaction, Amplification

    38) Product Images from "Tick-Borne Transmission of Murine Gammaherpesvirus 68"

    Article Title: Tick-Borne Transmission of Murine Gammaherpesvirus 68

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00458

    MHV68 detection in blood samples from mice infested with naturally infected nymphs or adult ticks. (A) Lanes N1-N7, blood samples of mouse 1-7 exposed to nymphs molted from larvae that had engorged on infected mice; lane N8, blood of mouse infested with uninfected nymphs. (B) Lane A1, blood of mouse infested with uninfected adult ticks; lanes A2-A14, blood samples of 13 mice exposed to adults molted from nymphs that had engorged on infected mice. All blood samples were collected 15 days after tick infestation. Lanes L1, L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene PCR product of 382 base pairs.
    Figure Legend Snippet: MHV68 detection in blood samples from mice infested with naturally infected nymphs or adult ticks. (A) Lanes N1-N7, blood samples of mouse 1-7 exposed to nymphs molted from larvae that had engorged on infected mice; lane N8, blood of mouse infested with uninfected nymphs. (B) Lane A1, blood of mouse infested with uninfected adult ticks; lanes A2-A14, blood samples of 13 mice exposed to adults molted from nymphs that had engorged on infected mice. All blood samples were collected 15 days after tick infestation. Lanes L1, L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene PCR product of 382 base pairs.

    Techniques Used: Mouse Assay, Infection, Polymerase Chain Reaction

    MHV68 detection in lung and spleen samples from mice infested with F1 i adults and in F1 i female ticks. (A) Lung (a,c) and spleen (b,d) samples of mice infested with F1 i adults examined by nested PCR (a,b) and RT-PCR (c,d) . Lanes 1a 26 , 2a 17 , 3a 18 , 4a 28 , 5a 19 , 6a 20 , 7a 32 , 8a 33 , and 9a 24 sa mples of mice infested with F1 i adult ticks; A35, A36, samples of control mice infested with F1 c adults. Lanes L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs; ++ indicates MHV68 M3 gene nested RT-PCR product of 241 base pairs. (B) Semi-thin sections of frozen whole body of F1 i females fed for 4 days. (a,b) F1 i tick from mouse 3a 18 and 5a 19 stained with anti-MHV68 rabbit polyclonal serum; (c) uninfected tick (F6 generation of breeding) stained with anti-MHV68 rabbit polyclonal serum; (d) F1 i tick from mouse 3a 18 stained with rabbit polyclonal serum against PB1-F2 protein of influenza virus A (H1N1) (negative control). MD, cells of midgut diverticula; L, lumen of midgut diverculum. Scale bar, 200 μm.
    Figure Legend Snippet: MHV68 detection in lung and spleen samples from mice infested with F1 i adults and in F1 i female ticks. (A) Lung (a,c) and spleen (b,d) samples of mice infested with F1 i adults examined by nested PCR (a,b) and RT-PCR (c,d) . Lanes 1a 26 , 2a 17 , 3a 18 , 4a 28 , 5a 19 , 6a 20 , 7a 32 , 8a 33 , and 9a 24 sa mples of mice infested with F1 i adult ticks; A35, A36, samples of control mice infested with F1 c adults. Lanes L2, C1–C4 as for Figure 1A . ** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs; ++ indicates MHV68 M3 gene nested RT-PCR product of 241 base pairs. (B) Semi-thin sections of frozen whole body of F1 i females fed for 4 days. (a,b) F1 i tick from mouse 3a 18 and 5a 19 stained with anti-MHV68 rabbit polyclonal serum; (c) uninfected tick (F6 generation of breeding) stained with anti-MHV68 rabbit polyclonal serum; (d) F1 i tick from mouse 3a 18 stained with rabbit polyclonal serum against PB1-F2 protein of influenza virus A (H1N1) (negative control). MD, cells of midgut diverticula; L, lumen of midgut diverculum. Scale bar, 200 μm.

