Structured Review

Promega 16s rrna gene hypervariable regions
Rarefaction curves generated for <t>16S</t> <t>rRNA</t> gene sequences and Principal Coordinate Analysis (PCoA) based on the phylotypes identified from different PCR primer sets as well as from different samples (stool samples of infants and fecal samples of mothers). Panels a and c display the rarefaction curves and the PCoA from stool samples of infants. Panels b and d show the rarefaction curves and the PCoA from fecal samples of mothers. In panels c and d percentages shown along the axes represent the proportion of dissimilarities captured by the axes. Each symbol represents the 16S rRNA gene sequences from each sample which are displayed in a different colour and shape according to the PCR primer pair. In panels a and b the plots depicted on the left represent the rarefaction curves determined using the PD index whereas the plots show on the right constitute the rarefaction curves obtained using the Chao index. A 95% confidence intervals was added to the rarefaction curves.
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1) Product Images from "Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol"

Article Title: Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068739

Rarefaction curves generated for 16S rRNA gene sequences and Principal Coordinate Analysis (PCoA) based on the phylotypes identified from different PCR primer sets as well as from different samples (stool samples of infants and fecal samples of mothers). Panels a and c display the rarefaction curves and the PCoA from stool samples of infants. Panels b and d show the rarefaction curves and the PCoA from fecal samples of mothers. In panels c and d percentages shown along the axes represent the proportion of dissimilarities captured by the axes. Each symbol represents the 16S rRNA gene sequences from each sample which are displayed in a different colour and shape according to the PCR primer pair. In panels a and b the plots depicted on the left represent the rarefaction curves determined using the PD index whereas the plots show on the right constitute the rarefaction curves obtained using the Chao index. A 95% confidence intervals was added to the rarefaction curves.
Figure Legend Snippet: Rarefaction curves generated for 16S rRNA gene sequences and Principal Coordinate Analysis (PCoA) based on the phylotypes identified from different PCR primer sets as well as from different samples (stool samples of infants and fecal samples of mothers). Panels a and c display the rarefaction curves and the PCoA from stool samples of infants. Panels b and d show the rarefaction curves and the PCoA from fecal samples of mothers. In panels c and d percentages shown along the axes represent the proportion of dissimilarities captured by the axes. Each symbol represents the 16S rRNA gene sequences from each sample which are displayed in a different colour and shape according to the PCR primer pair. In panels a and b the plots depicted on the left represent the rarefaction curves determined using the PD index whereas the plots show on the right constitute the rarefaction curves obtained using the Chao index. A 95% confidence intervals was added to the rarefaction curves.

Techniques Used: Generated, Polymerase Chain Reaction

Heat map showing the order classification rates for optimal choice of primers. Cells are colored on a gradient from 0% to 100% matches of the primer with the 16S rRNA gene target sequences of the indicated microbial order. The size of the amplicons generated by the various primer pairs are indicated. Furthermore, the percentage of the amplification rate from each primer pair on the various microbial taxa here described is indicated at the top of the heat map.
Figure Legend Snippet: Heat map showing the order classification rates for optimal choice of primers. Cells are colored on a gradient from 0% to 100% matches of the primer with the 16S rRNA gene target sequences of the indicated microbial order. The size of the amplicons generated by the various primer pairs are indicated. Furthermore, the percentage of the amplification rate from each primer pair on the various microbial taxa here described is indicated at the top of the heat map.

Techniques Used: Generated, Amplification

Fecal levels of different microorganisms as analyzed by PCR using different 16S rRNA gene-based primers. Panel a depicts PCR amplification targeting the 16S rRNA gene of different enteric bifidobacteria as well as key intestinal bacteria using the primers described by [22] [27] , [32] , [33] , [43] and the primer set Probio_Uni/Probio_Rev. Panel b. displays the microbial profile of the amplicons generated by the primer sets described by [22] , [32] , [33] according to the following taxa: Bifidobacteriaceae , Enterobacteriaceae , Enterococcaceae , Lactobacillales , Bacteroidetes group, and Atopobium group.
Figure Legend Snippet: Fecal levels of different microorganisms as analyzed by PCR using different 16S rRNA gene-based primers. Panel a depicts PCR amplification targeting the 16S rRNA gene of different enteric bifidobacteria as well as key intestinal bacteria using the primers described by [22] [27] , [32] , [33] , [43] and the primer set Probio_Uni/Probio_Rev. Panel b. displays the microbial profile of the amplicons generated by the primer sets described by [22] , [32] , [33] according to the following taxa: Bifidobacteriaceae , Enterobacteriaceae , Enterococcaceae , Lactobacillales , Bacteroidetes group, and Atopobium group.

Techniques Used: Polymerase Chain Reaction, Amplification, Generated

2) Product Images from "Establishment of a versatile cell line for juvenile hormone signaling analysis in Tribolium castaneum"

Article Title: Establishment of a versatile cell line for juvenile hormone signaling analysis in Tribolium castaneum

Journal: Scientific Reports

doi: 10.1038/srep01570

Induction of TcKr-h1 transcripts by JH in Tc81 cells. Transcript levels of TcKr-h1 were determined by qPCR (mean ± SD, n = 3). (A) Cells were treated with 10 μM methoprene (JHA), and temporal changes in TcKr-h1 expression were monitored. (B) Cells were treated with different concentrations of JH I, JH III, JHA, or farnesoic acid (FA), and the expression levels of TcKr-h1 were determined 2 h after treatment.
Figure Legend Snippet: Induction of TcKr-h1 transcripts by JH in Tc81 cells. Transcript levels of TcKr-h1 were determined by qPCR (mean ± SD, n = 3). (A) Cells were treated with 10 μM methoprene (JHA), and temporal changes in TcKr-h1 expression were monitored. (B) Cells were treated with different concentrations of JH I, JH III, JHA, or farnesoic acid (FA), and the expression levels of TcKr-h1 were determined 2 h after treatment.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing

Prediction and functional determination of JHREs of TcKr-h1 . (A) Schematic representation of core JHRE ( k JHREc)-like sequences present upstream and within the first intron of Kr-h1 genes from 4 insect species. Tc , T. castaneum ; Bm , B. mori ; Am , A. mellifera ; Dm , D. melanogaster . White boxes, black boxes, and arrows represent the 5′-UTRs, ORFs, and transcription start sites, respectively. The numbers indicate distances from the transcription start site. Red ellipses represent the authentic k JHREc (-2079/-2068) in B. mori 26 and its homologs. The asterisk shows a gap in the genomic sequence. (B) Alignment of the putative k JHREc sequences is shown in (A). (C) Functional characterization of putative k JHREcs of TcKr-h1. Tc81 cells were cotransfected with reporter plasmids that express firefly luciferase under the regulation of indicated regions and a reference reporter plasmid carrying Renilla luciferase . Red Xs indicate mutations in putative k JHREc sequences (see Materials and Methods for details). Cells were treated with 10 μM JHA for 24 h, and reporter activity was measured using a dual-luciferase reporter assay system. The activity of the firefly luciferase reporter was normalized against that of the Renilla luciferase reporter in the same samples. Data represent means ± SD (n = 3). Means with the same letter are not significantly different (Tukey–Kramer test, P
Figure Legend Snippet: Prediction and functional determination of JHREs of TcKr-h1 . (A) Schematic representation of core JHRE ( k JHREc)-like sequences present upstream and within the first intron of Kr-h1 genes from 4 insect species. Tc , T. castaneum ; Bm , B. mori ; Am , A. mellifera ; Dm , D. melanogaster . White boxes, black boxes, and arrows represent the 5′-UTRs, ORFs, and transcription start sites, respectively. The numbers indicate distances from the transcription start site. Red ellipses represent the authentic k JHREc (-2079/-2068) in B. mori 26 and its homologs. The asterisk shows a gap in the genomic sequence. (B) Alignment of the putative k JHREc sequences is shown in (A). (C) Functional characterization of putative k JHREcs of TcKr-h1. Tc81 cells were cotransfected with reporter plasmids that express firefly luciferase under the regulation of indicated regions and a reference reporter plasmid carrying Renilla luciferase . Red Xs indicate mutations in putative k JHREc sequences (see Materials and Methods for details). Cells were treated with 10 μM JHA for 24 h, and reporter activity was measured using a dual-luciferase reporter assay system. The activity of the firefly luciferase reporter was normalized against that of the Renilla luciferase reporter in the same samples. Data represent means ± SD (n = 3). Means with the same letter are not significantly different (Tukey–Kramer test, P

Techniques Used: Functional Assay, Sequencing, Luciferase, Plasmid Preparation, Activity Assay, Reporter Assay

Effects of RNAi-mediated knockdown of TcMet and TcSRC on the JH-dependent induction of TcKr-h1 in Tc81 cells. Cells were soaked in 50 ng/μL dsRNA for MalE , TcMet , or TcSRC for 60 h. Control cells (−) were not treated with dsRNA. Cells were then incubated in media containing 10 μM JHA (JHA+) or JHA-free media (JHA-) for 2 h, and TcMet (A), TcSRC (B), and TcKr-h1 (C) transcript levels were determined by qPCR (mean ± SD, n = 3). Data were analyzed using Student's t -tests (*** P
Figure Legend Snippet: Effects of RNAi-mediated knockdown of TcMet and TcSRC on the JH-dependent induction of TcKr-h1 in Tc81 cells. Cells were soaked in 50 ng/μL dsRNA for MalE , TcMet , or TcSRC for 60 h. Control cells (−) were not treated with dsRNA. Cells were then incubated in media containing 10 μM JHA (JHA+) or JHA-free media (JHA-) for 2 h, and TcMet (A), TcSRC (B), and TcKr-h1 (C) transcript levels were determined by qPCR (mean ± SD, n = 3). Data were analyzed using Student's t -tests (*** P

Techniques Used: Incubation, Real-time Polymerase Chain Reaction

3) Product Images from "Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products"

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsn004

ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.
Figure Legend Snippet: ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.

Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, Binding Assay, Expressing

4) Product Images from "Detection of Viral Hemorrhagic Septicemia virus (VHSV) from the leech Myzobdella lugubris Leidy, 1851"

Article Title: Detection of Viral Hemorrhagic Septicemia virus (VHSV) from the leech Myzobdella lugubris Leidy, 1851

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-2-45

Agarose gel showing the bands from RT-PCR, used for the detection of VHSV (811 base pair) . Pooled leech samples (#66, 69, 71, 75, 79, 80, 83-86, and 90) are representative VHSV-positive samples. The marker (M) used was 1.0 kb plus (Invitrogen).
Figure Legend Snippet: Agarose gel showing the bands from RT-PCR, used for the detection of VHSV (811 base pair) . Pooled leech samples (#66, 69, 71, 75, 79, 80, 83-86, and 90) are representative VHSV-positive samples. The marker (M) used was 1.0 kb plus (Invitrogen).

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

5) 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

6) Product Images from "Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices"

Article Title: Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices

Journal: BMC Biotechnology

doi: 10.1186/s12896-015-0191-3

Application of the bidirectional p35S and tNOS DNA walking methods on GM maize matrices. a Visualisation of the obtained amplicons using the p35S and tNOS DNA walking methods applied on 100 ng of the GM MON863 maize CRM (9.85 %). For each method, four different DRT primer mixes (A-D) have been used. The analyzed amplicons are indicated by a numerotation going from 1 to 16. b For each DNA walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the GM maize MON863, is illustatred by the black arrows. Below the transgenic cassette, the sequence covering of the selected amplicons from the GM MON863 maize CRM (9.85 %) and the GeMMA proficiency test food matrix (GeMMA SU35-A) is schematically represented by rectangles. The corresponding amplicon numbering is indicated in the Fig. 2a and Additional file 6 . LB (left border); p35S (CaMV 35S promoter); nptII (neomycin phosphotransferase II gene); tNOS (Agrobacterium tumefaciens nopaline synthase terminator); p4-AS1 (modified CaMV 35S promoter); wtCAB (Wheat major chlorophyll a/b binding protein gene); rAct (Rice Actin intron); Cry3Bb1 (synthetic Cry3Bb1 gene); tahsp17 (Wheat heat shock protein terminator); RB (right border); maize (maize genome) [Schema adapted from 27 and 30]
Figure Legend Snippet: Application of the bidirectional p35S and tNOS DNA walking methods on GM maize matrices. a Visualisation of the obtained amplicons using the p35S and tNOS DNA walking methods applied on 100 ng of the GM MON863 maize CRM (9.85 %). For each method, four different DRT primer mixes (A-D) have been used. The analyzed amplicons are indicated by a numerotation going from 1 to 16. b For each DNA walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the GM maize MON863, is illustatred by the black arrows. Below the transgenic cassette, the sequence covering of the selected amplicons from the GM MON863 maize CRM (9.85 %) and the GeMMA proficiency test food matrix (GeMMA SU35-A) is schematically represented by rectangles. The corresponding amplicon numbering is indicated in the Fig. 2a and Additional file 6 . LB (left border); p35S (CaMV 35S promoter); nptII (neomycin phosphotransferase II gene); tNOS (Agrobacterium tumefaciens nopaline synthase terminator); p4-AS1 (modified CaMV 35S promoter); wtCAB (Wheat major chlorophyll a/b binding protein gene); rAct (Rice Actin intron); Cry3Bb1 (synthetic Cry3Bb1 gene); tahsp17 (Wheat heat shock protein terminator); RB (right border); maize (maize genome) [Schema adapted from 27 and 30]

Techniques Used: Transgenic Assay, Sequencing, Gas Phase Electrophoretic Molecular Mobility Analysis, Amplification, Modification, Binding Assay

Development of the bidirectional p35S and tNOS DNA walking methods on 100 % Bt rice. a Visualisation of the obtained amplicons, numeroted from 1 to 94, using the p35S and tNOS DNA walking methods applied on 100 ng of 100 % Bt rice and WT rice. For each method, four different DRT primer mixes (A-D) have been used. b For each DNA walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the Bt rice, is illustatred by the black arrows. Below the transgenic cassette, the sequence covering of the obtained amplicons from the 100 % Bt rice is schematically represented by rectangles. The corresponding amplicon numbering is indicated in the Fig. 1a. LB (left border); t35S (CaMV 35S terminator); hpt (hygromycin phosphotransferase gene); p35S (CaMV 35S promoter); lacZ (LacZ alpha fragment); pUBI (maize ubiquitin promoter); Cry1B (synthetic Cry1B gene); tNOS (Agrobacterium tumefaciens nopaline synthase terminator); RB (right border); rice (rice genome) [Schema adapted from 24]
Figure Legend Snippet: Development of the bidirectional p35S and tNOS DNA walking methods on 100 % Bt rice. a Visualisation of the obtained amplicons, numeroted from 1 to 94, using the p35S and tNOS DNA walking methods applied on 100 ng of 100 % Bt rice and WT rice. For each method, four different DRT primer mixes (A-D) have been used. b For each DNA walking method, a schematic representation of the potential start position and direction, applied on the transgenic cassette of the Bt rice, is illustatred by the black arrows. Below the transgenic cassette, the sequence covering of the obtained amplicons from the 100 % Bt rice is schematically represented by rectangles. The corresponding amplicon numbering is indicated in the Fig. 1a. LB (left border); t35S (CaMV 35S terminator); hpt (hygromycin phosphotransferase gene); p35S (CaMV 35S promoter); lacZ (LacZ alpha fragment); pUBI (maize ubiquitin promoter); Cry1B (synthetic Cry1B gene); tNOS (Agrobacterium tumefaciens nopaline synthase terminator); RB (right border); rice (rice genome) [Schema adapted from 24]

Techniques Used: Transgenic Assay, Sequencing, Amplification

7) Product Images from "Asymmetric parental genome engineering by Cas9 during mouse meiotic exit"

Article Title: Asymmetric parental genome engineering by Cas9 during mouse meiotic exit

Journal: Scientific Reports

doi: 10.1038/srep07621

Sequence analysis reveals allelic asymmetry in parental genome mII editing. (A) Sequences of reverse-transcriptase PCR products from embryonic day 4 (E4.0) blastocysts developing after the 1-step method of editing ( Fig. 1A ) in which Cas9 cRNA and eGFP gRNA were co-injected into mII oocytes with sperm from 129-eGFP single-copy hemizygotes ( eGFP single). The gRNA-target sequence (green typeface) plus adjacent 5′ sequence is displayed on the top row and mutants beneath (grey typeface), with the corresponding Cas9 cRNA (c) and eGFP gRNA (g) concentrations (ng/μl) that were injected to produced them. The proto-spacer adjacent motif (PAM) is highlighted in green. Mutations are indicated in red typeface. 5′ +, mutations detected 5′ (but not 3′) of the displayed sequence. (B) Sequences of editing mutants produced as per (A), by injecting sperm from 129-eGFP (upper) or Nanog-eGFP knock-in hemizygotes with wt oocytes, except by the sequential method ( Fig. 1A ). (C) Sequences of editing mutants as for (A), except that the transgenic alleles were maternal; wt 129 sperm were injected into mII oocytes obtained from 129-eGFP single copy (upper) or Nanog-eGFP knock-in hemizygotes. 3′ +, mutations were detected 3′ of the displayed sequence. Yellow highlighting indicates ambiguous calls presumptively produced by multiple targeting events. (D) Whole genome amplification of individual embryos collected 3 h after injection with sperm from a hemizygous transgenic line ( eGFP multi) carrying two copies of the eGFP transgene. The annotation used is as for (C). Red highlights provide at-a-glance indication of RNA concentrations in (A) to (D). (E) Merged confocal immunofluorescence images of single embryos at the times indicated (h) after ICSI, showing DNA labelled with propidium iodide (red) and antibody labeling (green) of tubulin-α (Tuba, upper panels) or histone H1 (H1). Both sperm and oocytes were wt. White arrowheads indicate paternal chromatin. Bar, 100 μm. (F) Schematic depicting a model for Cas9-mediated editing following injection of mII oocytes (mII). Limitted editing of maternal alleles during the gamete-to-embryo transition is inherent to the system, whereas limitted editing of paternal alleles in zygotes is because available targets have already been removed. Pb 2 , second polar body; pn, pronucleus.
Figure Legend Snippet: Sequence analysis reveals allelic asymmetry in parental genome mII editing. (A) Sequences of reverse-transcriptase PCR products from embryonic day 4 (E4.0) blastocysts developing after the 1-step method of editing ( Fig. 1A ) in which Cas9 cRNA and eGFP gRNA were co-injected into mII oocytes with sperm from 129-eGFP single-copy hemizygotes ( eGFP single). The gRNA-target sequence (green typeface) plus adjacent 5′ sequence is displayed on the top row and mutants beneath (grey typeface), with the corresponding Cas9 cRNA (c) and eGFP gRNA (g) concentrations (ng/μl) that were injected to produced them. The proto-spacer adjacent motif (PAM) is highlighted in green. Mutations are indicated in red typeface. 5′ +, mutations detected 5′ (but not 3′) of the displayed sequence. (B) Sequences of editing mutants produced as per (A), by injecting sperm from 129-eGFP (upper) or Nanog-eGFP knock-in hemizygotes with wt oocytes, except by the sequential method ( Fig. 1A ). (C) Sequences of editing mutants as for (A), except that the transgenic alleles were maternal; wt 129 sperm were injected into mII oocytes obtained from 129-eGFP single copy (upper) or Nanog-eGFP knock-in hemizygotes. 3′ +, mutations were detected 3′ of the displayed sequence. Yellow highlighting indicates ambiguous calls presumptively produced by multiple targeting events. (D) Whole genome amplification of individual embryos collected 3 h after injection with sperm from a hemizygous transgenic line ( eGFP multi) carrying two copies of the eGFP transgene. The annotation used is as for (C). Red highlights provide at-a-glance indication of RNA concentrations in (A) to (D). (E) Merged confocal immunofluorescence images of single embryos at the times indicated (h) after ICSI, showing DNA labelled with propidium iodide (red) and antibody labeling (green) of tubulin-α (Tuba, upper panels) or histone H1 (H1). Both sperm and oocytes were wt. White arrowheads indicate paternal chromatin. Bar, 100 μm. (F) Schematic depicting a model for Cas9-mediated editing following injection of mII oocytes (mII). Limitted editing of maternal alleles during the gamete-to-embryo transition is inherent to the system, whereas limitted editing of paternal alleles in zygotes is because available targets have already been removed. Pb 2 , second polar body; pn, pronucleus.