    Techniques Used: Mouse Assay, Nested PCR, Reverse Transcription Polymerase Chain Reaction, Staining, Negative Control

    Tick-borne virus transmission and tick infection following injection of ticks with MHV68. (A) Virus detected by nested PCR. Lanes S1-S7, salivary glands of virus-injected ticks fed on uninfected mice for 5 days; lanes B1-B5, blood samples of five mice 15 days after tick infestation; L1, HyperLadder; L2, GeneRuler DNA ladder; lane C1, MHV68 DNA (positive control); lane C2, nested PCR without template (negative control); lane C3, MHV68 DNA nested PCR 1st round product with outer primers (positive control); lane C4, 1st PCR round with outer primers without template (negative control). ** Indicates MHV68 ORF50 gene PCR product of 382 base pairs. (B) Infectivity as determined by plaque formation in BHK-21 cells. Cells inoculated with homogenate of virus infected tick and observed by (a) light microscopy (magnification x50) and (b–d) specific immunofluorescence staining (magnification x200). (b) Single plaque shown of a maximum 5 plaques observed per tick; (c) control, uninfected cells; and (d) cells inoculated with homogenate of uninfected tick.
    Figure Legend Snippet: Tick-borne virus transmission and tick infection following injection of ticks with MHV68. (A) Virus detected by nested PCR. Lanes S1-S7, salivary glands of virus-injected ticks fed on uninfected mice for 5 days; lanes B1-B5, blood samples of five mice 15 days after tick infestation; L1, HyperLadder; L2, GeneRuler DNA ladder; lane C1, MHV68 DNA (positive control); lane C2, nested PCR without template (negative control); lane C3, MHV68 DNA nested PCR 1st round product with outer primers (positive control); lane C4, 1st PCR round with outer primers without template (negative control). ** Indicates MHV68 ORF50 gene PCR product of 382 base pairs. (B) Infectivity as determined by plaque formation in BHK-21 cells. Cells inoculated with homogenate of virus infected tick and observed by (a) light microscopy (magnification x50) and (b–d) specific immunofluorescence staining (magnification x200). (b) Single plaque shown of a maximum 5 plaques observed per tick; (c) control, uninfected cells; and (d) cells inoculated with homogenate of uninfected tick.

    Techniques Used: Transmission Assay, Infection, Injection, Nested PCR, Mouse Assay, Positive Control, Negative Control, Polymerase Chain Reaction, Light Microscopy, Immunofluorescence, Staining

    MHV68 detection in blood samples from mice infested with F0 females or F1 nymphs. (A) Lanes A15-A34, blood samples of 20 mice infested with infected F0 females; blood collected 15 days after tick infestation. ** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs. (B) Lanes 1n 26 , 2n 17 , 3n 18 , 4n 28 , 5n 19 , 6n 20 , 7n 32 , 8n 33 , and 9n 24 , blood samples of mice infested with F1 i nymphs; N9, N10 blood samples of control mice infested with F1 c nymphs. + Indicates MHV68 M3 gene one step RT-PCR product of 520 base pairs. Lanes L2, C1–C4 as for Figure 1A .
    Figure Legend Snippet: MHV68 detection in blood samples from mice infested with F0 females or F1 nymphs. (A) Lanes A15-A34, blood samples of 20 mice infested with infected F0 females; blood collected 15 days after tick infestation. ** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs. (B) Lanes 1n 26 , 2n 17 , 3n 18 , 4n 28 , 5n 19 , 6n 20 , 7n 32 , 8n 33 , and 9n 24 , blood samples of mice infested with F1 i nymphs; N9, N10 blood samples of control mice infested with F1 c nymphs. + Indicates MHV68 M3 gene one step RT-PCR product of 520 base pairs. Lanes L2, C1–C4 as for Figure 1A .

    Techniques Used: Mouse Assay, Infection, Nested PCR, Reverse Transcription Polymerase Chain Reaction

    39) Product Images from "Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases"

    Article Title: Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

    Journal: Genetics

    doi: 10.1534/genetics.112.147645

    Dose-dependent mutagenesis by DJ1-TALENs. (A) MultiNA gel images of Hae III digestion. A PCR fragment containing the TALEN target site was digested with Hae III. Gel images from two representative embryos injected with 0–300 ng/µl RNA for
    Figure Legend Snippet: Dose-dependent mutagenesis by DJ1-TALENs. (A) MultiNA gel images of Hae III digestion. A PCR fragment containing the TALEN target site was digested with Hae III. Gel images from two representative embryos injected with 0–300 ng/µl RNA for

    Techniques Used: Mutagenesis, TALENs, Polymerase Chain Reaction, Injection

    40) Product Images from "The telomeric transcriptome of Schizosaccharomyces pombe"