Techniques Used: Sequencing, Polymerase Chain Reaction, Injection, Produced, Knock-In, Transgenic Assay, Whole Genome Amplification, Immunofluorescence, Antibody Labeling

8) Product Images from "An outbreak of bovine trypanosomiasis in the Blue Nile State, Sudan"

Article Title: An outbreak of bovine trypanosomiasis in the Blue Nile State, Sudan

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-4-74

Rooted phylogenetic tree showing the relationship of the T. simiae identified in this study with reference sequences of T. simiae, T congoloense and T. brucei . The relationship was determined using the ITS1 of rRNA gene sequences by neighbor joining with 1,000 bootstrap. T. simiae samples identified in this study were depicted in bold letters. Trypanosomes sequences from GenBank were shown both by their accession numbers and parasites names). Scale bar used was nucleotide substitutions per position
Figure Legend Snippet: Rooted phylogenetic tree showing the relationship of the T. simiae identified in this study with reference sequences of T. simiae, T congoloense and T. brucei . The relationship was determined using the ITS1 of rRNA gene sequences by neighbor joining with 1,000 bootstrap. T. simiae samples identified in this study were depicted in bold letters. Trypanosomes sequences from GenBank were shown both by their accession numbers and parasites names). Scale bar used was nucleotide substitutions per position

Techniques Used:

9) Product Images from "Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing"

Article Title: Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/err188

Diagrammatic representation of the RNA silencing constructs (not drawn to scale). (A) A 21 nucleotide artificial microRNA (ami-BEIIb)-based osa -miR528 was synthesized by fusion PCR and cloned into Vec8, a Ti binary vector with a wheat high molecular weight glutenin promoter (wHMGPro) and nopaline synthase terminator (NOS). (B) The secondary structure of the osa -miR528 backbone as predicted by RNAfold, including information on ami-BEIIb (reverse complement). The predicted target cleavage site (arrow with sequence bold and highlighted) is located between positions 10 and 11 of the amiRNA, while the two mismatches (grey highlight) are located at positions 1 and 21. (C) The hairpin RNA (hp-BEIIb) was cloned in Vec8 by inserting a 397 bp BEIIb fragment in the sense (BEIIb→) and antisense (BEIIb←) orientations. The two fragments are flanked by two rice introns, Rint4 and Rint9, which form a hairpin loop. The amiRNA and hp-RNA fragments were directionally cloned using several restriction sites (H, Hin dIII; B, Bam HI; K, Kpn I; N, Not I; E Eco RI; S, Spe I).
Figure Legend Snippet: Diagrammatic representation of the RNA silencing constructs (not drawn to scale). (A) A 21 nucleotide artificial microRNA (ami-BEIIb)-based osa -miR528 was synthesized by fusion PCR and cloned into Vec8, a Ti binary vector with a wheat high molecular weight glutenin promoter (wHMGPro) and nopaline synthase terminator (NOS). (B) The secondary structure of the osa -miR528 backbone as predicted by RNAfold, including information on ami-BEIIb (reverse complement). The predicted target cleavage site (arrow with sequence bold and highlighted) is located between positions 10 and 11 of the amiRNA, while the two mismatches (grey highlight) are located at positions 1 and 21. (C) The hairpin RNA (hp-BEIIb) was cloned in Vec8 by inserting a 397 bp BEIIb fragment in the sense (BEIIb→) and antisense (BEIIb←) orientations. The two fragments are flanked by two rice introns, Rint4 and Rint9, which form a hairpin loop. The amiRNA and hp-RNA fragments were directionally cloned using several restriction sites (H, Hin dIII; B, Bam HI; K, Kpn I; N, Not I; E Eco RI; S, Spe I).

Techniques Used: Construct, Synthesized, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Molecular Weight, Sequencing

10) Product Images from "Distinct Expression Profiles and Different Functions of Odorant Binding Proteins in Nilaparvata lugens St?l"

Article Title: Distinct Expression Profiles and Different Functions of Odorant Binding Proteins in Nilaparvata lugens St?l

Journal: PLoS ONE

doi: 10.1371/journal.pone.0028921

Effects of feeding NlugOBP3 dsRNA on NlugOBP3 mRNA level (A), survival rate (B), jumping ability (C), and response to rice seedlings (D-E) of BPH nymphs. CK, nymph fed with normal diet; dsGFP, fed with diet mixed with dsRNA of green fluorescent protein (0.5 mg/ml); dsOBP3, fed with dsRNA of NlugOBP3 (0.5 mg/ml). Data topped with different letters are significant different as determined using a one-way ANOVA (Duncan’s multiple range test, P
Figure Legend Snippet: Effects of feeding NlugOBP3 dsRNA on NlugOBP3 mRNA level (A), survival rate (B), jumping ability (C), and response to rice seedlings (D-E) of BPH nymphs. CK, nymph fed with normal diet; dsGFP, fed with diet mixed with dsRNA of green fluorescent protein (0.5 mg/ml); dsOBP3, fed with dsRNA of NlugOBP3 (0.5 mg/ml). Data topped with different letters are significant different as determined using a one-way ANOVA (Duncan’s multiple range test, P

Techniques Used:

11) Product Images from "Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)"

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

Journal: Current protocols in microbiology

doi: 10.1002/9780471729259.mc09d03s30

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.
Figure Legend 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.

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

12) Product Images from "Molecular characterization of orf virus from sheep and goats in Ethiopia, 2008–2013"

Article Title: Molecular characterization of orf virus from sheep and goats in Ethiopia, 2008–2013

Journal: Virology Journal

doi: 10.1186/s12985-016-0489-3

Phylogenetic tree of the A32L gene nucleotide sequence (825 nt) of Ethiopian isolates orf virus isolates. Eighteen Ethiopian orf virus isolates and ten foreign orf viruses were compared. Additionally, the homologue gene sequence of BPSV (AY386265) and PCPV (GQ329670) were used as out-groups. The numbers displayed on the branches represent the posterior probabilities. The sequences retrieved from GenBank are indicated with accession number under bracket and those of this study were highlighted with red bubbles
Figure Legend Snippet: Phylogenetic tree of the A32L gene nucleotide sequence (825 nt) of Ethiopian isolates orf virus isolates. Eighteen Ethiopian orf virus isolates and ten foreign orf viruses were compared. Additionally, the homologue gene sequence of BPSV (AY386265) and PCPV (GQ329670) were used as out-groups. The numbers displayed on the branches represent the posterior probabilities. The sequences retrieved from GenBank are indicated with accession number under bracket and those of this study were highlighted with red bubbles

Techniques Used: Sequencing

Multiple sequence alignment of the deduced amino acid sequence of the A32L gene of the Ethiopian orf virus isolates. The deduced amino acid sequence of the A32L gene of twenty four foreign orf virus strains retrieved from GenBank were added of comparison. The analysis revealed a high divergence in the carboxyl terminal region of ATPase protein sequence unlike the N-terminal region. The three repeats of KGD amino acid residues are highlighted with red color box. The two green color boxes indicate the presence of RGD sequences at two sites
Figure Legend Snippet: Multiple sequence alignment of the deduced amino acid sequence of the A32L gene of the Ethiopian orf virus isolates. The deduced amino acid sequence of the A32L gene of twenty four foreign orf virus strains retrieved from GenBank were added of comparison. The analysis revealed a high divergence in the carboxyl terminal region of ATPase protein sequence unlike the N-terminal region. The three repeats of KGD amino acid residues are highlighted with red color box. The two green color boxes indicate the presence of RGD sequences at two sites

Techniques Used: Sequencing

13) Product Images from "Mechanistic studies of DepR in regulating FK228 biosynthesis in Chromobacterium violaceum no. 968"

Article Title: Mechanistic studies of DepR in regulating FK228 biosynthesis in Chromobacterium violaceum no. 968

Journal: PLoS ONE

doi: 10.1371/journal.pone.0196173

Relative levels of FK228 productions by C . violaceum strains detected and quantified by LC-MS. No. 968, the wild-type strain; CvΔdepR, depR gene deletion mutant strain. Eight complementation strains: depR , CvΔdepR (pBMTL-3- depR) ; C199A, CvΔdepR (pWHU3057); C199S, CvΔdepR (p3T9); C199T, CvΔdepR (pWHU3058); T232A, CvΔdepR (pWHU3060); R278A, CvΔdepR (pWHU3062); S299A, CvΔdepR (pWHU3059); T244A, CvΔdepR (pWHU3061). An overproduction strain of orf22 , No. 968 (pWHU3070). Data are mean values of results from duplicate experiments with error bars indicating standard deviation (n = 3).
Figure Legend Snippet: Relative levels of FK228 productions by C . violaceum strains detected and quantified by LC-MS. No. 968, the wild-type strain; CvΔdepR, depR gene deletion mutant strain. Eight complementation strains: depR , CvΔdepR (pBMTL-3- depR) ; C199A, CvΔdepR (pWHU3057); C199S, CvΔdepR (p3T9); C199T, CvΔdepR (pWHU3058); T232A, CvΔdepR (pWHU3060); R278A, CvΔdepR (pWHU3062); S299A, CvΔdepR (pWHU3059); T244A, CvΔdepR (pWHU3061). An overproduction strain of orf22 , No. 968 (pWHU3070). Data are mean values of results from duplicate experiments with error bars indicating standard deviation (n = 3).

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Standard Deviation

14) Product Images from "Efficacy of a Doxycycline Treatment Regimen Initiated during Three Different Phases of Experimental Ehrlichiosis ▿"

Article Title: Efficacy of a Doxycycline Treatment Regimen Initiated during Three Different Phases of Experimental Ehrlichiosis ▿

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01622-09

Sequences of selected PCR products amplified from canine and acarine hosts after treatment with doxycycline. Multiple sequence alignment of amplicons from PCR-positive tick and buffy coat samples collected after doxycycline treatment of dogs. Sequences
Figure Legend Snippet: Sequences of selected PCR products amplified from canine and acarine hosts after treatment with doxycycline. Multiple sequence alignment of amplicons from PCR-positive tick and buffy coat samples collected after doxycycline treatment of dogs. Sequences

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

15) Product Images from "Congruent Strain Specific Intestinal Persistence of Lactobacillus plantarum in an Intestine-Mimicking In Vitro System and in Human Volunteers"

Article Title: Congruent Strain Specific Intestinal Persistence of Lactobacillus plantarum in an Intestine-Mimicking In Vitro System and in Human Volunteers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044588

Strain-specific L. plantarum relative abundance after human consumption as detected by pyrosequencing. Relative strain abundances of the bacterial preparations consumed by the volunteers are depicted in black diamonds and those determined in time-specified post-consumption fecal material from the subjects 1 to 5 in red, green, blue, purple, and yellow diamonds, respectively. The graphs represent the number of strain specific sequences in the amplicons generated from DNA derived from fecal samples, divided by the number of strain-specific sequences identified in the input mixture amplicon. The total number of sequences per sample was set at 1 for normalization purposes. Axis-scaling in all the graphs is the same as depicted for strain WCFS1.
Figure Legend Snippet: Strain-specific L. plantarum relative abundance after human consumption as detected by pyrosequencing. Relative strain abundances of the bacterial preparations consumed by the volunteers are depicted in black diamonds and those determined in time-specified post-consumption fecal material from the subjects 1 to 5 in red, green, blue, purple, and yellow diamonds, respectively. The graphs represent the number of strain specific sequences in the amplicons generated from DNA derived from fecal samples, divided by the number of strain-specific sequences identified in the input mixture amplicon. The total number of sequences per sample was set at 1 for normalization purposes. Axis-scaling in all the graphs is the same as depicted for strain WCFS1.

Techniques Used: Generated, Derivative Assay, Amplification

Schematic representation of the 339-IR-340 region of L. plantarum strains. Panel A: Schematic representation of the variable region (grey area) between the lp_0339 and lp_0340 genes (white open arrows) of L. plantarum WCFS1 with the single nucleotide polymorphism positions (yellow areas) detected in the other strains. Primers used to generate amplicons for sequencing are displayed. Panel B: Sequence comparison of the 10 sequence variations in the 339-IR-340 intergenic region. Yellow circles indicate the nucleotide(s) that distinguish the 339-IR-340 sequence types.
Figure Legend Snippet: Schematic representation of the 339-IR-340 region of L. plantarum strains. Panel A: Schematic representation of the variable region (grey area) between the lp_0339 and lp_0340 genes (white open arrows) of L. plantarum WCFS1 with the single nucleotide polymorphism positions (yellow areas) detected in the other strains. Primers used to generate amplicons for sequencing are displayed. Panel B: Sequence comparison of the 10 sequence variations in the 339-IR-340 intergenic region. Yellow circles indicate the nucleotide(s) that distinguish the 339-IR-340 sequence types.

Techniques Used: Sequencing

L. plantarum strain WCFS1, NCTH19-2, and NC8 relative abundance after human consumption as assessed by pyrosequencing. Relative strain abundances from subjects 1 to 10 are depicted in red, green, blue, purple, yellow, pink, brown, orange, white and grey diamonds, respectively. The graphs represent the number of strain specific sequences in the fecal amplicons, divided by the number of strain-specific sequences identified in the input mixture amplicon. The total number of sequences per sample was set at 1 for normalization purposes. Axis-scaling in all the graphs is the same as depicted for strain WCFS1.
Figure Legend Snippet: L. plantarum strain WCFS1, NCTH19-2, and NC8 relative abundance after human consumption as assessed by pyrosequencing. Relative strain abundances from subjects 1 to 10 are depicted in red, green, blue, purple, yellow, pink, brown, orange, white and grey diamonds, respectively. The graphs represent the number of strain specific sequences in the fecal amplicons, divided by the number of strain-specific sequences identified in the input mixture amplicon. The total number of sequences per sample was set at 1 for normalization purposes. Axis-scaling in all the graphs is the same as depicted for strain WCFS1.

Techniques Used: Amplification

16) Product Images from "Taxonomically-linked growth phenotypes during arsenic stress among arsenic resistant bacteria isolated from soils overlying the Centralia coal seam fire"

Article Title: Taxonomically-linked growth phenotypes during arsenic stress among arsenic resistant bacteria isolated from soils overlying the Centralia coal seam fire

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191893

Comparison of AsRG sequences and 16S rRNA gene sequences from As resistant isolates. Maximum likelihood trees for AsRGs (left panel) (A) arsB , (B) ACR3(2) , and (C) arsC are shown alongside trees of corresponding 16S rRNA genes (right panel). Incongruence is highlighted with grey lines between the two trees. Scale bars indicate the percent difference in nucleotide sequence. Bootstrap values greater than 50% are indicated at the corresponding node, and boxes are colored based on isolate genus.
Figure Legend Snippet: Comparison of AsRG sequences and 16S rRNA gene sequences from As resistant isolates. Maximum likelihood trees for AsRGs (left panel) (A) arsB , (B) ACR3(2) , and (C) arsC are shown alongside trees of corresponding 16S rRNA genes (right panel). Incongruence is highlighted with grey lines between the two trees. Scale bars indicate the percent difference in nucleotide sequence. Bootstrap values greater than 50% are indicated at the corresponding node, and boxes are colored based on isolate genus.

Techniques Used: Sequencing

Phylogenetic tree of 16S rRNA sequences from Centralia As resistant isolates. Isolates from this study were compared with isolates from other studies that cultivated As resistant isolates from soil. (A) Actinobacteria, Proteobacteria, and Sphingobacteria. (B) Firmicutes. Scale bars indicate the percent difference in nucleotide sequence.
Figure Legend Snippet: Phylogenetic tree of 16S rRNA sequences from Centralia As resistant isolates. Isolates from this study were compared with isolates from other studies that cultivated As resistant isolates from soil. (A) Actinobacteria, Proteobacteria, and Sphingobacteria. (B) Firmicutes. Scale bars indicate the percent difference in nucleotide sequence.