    Article Title: The telomeric transcriptome of Schizosaccharomyces pombe

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1153

    ( A ) Chromatin isolated from wt and Δ rap1 cells was immuno-precipitated using antibodies against RNAPII C terminal domain repeats either unmodified (αtotal) or phosphorylated at Serine 2 (αpS2) or Serine 5 (αpS5). Quantitative real-time PCR was performed using primers flanking TERRA TSS (left graph) or amplifying a fragment from the highly transcribed RNAPII substrate gene act1 (right graph; positive control). Graphs show the fraction of input DNA retrieved in the different samples, after subtraction of the background signal measured for control reactions performed using only beads. Bars and error bars are averages and standard deviations from three independent experiments. ( B ) Total proteins were extracted from wt and Δ rap1 strains and analyzed by western blot with the same antibodies used for ChIP. Act1 was used as loading control.
    Figure Legend Snippet: ( A ) Chromatin isolated from wt and Δ rap1 cells was immuno-precipitated using antibodies against RNAPII C terminal domain repeats either unmodified (αtotal) or phosphorylated at Serine 2 (αpS2) or Serine 5 (αpS5). Quantitative real-time PCR was performed using primers flanking TERRA TSS (left graph) or amplifying a fragment from the highly transcribed RNAPII substrate gene act1 (right graph; positive control). Graphs show the fraction of input DNA retrieved in the different samples, after subtraction of the background signal measured for control reactions performed using only beads. Bars and error bars are averages and standard deviations from three independent experiments. ( B ) Total proteins were extracted from wt and Δ rap1 strains and analyzed by western blot with the same antibodies used for ChIP. Act1 was used as loading control.

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Positive Control, Western Blot, Chromatin Immunoprecipitation

    Related Articles

    Clone Assay:

    Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
    Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

    Amplification:

    Article Title: SRY Induced TCF21 Genome-Wide Targets and Cascade of bHLH Factors During Sertoli Cell Differentiation and Male Sex Determination in Rats 1
    Article Snippet: .. Pooled whole-genome amplified DNA was purified by using the Wizard SV40 PCR Clean-up System (A9281; Promega). .. Purified DNA was checked on the gel and sent to Roche NimbleGen for ChIP-chip hybridization, and a three-plex promoter array was used for competitive hybridizations.

    Agarose Gel Electrophoresis:

    Article Title: CTNND1 755 T > G Promoter Polymorphism and Risk of Pancreatic Carcinoma in Chinese
    Article Snippet: .. The PCR fragments were recovered from agarose gel followed by purification with a DNA clean‐up kit (Wizard SV Gel and PCR Clean‐up System, Promega). .. DNA sequences of the PCR products were determined using the PCR sense primer with an Applied Biosystems model 377 sequencer (PE Applied Biosystems, Warrington, UK).

    Mutagenesis:

    Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
    Article Snippet: .. Genomic DNA of GAS osKaR mutant (see Basic Protocol 1) Primers oPCR1, Deg3, Anchor1, Deg4 and Anchor2 (see recipe) Reagents and equipment for PCR Gel and PCR Clean-Up System kit (Wizard SV, Promega Cat. No. A9282) .. 1 Set up the first PCR (AP-PCR #1) using 1 μl of genomic DNA, 1 μl of primers oPCR1 and Deg3 (10 μM each) using Taq .

    Purification:

    Article Title: SRY Induced TCF21 Genome-Wide Targets and Cascade of bHLH Factors During Sertoli Cell Differentiation and Male Sex Determination in Rats 1
    Article Snippet: .. Pooled whole-genome amplified DNA was purified by using the Wizard SV40 PCR Clean-up System (A9281; Promega). .. Purified DNA was checked on the gel and sent to Roche NimbleGen for ChIP-chip hybridization, and a three-plex promoter array was used for competitive hybridizations.

    Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
    Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

    Article Title: CTNND1 755 T > G Promoter Polymorphism and Risk of Pancreatic Carcinoma in Chinese
    Article Snippet: .. The PCR fragments were recovered from agarose gel followed by purification with a DNA clean‐up kit (Wizard SV Gel and PCR Clean‐up System, Promega). .. DNA sequences of the PCR products were determined using the PCR sense primer with an Applied Biosystems model 377 sequencer (PE Applied Biosystems, Warrington, UK).

    Article Title: Substantial Variation in the Extent of Mitochondrial Genome Fragmentation among Blood-Sucking Lice of Mammals
    Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega). .. Next-Generation Sequencing of the Coding Regions of mt Minichromosomes Purified PCR amplicons generated above with primers PLF1 and PLR from the coding regions of the mt minichromosomes of the domestic pig louse and the wild pig louse were sequenced initially with Roche GS FLX (454) platform at the AGRF and then with Illumina Hiseq 2000 platform at the Beijing Genomics Institute (BGI) for deeper coverages.

    Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points
    Article Snippet: .. DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (A9282; Promega) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent Technologies 2100 Bioanalyzer by using the DNA 1000 kit. .. The Bioanalyzer determines the quantity of DNA on the basis of fluorescence intensity.

    Real-time Polymerase Chain Reaction:

    Article Title: IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner
    Article Snippet: .. DNA was finally eluted using the WIZARD® SV Gel & PCR Clean-Up System (Promega A9281) according to the manufacturer’s protocol and analyzed by quantitative real-time PCR (qPCR). .. RNA immunoprecipitation (RIP) and quantitative RT-PCR analyses were performed essentially as recently described ( ).

    Polymerase Chain Reaction:

    Article Title: IGF2BP1 promotes SRF-dependent transcription in cancer in a m6A- and miRNA-dependent manner
    Article Snippet: .. DNA was finally eluted using the WIZARD® SV Gel & PCR Clean-Up System (Promega A9281) according to the manufacturer’s protocol and analyzed by quantitative real-time PCR (qPCR). .. RNA immunoprecipitation (RIP) and quantitative RT-PCR analyses were performed essentially as recently described ( ).

    Article Title: SRY Induced TCF21 Genome-Wide Targets and Cascade of bHLH Factors During Sertoli Cell Differentiation and Male Sex Determination in Rats 1
    Article Snippet: .. Pooled whole-genome amplified DNA was purified by using the Wizard SV40 PCR Clean-up System (A9281; Promega). .. Purified DNA was checked on the gel and sent to Roche NimbleGen for ChIP-chip hybridization, and a three-plex promoter array was used for competitive hybridizations.

    Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
    Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

    Article Title: CTNND1 755 T > G Promoter Polymorphism and Risk of Pancreatic Carcinoma in Chinese
    Article Snippet: .. The PCR fragments were recovered from agarose gel followed by purification with a DNA clean‐up kit (Wizard SV Gel and PCR Clean‐up System, Promega). .. DNA sequences of the PCR products were determined using the PCR sense primer with an Applied Biosystems model 377 sequencer (PE Applied Biosystems, Warrington, UK).

    Article Title: Substantial Variation in the Extent of Mitochondrial Genome Fragmentation among Blood-Sucking Lice of Mammals
    Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega). .. Next-Generation Sequencing of the Coding Regions of mt Minichromosomes Purified PCR amplicons generated above with primers PLF1 and PLR from the coding regions of the mt minichromosomes of the domestic pig louse and the wild pig louse were sequenced initially with Roche GS FLX (454) platform at the AGRF and then with Illumina Hiseq 2000 platform at the Beijing Genomics Institute (BGI) for deeper coverages.

    Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)
    Article Snippet: .. Genomic DNA of GAS osKaR mutant (see Basic Protocol 1) Primers oPCR1, Deg3, Anchor1, Deg4 and Anchor2 (see recipe) Reagents and equipment for PCR Gel and PCR Clean-Up System kit (Wizard SV, Promega Cat. No. A9282) .. 1 Set up the first PCR (AP-PCR #1) using 1 μl of genomic DNA, 1 μl of primers oPCR1 and Deg3 (10 μM each) using Taq .

    DNA Purification:

    Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points
    Article Snippet: .. DNA was purified from proteinase K–treated samples using a DNA purification kit following the manufacturer instructions (A9282; Promega) and was subsequently analyzed either by running a 2% low melting agarose (APEX) gel or by an Agilent Technologies 2100 Bioanalyzer by using the DNA 1000 kit. .. The Bioanalyzer determines the quantity of DNA on the basis of fluorescence intensity.

    Sequencing:

    Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
    Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

    Article Title: Substantial Variation in the Extent of Mitochondrial Genome Fragmentation among Blood-Sucking Lice of Mammals
    Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega). .. Next-Generation Sequencing of the Coding Regions of mt Minichromosomes Purified PCR amplicons generated above with primers PLF1 and PLR from the coding regions of the mt minichromosomes of the domestic pig louse and the wild pig louse were sequenced initially with Roche GS FLX (454) platform at the AGRF and then with Illumina Hiseq 2000 platform at the Beijing Genomics Institute (BGI) for deeper coverages.