Techniques Used: Sequencing

17) Product Images from "IL-4 induces expression of TARC/CCL17 via two STAT6 binding sites"

Article Title: IL-4 induces expression of TARC/CCL17 via two STAT6 binding sites

Journal: European journal of immunology

doi: 10.1002/eji.200635972

(A) Genomic organization of the TARC/CCL17 locus. Filled boxes upstream of exon 1 represent GAS motifs A and B. The numbers indicate the positions of the GAS motifs relative to the translational start site. (B) Nucleotide sequences of motif A, motif B, the GAS motif found in the promoter region of Eotaxin-3/CCL26 and the mutated motif. The core sequence is shown in bold, the mutated nucleotides are underlined. Note that motif A of the TARC/CCL17 promoter is identical to the Eotaxin-3 motif. (C) Transcription start site as mapped by means of 5′ RACE. PCR products of the semi-nested PCR reaction were cloned into the pGL3 Basic vector for sequencing. The transcriptional start site was determined to be 64 ± 1 bp upstream of the start codon, by sequence analysis of positive clones. (D) Structures of the two TARC/CCL17 promoter luciferase (LUC) constructs used in this study. Filled boxes represent the GAS motifs A and B, ‘TATA’ designates the TATA box in the vector. ‘913 bp’ and ‘239 bp’ are the lengths of the TARC/CCL17 promoter fragments used.
Figure Legend Snippet: (A) Genomic organization of the TARC/CCL17 locus. Filled boxes upstream of exon 1 represent GAS motifs A and B. The numbers indicate the positions of the GAS motifs relative to the translational start site. (B) Nucleotide sequences of motif A, motif B, the GAS motif found in the promoter region of Eotaxin-3/CCL26 and the mutated motif. The core sequence is shown in bold, the mutated nucleotides are underlined. Note that motif A of the TARC/CCL17 promoter is identical to the Eotaxin-3 motif. (C) Transcription start site as mapped by means of 5′ RACE. PCR products of the semi-nested PCR reaction were cloned into the pGL3 Basic vector for sequencing. The transcriptional start site was determined to be 64 ± 1 bp upstream of the start codon, by sequence analysis of positive clones. (D) Structures of the two TARC/CCL17 promoter luciferase (LUC) constructs used in this study. Filled boxes represent the GAS motifs A and B, ‘TATA’ designates the TATA box in the vector. ‘913 bp’ and ‘239 bp’ are the lengths of the TARC/CCL17 promoter fragments used.

Techniques Used: Sequencing, Polymerase Chain Reaction, Nested PCR, Clone Assay, Plasmid Preparation, Luciferase, Construct

18) Product Images from "A Profibrotic Phenotype in Naïve and in Fibrotic Lung Myofibroblasts Is Governed by Modulations in Thy-1 Expression and Activation"

Article Title: A Profibrotic Phenotype in Naïve and in Fibrotic Lung Myofibroblasts Is Governed by Modulations in Thy-1 Expression and Activation

Journal: Mediators of Inflammation

doi: 10.1155/2018/4638437

Inverse correlation between Thy1 expression and fibroblast proliferation. (a) 24 h following transfection, cell mass was measured by colorimetric assay using methylene blue staining in Mlg cells transfected with Thy1 expression vector and in control plasmid-containing Thy1 cDNA in antisense orientation. Results are presented as fold change from day 0 ( n = 4 − 5 in each group) ( ∗ p
Figure Legend Snippet: Inverse correlation between Thy1 expression and fibroblast proliferation. (a) 24 h following transfection, cell mass was measured by colorimetric assay using methylene blue staining in Mlg cells transfected with Thy1 expression vector and in control plasmid-containing Thy1 cDNA in antisense orientation. Results are presented as fold change from day 0 ( n = 4 − 5 in each group) ( ∗ p

Techniques Used: Expressing, Transfection, Colorimetric Assay, Staining, Plasmid Preparation

Thy1 gene expression following genetic manipulation, using Thy1 knockout mice, Thy1 siRNA, and Thy1 cDNA expression vector. (a) Flow cytometry Thy-FITC mAb staining in spleen cells from wild-type (WT) C57BL/6 mice and Thy1-deficient mice, respectively. (b) Downregulation of Thy1 by siRNA was performed. Thy1 downregulation was detected 48 h after transfection by FACS analysis (c) Mlg cells were transfected with Thy1 expression vector. Plasmid-containing Thy1 cDNA in antisense orientation serves as a control. Thy1 expression was detected 24 h after the transfection by FACS analysis.
Figure Legend Snippet: Thy1 gene expression following genetic manipulation, using Thy1 knockout mice, Thy1 siRNA, and Thy1 cDNA expression vector. (a) Flow cytometry Thy-FITC mAb staining in spleen cells from wild-type (WT) C57BL/6 mice and Thy1-deficient mice, respectively. (b) Downregulation of Thy1 by siRNA was performed. Thy1 downregulation was detected 48 h after transfection by FACS analysis (c) Mlg cells were transfected with Thy1 expression vector. Plasmid-containing Thy1 cDNA in antisense orientation serves as a control. Thy1 expression was detected 24 h after the transfection by FACS analysis.

Techniques Used: Expressing, Knock-Out, Mouse Assay, Plasmid Preparation, Flow Cytometry, Cytometry, Staining, Transfection, FACS

Thy1 expression is not involved in the regulation of fibroblast apoptosis. (a) Western blots determining the extent of pro-caspase 3 cleavage (apoptosis) in Thy1 + or Thy1 − sorted fibroblasts from the lungs of bleomycin-treated mice and in a lung fibroblast cell line (Mlg) transfected with a Thy1.2 expression vector or plasmid-containing Thy1 cDNA in antisense orientation and in murine primary lung myofibroblasts transfected with Thy1 siRNA or control siRNA. (b) Western blot of pro-caspase 3 cleavage. (c) DNA ladder in lung fibroblast primary cultures following stimulation with G7 anti-Thy1 mAb (5 μ g/ml) or IgG isotype match control for 24–72 h.
Figure Legend Snippet: Thy1 expression is not involved in the regulation of fibroblast apoptosis. (a) Western blots determining the extent of pro-caspase 3 cleavage (apoptosis) in Thy1 + or Thy1 − sorted fibroblasts from the lungs of bleomycin-treated mice and in a lung fibroblast cell line (Mlg) transfected with a Thy1.2 expression vector or plasmid-containing Thy1 cDNA in antisense orientation and in murine primary lung myofibroblasts transfected with Thy1 siRNA or control siRNA. (b) Western blot of pro-caspase 3 cleavage. (c) DNA ladder in lung fibroblast primary cultures following stimulation with G7 anti-Thy1 mAb (5 μ g/ml) or IgG isotype match control for 24–72 h.

Techniques Used: Expressing, Western Blot, Mouse Assay, Transfection, Plasmid Preparation

19) Product Images from "Human RFP2 gene promoter: Unique structure and unusual strength"

Article Title: Human RFP2 gene promoter: Unique structure and unusual strength

Journal: Biochemical and biophysical research communications

doi: 10.1016/j.bbrc.2006.01.187

Computationally predicted promoter elements located within the sequence of the promoter area of the human RFP2 gene. DNA fragment corresponding to insert in pGLM6 most active in luciferase assay is highlighted by blue background. First exon of the human RFP2 gene is in the underlined letters in red color, shorter isoform of this exon is shown in bold. Bold lines depict locations of the computationally predicted promoter elements. Results of the human first exon finder are underlined in blue, results of the promoter scan—in green, and NNPP prediction algorithm results in brown. Nucleotides shown in capital red letters indicate transcription start sites predicted by NNPP. Transcription Start Sites predicted by Markov chain promoter finder and by Promoter 2.0 Prediction Server are shown by green and blue arrows, respectively. Nucleotide positions shown in bold blue letters and in bold green letters are occupied by imperfect repeats forming quadruplexes at non-coding and coding DNA strands, respectively.
Figure Legend Snippet: Computationally predicted promoter elements located within the sequence of the promoter area of the human RFP2 gene. DNA fragment corresponding to insert in pGLM6 most active in luciferase assay is highlighted by blue background. First exon of the human RFP2 gene is in the underlined letters in red color, shorter isoform of this exon is shown in bold. Bold lines depict locations of the computationally predicted promoter elements. Results of the human first exon finder are underlined in blue, results of the promoter scan—in green, and NNPP prediction algorithm results in brown. Nucleotides shown in capital red letters indicate transcription start sites predicted by NNPP. Transcription Start Sites predicted by Markov chain promoter finder and by Promoter 2.0 Prediction Server are shown by green and blue arrows, respectively. Nucleotide positions shown in bold blue letters and in bold green letters are occupied by imperfect repeats forming quadruplexes at non-coding and coding DNA strands, respectively.

Techniques Used: Sequencing, Luciferase

Human RFP2 promoter nucleosome binding profile (A) and the profile of electrostatic field magnitudes induced by sequence-dependent dipoles (B) for free DNA double helix produced by RFP2 promoter nucleotide sequence.
Figure Legend Snippet: Human RFP2 promoter nucleosome binding profile (A) and the profile of electrostatic field magnitudes induced by sequence-dependent dipoles (B) for free DNA double helix produced by RFP2 promoter nucleotide sequence.

Techniques Used: Binding Assay, Sequencing, Produced

20) Product Images from "Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)"

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

Journal: Current protocols in microbiology

doi: 10.1002/9780471729259.mc09d03s30

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.
Figure Legend 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.