    Plasmid Preparation:

    Article Title: Evolution of Extensively Fragmented Mitochondrial Genomes in the Lice of Humans
    Article Snippet: .. PCR amplicons used for sequencing were purified with Wizard SV Gel/PCR Clean-up System (Promega); cloning was with pGEM-T Easy Vector System (Promega). .. Sequence-reads were assembled into contigs with Sequencher 5.0 (Gene Codes); the parameters for assembly were minimum match 90% and minimum overlap 100 bp. tRNA genes were identified with tRNA-Scan ( ) and ARWEN ( ).

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    Promega pcr clean up kit
    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by <t>PCR</t> using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic <t>DNA</t> level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).
    Pcr Clean Up Kit, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pcr clean up system
    Comparison of the sensitivities for bla L1 gene detection by LAMP and conventional <t>PCR</t> methods. Pure genomic <t>DNA</t> extracted from S. maltophilia- K279a was diluted tenfold (379.0 ng/μl to 0.00379 pg/μl) and the DNA assayed by LAMP (A,B) and PCR (C) . (A) Turbidity was monitored using the Loopamp real-time turbidimeter and the OD recorded at 650 nm, at 6 s intervals. (B) Visual inspection of the color change, post-LAMP assay, and in the presence of calcein/Mn 2+ complex. (C) PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. The DNA marker is D2000 DNA Marker (Tiangen Biotech Co., Ltd.) The size is about 179 bp.
    Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3067 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up system/product/Promega
    Average 99 stars, based on 3067 article reviews
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    Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by PCR using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic DNA level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).

    Journal: BioMed Research International

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico

    doi: 10.1155/2017/5170680

    Figure Lengend Snippet: Amplification, sequencing and analysis of exons 2, 5, 7, and 26 . Exons 2, 5, 7, and 26 were amplified by PCR using primers specific to these regions (a). L-100 bp ladder. PCR products were sequenced by Sangers method. All the 4 nucleotide differences were also present at genomic DNA level. Representative image of exon specific amplification (b). Alignment of exon specific sequencing results with human, mouse, and rat (predicted) sequence shows 2 amino acid residue changes (c).

    Article Snippet: PCR amplified DNA was cleaned using PCR clean-up kit following the kit protocol (Promega, Wisconsin, USA), sequenced, and verified using nucleotide BLAST tool.

    Techniques: Amplification, Sequencing, Polymerase Chain Reaction

    Disruption of HBV X gene via deletion using DNA fragments derived from 4 g, 8 g and 12 g cosmids in 293 cells. (A) Cleavage sites of gRNAs included in the 4 g (4gRNA, red) and 8 g (8gRNA, green) fragments. The 12 g fragment includes both 4gRNA and 8 gRNA cleavage sites. The 20‐nucleotide recognition sequences of individual guide RNAs are boxed. The coding region is indicated by thick blue lines. HBV poly(a) sequences are disrupted and replaced by chicken β‐globin poly(a) sequences to elongate the half‐life of HBV mRNAs. 18 , 21 (B) Specific cleavages using 4 g, 8 g and 12 g DNA fragments. The 293 cells were transfected with the above fragments together with the target plasmid psCM103G and the nuclear DNAs were amplified using HBV‐X F and β‐globin poly(a) R primers (shown in A), 3 days post transfection. A short exposure of the photograph of the unprocessed PCR product of 0.6 kb is also shown. Control, 293 cells transfected only with psCM103G. (C) Schematic representation of possible gRNA cleavage sites in the PCR products. Cleavage sites of 4gRNA and 8 gRNA are shown in red and green, respectively

    Journal: The Journal of Gene Medicine

    Article Title: Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure, et al. Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure

    doi: 10.1002/jgm.3115

    Figure Lengend Snippet: Disruption of HBV X gene via deletion using DNA fragments derived from 4 g, 8 g and 12 g cosmids in 293 cells. (A) Cleavage sites of gRNAs included in the 4 g (4gRNA, red) and 8 g (8gRNA, green) fragments. The 12 g fragment includes both 4gRNA and 8 gRNA cleavage sites. The 20‐nucleotide recognition sequences of individual guide RNAs are boxed. The coding region is indicated by thick blue lines. HBV poly(a) sequences are disrupted and replaced by chicken β‐globin poly(a) sequences to elongate the half‐life of HBV mRNAs. 18 , 21 (B) Specific cleavages using 4 g, 8 g and 12 g DNA fragments. The 293 cells were transfected with the above fragments together with the target plasmid psCM103G and the nuclear DNAs were amplified using HBV‐X F and β‐globin poly(a) R primers (shown in A), 3 days post transfection. A short exposure of the photograph of the unprocessed PCR product of 0.6 kb is also shown. Control, 293 cells transfected only with psCM103G. (C) Schematic representation of possible gRNA cleavage sites in the PCR products. Cleavage sites of 4gRNA and 8 gRNA are shown in red and green, respectively

    Article Snippet: In total, 10 μg of s‐b cosmid was digested with Sal I (Figure , asterisks) and electrophoresed overnight in a 14‐cm long 0.8% Tris‐acetate‐ethylenediaminetetraacetic acid (TAE) agarose gel at 35 V. The DNA fragment was purified using the Wizard SV Gel and PCR Clean‐up kit as described above and was self‐ligated.