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

21) Product Images from "P2X7 ionotropic receptor is functionally expressed in rabbit articular chondrocytes and mediates extracellular ATP cytotoxicity"

Article Title: P2X7 ionotropic receptor is functionally expressed in rabbit articular chondrocytes and mediates extracellular ATP cytotoxicity

Journal: Purinergic Signalling

doi: 10.1007/s11302-018-9611-x

RT-PCR cloning, sequencing, and functional characterization of recombinant rabbit P2X7 receptor. A P2X7 mRNA expression in rabbit articular chondrocytes. Ethidium bromide-stained agarose gel showing the ~ 1800 bp RT-PCR product of P2X7 transcripts. B Amino acid sequences of rat, rabbit, and human P2X7. Consensus residues are shaded gray. The residue at 95 (indicated by a box) is known to be responsible for the difference between human and rat P2X7 in the sensitivity to KN-62. C Functional reconstitution of rabbit P2X7 in HEK293 cells. Representative current traces during exposure to 300 μM ATP recorded from HEK cells transfected with GFP alone ( a ) or GFP plus P2X7 ( b ). The I – V relationship of the ATP-induced P2X7 current is shown in c . D Inhibition of rabbit P2X7 current by KN-62. Currents were repeatedly activated by 300 μM ATP with a resting interval of > 3 min in the absence ( upper trace ) and presence ( lower trace ) of 1 μM KN-62
Figure Legend Snippet: RT-PCR cloning, sequencing, and functional characterization of recombinant rabbit P2X7 receptor. A P2X7 mRNA expression in rabbit articular chondrocytes. Ethidium bromide-stained agarose gel showing the ~ 1800 bp RT-PCR product of P2X7 transcripts. B Amino acid sequences of rat, rabbit, and human P2X7. Consensus residues are shaded gray. The residue at 95 (indicated by a box) is known to be responsible for the difference between human and rat P2X7 in the sensitivity to KN-62. C Functional reconstitution of rabbit P2X7 in HEK293 cells. Representative current traces during exposure to 300 μM ATP recorded from HEK cells transfected with GFP alone ( a ) or GFP plus P2X7 ( b ). The I – V relationship of the ATP-induced P2X7 current is shown in c . D Inhibition of rabbit P2X7 current by KN-62. Currents were repeatedly activated by 300 μM ATP with a resting interval of > 3 min in the absence ( upper trace ) and presence ( lower trace ) of 1 μM KN-62

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Sequencing, Functional Assay, Recombinant, Expressing, Staining, Agarose Gel Electrophoresis, Transfection, Inhibition

22) Product Images from "Occurrence and abundance of a mariner-like element in freshwater and terrestrial planarians (Platyhelminthes, Tricladida) from southern Brazil"

Article Title: Occurrence and abundance of a mariner-like element in freshwater and terrestrial planarians (Platyhelminthes, Tricladida) from southern Brazil

Journal: Genetics and Molecular Biology

doi: 10.1590/S1415-47572009005000072

Karyotype of (A) Girardia schubarti (2n = 8) and (B) G. tigrina (2n = 16).
Figure Legend Snippet: Karyotype of (A) Girardia schubarti (2n = 8) and (B) G. tigrina (2n = 16).

Techniques Used:

23) Product Images from "An outbreak of bovine trypanosomiasis in the Blue Nile State, Sudan"

Article Title: An outbreak of bovine trypanosomiasis in the Blue Nile State, Sudan

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-4-74

Rooted phylogenetic tree showing the relationship of the T. simiae identified in this study with reference sequences of T. simiae, T congoloense and T. brucei . The relationship was determined using the ITS1 of rRNA gene sequences by neighbor joining with 1,000 bootstrap. T. simiae samples identified in this study were depicted in bold letters. Trypanosomes sequences from GenBank were shown both by their accession numbers and parasites names). Scale bar used was nucleotide substitutions per position
Figure Legend Snippet: Rooted phylogenetic tree showing the relationship of the T. simiae identified in this study with reference sequences of T. simiae, T congoloense and T. brucei . The relationship was determined using the ITS1 of rRNA gene sequences by neighbor joining with 1,000 bootstrap. T. simiae samples identified in this study were depicted in bold letters. Trypanosomes sequences from GenBank were shown both by their accession numbers and parasites names). Scale bar used was nucleotide substitutions per position

Techniques Used:

24) Product Images from "Congruent Strain Specific Intestinal Persistence of Lactobacillus plantarum in an Intestine-Mimicking In Vitro System and in Human Volunteers"

Article Title: Congruent Strain Specific Intestinal Persistence of Lactobacillus plantarum in an Intestine-Mimicking In Vitro System and in Human Volunteers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0044588

Schematic representation of the 339-IR-340 region of L. plantarum strains. Panel A: Schematic representation of the variable region (grey area) between the lp_0339 and lp_0340 genes (white open arrows) of L. plantarum WCFS1 with the single nucleotide polymorphism positions (yellow areas) detected in the other strains. Primers used to generate amplicons for sequencing are displayed. Panel B: Sequence comparison of the 10 sequence variations in the 339-IR-340 intergenic region. Yellow circles indicate the nucleotide(s) that distinguish the 339-IR-340 sequence types.
Figure Legend Snippet: Schematic representation of the 339-IR-340 region of L. plantarum strains. Panel A: Schematic representation of the variable region (grey area) between the lp_0339 and lp_0340 genes (white open arrows) of L. plantarum WCFS1 with the single nucleotide polymorphism positions (yellow areas) detected in the other strains. Primers used to generate amplicons for sequencing are displayed. Panel B: Sequence comparison of the 10 sequence variations in the 339-IR-340 intergenic region. Yellow circles indicate the nucleotide(s) that distinguish the 339-IR-340 sequence types.

Techniques Used: Sequencing

25) Product Images from "The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed"

Article Title: The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed

Journal: Virus Evolution

doi: 10.1093/ve/vev002

(A) FISH on D. alata chromosomes with a 2.6 kb EGV1 probe from D. alata (detected in green) and a rDNA 45 S probe used as control (detected in red). Chromosomes are counterstained with DAPI, in blue. Scale bar = 5 µm. The green arrows indicate four hybridization signals potentially corresponding to the presence of tandem repeats of EGV located on both chromatids of two chromosome pairs. The white arrows indicate faint signals potentially corresponding to the presence of copies of the rep gene scattered around the genome of D. alata . (B) Long template PCRs enabled the amplification of two DNA fragments (444 bp and 3,086 bp) from D. alata acc. 313. Sequencing of 444 and 3,086 fragments revealed a partial tandem repeat genomic organization with two partial rep genes, a ren gene, and an intergenic region containing a GC-rich stem carrying a nonanucleotide loop TAATATTAC. The size of one repeat is 2,642 bp. (C) Representation of a typical linearized begomovirus genome in the virion sense orientation starting from the origin of replication (mp, movement protein; cp, capsid protein; ren, replication enhancer; trap, transactivator protein; rep, replication-associated protein; C4 ORF has been shown to suppress transcriptional gene silencing).
Figure Legend Snippet: (A) FISH on D. alata chromosomes with a 2.6 kb EGV1 probe from D. alata (detected in green) and a rDNA 45 S probe used as control (detected in red). Chromosomes are counterstained with DAPI, in blue. Scale bar = 5 µm. The green arrows indicate four hybridization signals potentially corresponding to the presence of tandem repeats of EGV located on both chromatids of two chromosome pairs. The white arrows indicate faint signals potentially corresponding to the presence of copies of the rep gene scattered around the genome of D. alata . (B) Long template PCRs enabled the amplification of two DNA fragments (444 bp and 3,086 bp) from D. alata acc. 313. Sequencing of 444 and 3,086 fragments revealed a partial tandem repeat genomic organization with two partial rep genes, a ren gene, and an intergenic region containing a GC-rich stem carrying a nonanucleotide loop TAATATTAC. The size of one repeat is 2,642 bp. (C) Representation of a typical linearized begomovirus genome in the virion sense orientation starting from the origin of replication (mp, movement protein; cp, capsid protein; ren, replication enhancer; trap, transactivator protein; rep, replication-associated protein; C4 ORF has been shown to suppress transcriptional gene silencing).

Techniques Used: Fluorescence In Situ Hybridization, Hybridization, Amplification, Sequencing

26) Product Images from "Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles"

Article Title: Modulation of cyclobutane thymine photodimer formation in T11-tracts in rotationally phased nucleosome core particles and DNA minicircles