    Techniques: Derivative Assay, Transfection, Plasmid Preparation, Amplification, Polymerase Chain Reaction

    Comparison of the sensitivities for bla L1 gene detection by LAMP and conventional PCR methods. Pure genomic DNA extracted from S. maltophilia- K279a was diluted tenfold (379.0 ng/μl to 0.00379 pg/μl) and the DNA assayed by LAMP (A,B) and PCR (C) . (A) Turbidity was monitored using the Loopamp real-time turbidimeter and the OD recorded at 650 nm, at 6 s intervals. (B) Visual inspection of the color change, post-LAMP assay, and in the presence of calcein/Mn 2+ complex. (C) PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. The DNA marker is D2000 DNA Marker (Tiangen Biotech Co., Ltd.) The size is about 179 bp.

    Journal: Frontiers in Microbiology

    Article Title: Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-β-lactamase blaL1 in Beijing, China

    doi: 10.3389/fmicb.2014.00692

    Figure Lengend Snippet: Comparison of the sensitivities for bla L1 gene detection by LAMP and conventional PCR methods. Pure genomic DNA extracted from S. maltophilia- K279a was diluted tenfold (379.0 ng/μl to 0.00379 pg/μl) and the DNA assayed by LAMP (A,B) and PCR (C) . (A) Turbidity was monitored using the Loopamp real-time turbidimeter and the OD recorded at 650 nm, at 6 s intervals. (B) Visual inspection of the color change, post-LAMP assay, and in the presence of calcein/Mn 2+ complex. (C) PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. The DNA marker is D2000 DNA Marker (Tiangen Biotech Co., Ltd.) The size is about 179 bp.

    Article Snippet: The DNA was purified with the SV GEL and PCR Clean-Up System (Promega Co., USA).

    Techniques: Polymerase Chain Reaction, Lamp Assay, Agarose Gel Electrophoresis, Staining, Marker

    Identification of osKaR insertion site Arbitrary-primed PCR (AP-PCR) is a quick method to precisely identify the genomic region where a transposon has inserted. The following is specific for osKaR insertions: genomic DNA of the osKaR mutant is extracted (see Basic Protocol 1) and used for a semi-random PCR using the osKaR -specific primer oPCR1 and the random primer Deg3. The Deg3 primer consists of an 11-nucleotide random primer (in blue) with a 25-nucleotide specific tail (in red). The resulting PCR product is purified and used for a 2 nd PCR using the osKaR -specific primer Anchor1 and the Deg3-tail specific primer Deg4. The resulting PCR product is purified and DNA sequencing is performed using the osKaR -specific primer Anchor2.

    Journal: Current protocols in microbiology

    Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)

    doi: 10.1002/9780471729259.mc09d03s30

    Figure Lengend Snippet: Identification of osKaR insertion site Arbitrary-primed PCR (AP-PCR) is a quick method to precisely identify the genomic region where a transposon has inserted. The following is specific for osKaR insertions: genomic DNA of the osKaR mutant is extracted (see Basic Protocol 1) and used for a semi-random PCR using the osKaR -specific primer oPCR1 and the random primer Deg3. The Deg3 primer consists of an 11-nucleotide random primer (in blue) with a 25-nucleotide specific tail (in red). The resulting PCR product is purified and used for a 2 nd PCR using the osKaR -specific primer Anchor1 and the Deg3-tail specific primer Deg4. The resulting PCR product is purified and DNA sequencing is performed using the osKaR -specific primer Anchor2.

    Article Snippet: Genomic DNA of GAS osKaR mutant (see Basic Protocol 1) Primers oPCR1, Deg3, Anchor1, Deg4 and Anchor2 (see recipe) Reagents and equipment for PCR Gel and PCR Clean-Up System kit (Wizard SV, Promega Cat. No. A9282)

    Techniques: Polymerase Chain Reaction, Mutagenesis, Purification, DNA Sequencing