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx427

Circular permutation polymerase chain reaction (PCR) strategy used for preparing nucleosomal DNA with T 11 -tracts at specific superhelix locations (SHLs). ( A ) A 168-mer DNA duplex was designed to have a centrally located T 11 -tract (underlined), terminal EcoRI restriction sites (green italic), phased minor groove bending (T/A) 3 sequences in red and major groove bending (G/C) 3 sequences in blue. The T in orange in the T 11 -tract corresponds to the position at which the major groove is expected to bend toward the histone surface at the dyad axis. ( B ) The 168-mer DNA duplex was prepared by primer extension of two overlapping 95 and 96-mers, cleaved with EcoRI and multimerized with T4 DNA ligase and adenosine triphosphate. The dimer was then excised from a gel and cloned. Nucleosomal DNAs with T 11 -tracts at specific SHLs were prepared from the clone by PCR using specific pairs of forward and reverse primers ( Supplementary Figure S3 ) whose positions are shown as solid and dashed arrows respectively on the sequence in panel A. The SHLs are identified by the number of helical turns from the dyad axis on the blue strand containing the T 11 -tract. They are negative to correspond with the negative numbers assigned to nucleotides on the T 11 -tract strand that are 5΄-to the T at the dyad axis which is assigned as 0. The −3 (inside) and +3 (outside) nucleotide positions are shown in blue. The positions at which the major and minor grooves face the histone surface are indicated by M and m, respectively, and are colored coded blue and red to match the major groove and minor groove bending motifs in panel A.
Figure Legend Snippet: Circular permutation polymerase chain reaction (PCR) strategy used for preparing nucleosomal DNA with T 11 -tracts at specific superhelix locations (SHLs). ( A ) A 168-mer DNA duplex was designed to have a centrally located T 11 -tract (underlined), terminal EcoRI restriction sites (green italic), phased minor groove bending (T/A) 3 sequences in red and major groove bending (G/C) 3 sequences in blue. The T in orange in the T 11 -tract corresponds to the position at which the major groove is expected to bend toward the histone surface at the dyad axis. ( B ) The 168-mer DNA duplex was prepared by primer extension of two overlapping 95 and 96-mers, cleaved with EcoRI and multimerized with T4 DNA ligase and adenosine triphosphate. The dimer was then excised from a gel and cloned. Nucleosomal DNAs with T 11 -tracts at specific SHLs were prepared from the clone by PCR using specific pairs of forward and reverse primers ( Supplementary Figure S3 ) whose positions are shown as solid and dashed arrows respectively on the sequence in panel A. The SHLs are identified by the number of helical turns from the dyad axis on the blue strand containing the T 11 -tract. They are negative to correspond with the negative numbers assigned to nucleotides on the T 11 -tract strand that are 5΄-to the T at the dyad axis which is assigned as 0. The −3 (inside) and +3 (outside) nucleotide positions are shown in blue. The positions at which the major and minor grooves face the histone surface are indicated by M and m, respectively, and are colored coded blue and red to match the major groove and minor groove bending motifs in panel A.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Sequencing

27) 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

28) 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

29) 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

30) 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

31) 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

32) 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

33) Product Images from "Production of a unique pneumococcal capsule serotype belonging to serogroup 6"

Article Title: Production of a unique pneumococcal capsule serotype belonging to serogroup 6

Journal:

doi: 10.1099/mic.0.024521-0

PCR and DNA sequencing
Figure Legend Snippet: PCR and DNA sequencing

Techniques Used: Polymerase Chain Reaction, DNA Sequencing

34) Product Images from "Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates"

Article Title: Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq674

Trapping a dye-labeled supercoiled Holliday junction recombination intermediate and its relaxation by site-specific nicking with the restriction enzyme Nb.BbvCI. Recombination reactions with the HJ-trapping att sites diagramed in Figure 4 were carried out with dye-labeled supercoiled att P and linear 32 P-labeled att B, and analyzed by agarose gel electrophoresis, as described in ‘Materials and Methods’ section. ( A ) Visualization of the gel by 488-nm excitation of fluorescein in att P. ( B ) Visualization of the gel by autoradiography of the 32 P-radiolabel in att B. Recombination reactions, without (lanes 1 and 2), or with (lanes 3 and 4), Int were incubated for 1 h, after which MgCl 2 (10 mM final) and the nicking enzyme, Nb.BbvCI were added to reactions 2 and 4, and all four reactions were incubated for an additional hour before loading onto a native agarose gel. No full recombination product is formed under these conditions. In control reactions with canonical att sites, the nicking enzyme does not alter the mobility of the full linear recombination product (data not shown).
Figure Legend Snippet: Trapping a dye-labeled supercoiled Holliday junction recombination intermediate and its relaxation by site-specific nicking with the restriction enzyme Nb.BbvCI. Recombination reactions with the HJ-trapping att sites diagramed in Figure 4 were carried out with dye-labeled supercoiled att P and linear 32 P-labeled att B, and analyzed by agarose gel electrophoresis, as described in ‘Materials and Methods’ section. ( A ) Visualization of the gel by 488-nm excitation of fluorescein in att P. ( B ) Visualization of the gel by autoradiography of the 32 P-radiolabel in att B. Recombination reactions, without (lanes 1 and 2), or with (lanes 3 and 4), Int were incubated for 1 h, after which MgCl 2 (10 mM final) and the nicking enzyme, Nb.BbvCI were added to reactions 2 and 4, and all four reactions were incubated for an additional hour before loading onto a native agarose gel. No full recombination product is formed under these conditions. In control reactions with canonical att sites, the nicking enzyme does not alter the mobility of the full linear recombination product (data not shown).

Techniques Used: Labeling, Agarose Gel Electrophoresis, Autoradiography, Incubation

Overlap regions of the att P and att B sites showing the bubble–heterology sequences used to trap Holliday junction recombination intermediates. The altered base pairs of the 7-bp overlap region are indicated in bold upper case letters, the Int cleavage sites flanking each overlap region are indicated by curved arrows, and the strands conventionally referred to as the top- and bottom-strands are labeled TS and BS, respectively; numbering is the same as in Figure 1 . To highlight the salient features of these sites, three modifications to the conventional depictions have been made: (a) the two att sites are aligned in a parallel rather than antiparallel orientation; (b) the att P site is drawn with the ‘top strand’ (TS) on the bottom; and (c) as a result of the parallel alignment of the att sites, the exchanged strands in the Holliday junction (HJ) are crossed. Following the first pair of Int cleavages the top strands are swapped to form the Holliday junction, simultaneously converting the unpaired (bubble) bases to the more stable duplex DNA. The second pair of Int cleavages on the bottom strands that would normally resolve the Holliday junction to recombinant products (attL and attR) is strongly disfavored because the sequence differences between the two overlap regions would generate unpaired bubbles in both att L and att R after strand exchange. The resulting Holliday intermediate is thus stabilized with respect to both the reverse and forward reaction products.
Figure Legend Snippet: Overlap regions of the att P and att B sites showing the bubble–heterology sequences used to trap Holliday junction recombination intermediates. The altered base pairs of the 7-bp overlap region are indicated in bold upper case letters, the Int cleavage sites flanking each overlap region are indicated by curved arrows, and the strands conventionally referred to as the top- and bottom-strands are labeled TS and BS, respectively; numbering is the same as in Figure 1 . To highlight the salient features of these sites, three modifications to the conventional depictions have been made: (a) the two att sites are aligned in a parallel rather than antiparallel orientation; (b) the att P site is drawn with the ‘top strand’ (TS) on the bottom; and (c) as a result of the parallel alignment of the att sites, the exchanged strands in the Holliday junction (HJ) are crossed. Following the first pair of Int cleavages the top strands are swapped to form the Holliday junction, simultaneously converting the unpaired (bubble) bases to the more stable duplex DNA. The second pair of Int cleavages on the bottom strands that would normally resolve the Holliday junction to recombinant products (attL and attR) is strongly disfavored because the sequence differences between the two overlap regions would generate unpaired bubbles in both att L and att R after strand exchange. The resulting Holliday intermediate is thus stabilized with respect to both the reverse and forward reaction products.

Techniques Used: Labeling, Recombinant, Sequencing

Comparison of recombination efficiencies of supercoiled att P as plasmid or dye-labeled phage DNA. Recombination reactions between a 60-bp 32 P-radiolabeled att B (run off the gel) and supercoiled att P, either as plasmid DNA ( A ) or as fluorescein- and TAMRA-labeled phage DNA ( B ) were carried out with the indicated concentrations of Int at 25°C for 1 h and then resolved by gel electrophoresis (1% agarose and 0.4% Synergel), as described in ‘Materials and Methods’ section. ( C ) Gels containing the dye-labeled phage DNA were scanned for fluorescence by fluorescein (excited at 488 nm) and by TAMRA (excited at 532 nm) (lower left panels); then all of the gels were stained with ethidium bromide (top left panels). The 32 P label from att B was visualized by autoradiography and the fluorescence intensity and the amount of 32 P were quantified using Multigauge software, as described in ‘Materials and Methods’ section, and in the text.
Figure Legend Snippet: Comparison of recombination efficiencies of supercoiled att P as plasmid or dye-labeled phage DNA. Recombination reactions between a 60-bp 32 P-radiolabeled att B (run off the gel) and supercoiled att P, either as plasmid DNA ( A ) or as fluorescein- and TAMRA-labeled phage DNA ( B ) were carried out with the indicated concentrations of Int at 25°C for 1 h and then resolved by gel electrophoresis (1% agarose and 0.4% Synergel), as described in ‘Materials and Methods’ section. ( C ) Gels containing the dye-labeled phage DNA were scanned for fluorescence by fluorescein (excited at 488 nm) and by TAMRA (excited at 532 nm) (lower left panels); then all of the gels were stained with ethidium bromide (top left panels). The 32 P label from att B was visualized by autoradiography and the fluorescence intensity and the amount of 32 P were quantified using Multigauge software, as described in ‘Materials and Methods’ section, and in the text.

Techniques Used: Plasmid Preparation, Labeling, Nucleic Acid Electrophoresis, Fluorescence, Staining, Autoradiography, Software

Preparing supercoiled DNA labeled with fluorescent dyes. ( A ) Schematic summary of the method. Single-strand circular att P DNA from Phagemid DNA A(–) is used as the template for a PCR reaction that will give rise to the ‘non-template’ strand that will define (flank) the gap over a selected portion of att P. The left side of the gap is defined by a primer that has a 5′ adapter sequence (not complementary to the template) encoding a BamHI site followed by a HindIII site and then DNA sequence complementary to the template on the left side of the gap. The right side of the gap is defined by a primer that has a 5′ adapter sequence encoding a BamHI site followed by a PstI site and then DNA sequence complementary to the template on the right side of the gap. The linear products of PCR amplification are cut with BamHI, circularized by ligating the annealed overhangs, and used to generate the B(–) family of phagemids. Circular single-strand DNA made from the B(–) family of phagemids [SSC Phage DNA B(-)] is linearized by annealing it with a 45-base oligonucleotide complementary to the HindIII–PstI region and cleaving with those two enzymes. The resulting linear single-strand DNA, which corresponds to the ‘top-strand’ of att P, is annealed to the single-strand circular DNA made from phagemid A(+), which corresponds to the ‘bottom-strand’ of att P [SSC Phage DNA A(+)]. Included in the annealing mixture are oligonucleotides that will fill in the designed gap and will also bring in the specified acceptor (A) and/or donor (D) fluorescent dyes. The resulting nicked circle is incubated with ligase to generate covalently closed circular DNA that is then supercoiled by incubation with DNA gyrase to generate the final dye-labeled DNA. ( B ) Electrophoresis of the DNA intermediates and products in a 1% agarose gel with (lanes 1–5), or without (lanes 6–8), ethidium bromide (0.5 μg/ml). Following electrophoresis the DNA was visualized by staining with ethidium bromide. A 1-kb ladder (NEB) (lane 1) and a supercoiled plasmid DNA (lane 8) serve as markers. Lane 2, single-strand circular phage DNA [A(+) SSC], encoding the bottom strand of att P (from pMM12); lane 3, linearized phage DNA [B(–) linear], encoding truncated top-strand sequences of att P (from pMM32); lane 4, the nicked circle resulting from annealing the linearized B(–) DNA and the circular A(+) DNA, along with the gap-filling oligonucleotides; lane 5, the mixture of covalently closed and nicked circles after incubation with ligase; lane 6, the DNA from lane 5 electrophoresed in the absence of ethidium bromide; lane 7, the mixture of supercoiled and nicked DNA following incubation with DNA gyrase.
Figure Legend Snippet: Preparing supercoiled DNA labeled with fluorescent dyes. ( A ) Schematic summary of the method. Single-strand circular att P DNA from Phagemid DNA A(–) is used as the template for a PCR reaction that will give rise to the ‘non-template’ strand that will define (flank) the gap over a selected portion of att P. The left side of the gap is defined by a primer that has a 5′ adapter sequence (not complementary to the template) encoding a BamHI site followed by a HindIII site and then DNA sequence complementary to the template on the left side of the gap. The right side of the gap is defined by a primer that has a 5′ adapter sequence encoding a BamHI site followed by a PstI site and then DNA sequence complementary to the template on the right side of the gap. The linear products of PCR amplification are cut with BamHI, circularized by ligating the annealed overhangs, and used to generate the B(–) family of phagemids. Circular single-strand DNA made from the B(–) family of phagemids [SSC Phage DNA B(-)] is linearized by annealing it with a 45-base oligonucleotide complementary to the HindIII–PstI region and cleaving with those two enzymes. The resulting linear single-strand DNA, which corresponds to the ‘top-strand’ of att P, is annealed to the single-strand circular DNA made from phagemid A(+), which corresponds to the ‘bottom-strand’ of att P [SSC Phage DNA A(+)]. Included in the annealing mixture are oligonucleotides that will fill in the designed gap and will also bring in the specified acceptor (A) and/or donor (D) fluorescent dyes. The resulting nicked circle is incubated with ligase to generate covalently closed circular DNA that is then supercoiled by incubation with DNA gyrase to generate the final dye-labeled DNA. ( B ) Electrophoresis of the DNA intermediates and products in a 1% agarose gel with (lanes 1–5), or without (lanes 6–8), ethidium bromide (0.5 μg/ml). Following electrophoresis the DNA was visualized by staining with ethidium bromide. A 1-kb ladder (NEB) (lane 1) and a supercoiled plasmid DNA (lane 8) serve as markers. Lane 2, single-strand circular phage DNA [A(+) SSC], encoding the bottom strand of att P (from pMM12); lane 3, linearized phage DNA [B(–) linear], encoding truncated top-strand sequences of att P (from pMM32); lane 4, the nicked circle resulting from annealing the linearized B(–) DNA and the circular A(+) DNA, along with the gap-filling oligonucleotides; lane 5, the mixture of covalently closed and nicked circles after incubation with ligase; lane 6, the DNA from lane 5 electrophoresed in the absence of ethidium bromide; lane 7, the mixture of supercoiled and nicked DNA following incubation with DNA gyrase.

Techniques Used: Labeling, Polymerase Chain Reaction, Sequencing, Amplification, Incubation, Electrophoresis, Agarose Gel Electrophoresis, Staining, Plasmid Preparation

Schematic summary of integrative and excisive site-specific recombination. Integrative recombination between supercoiled att P and linear att B requires virally-encoded Integrase (Int) and the host-encoded accessory DNA-bending protein (IHF) and gives rise to att L and att R products. The excisive recombination between att L and att R requires the accessory DNA-bending protein Xis and is stimulated by Fis. Both reactions proceed via two pairs of sequential strand exchanges that first generate, and then resolve, a Holliday junction recombination intermediate (HJ). The four core-type Int binding sites (C, C′, B and B′; inward-facing thick arrow heads) flanking the seven bp overlap region are bound by the CB and catalytic domains of Int and form a tetrameric complex in the HJ intermediate. The arm-type Int binding sites are differentially occupied (dark thin arrow heads) by the N-domain of Int during integrative (P1, P′1, P′2 and P′3) and excisive (P2, P′1 and P′2) reactions. The IHF binding sites are differentially occupied (dark diamonds) during integrative (H1, H2 and H′) and excisive (H2 and H′) reactions. The Xis binding sites (X1, X1.5 and X2; dark hexagons) and the Fis binding site (F; dark polygon) are occupied only during the excisive reaction. (Inset) Numbering of the donor Fluorescein-labeled T (+18, D), and the acceptor, TAMRA-labeled T (+63; A) is according to previous work, where the third base pair (T) in the overlap region (bold letters) has been assigned zero and bases to its left and right are assigned negative and positive numbers, respectively ( 22 ).
Figure Legend Snippet: Schematic summary of integrative and excisive site-specific recombination. Integrative recombination between supercoiled att P and linear att B requires virally-encoded Integrase (Int) and the host-encoded accessory DNA-bending protein (IHF) and gives rise to att L and att R products. The excisive recombination between att L and att R requires the accessory DNA-bending protein Xis and is stimulated by Fis. Both reactions proceed via two pairs of sequential strand exchanges that first generate, and then resolve, a Holliday junction recombination intermediate (HJ). The four core-type Int binding sites (C, C′, B and B′; inward-facing thick arrow heads) flanking the seven bp overlap region are bound by the CB and catalytic domains of Int and form a tetrameric complex in the HJ intermediate. The arm-type Int binding sites are differentially occupied (dark thin arrow heads) by the N-domain of Int during integrative (P1, P′1, P′2 and P′3) and excisive (P2, P′1 and P′2) reactions. The IHF binding sites are differentially occupied (dark diamonds) during integrative (H1, H2 and H′) and excisive (H2 and H′) reactions. The Xis binding sites (X1, X1.5 and X2; dark hexagons) and the Fis binding site (F; dark polygon) are occupied only during the excisive reaction. (Inset) Numbering of the donor Fluorescein-labeled T (+18, D), and the acceptor, TAMRA-labeled T (+63; A) is according to previous work, where the third base pair (T) in the overlap region (bold letters) has been assigned zero and bases to its left and right are assigned negative and positive numbers, respectively ( 22 ).

Techniques Used: Immunohistofluorescence, Binding Assay, Labeling

Comparison of FRET within the Holliday junction complex and the recombination product. Integrative recombination reactions between a pair of bubble/heterology att sites, to yield Holliday junction complexes ( A ), or a pair of canonical att sites, to yield full recombinant product ( B ), were carried out, and the resulting protein–DNA complexes were analyzed by agarose gel electrophoresis, as described in ‘Materials and Methods’ section. The supercoiled att P DNAs were labeled with fluorescein donor dye (D) at position +18, plus or minus TAMRA acceptor dye (A) at position +63 (see inset of Figure 1 ). A tt B was radiolabeled with 32 P at its 5′ termini. Different size aliquots (7, 9.5, 12.5 and 16 μl for the HJ and 4, 7, 10, 13 and 16 μl for the recombinant) were loaded onto each lane (rising wedge). ( C ) The donor fluorescence and 32 P radioactivity of the Holliday junction and recombinant complexes were quantified using Multigauge software as described in ‘Materials and Methods’ section, and in the text . The values on the y -axis, ( ), are the donor fluorescence in the donor plus acceptor complexes multiplied by the ratio of 32 P radioactivity in the donor only versus the donor plus acceptor complexes (averaged as described in Supplementary Data ). The values for the x -axis are the fluorescence of the donor in the corresponding donor only complexes, . The slope of the line is , where is the energy of transfer, and in our experiments , as described in ‘Materials and Methods’ section, and the text.
Figure Legend Snippet: Comparison of FRET within the Holliday junction complex and the recombination product. Integrative recombination reactions between a pair of bubble/heterology att sites, to yield Holliday junction complexes ( A ), or a pair of canonical att sites, to yield full recombinant product ( B ), were carried out, and the resulting protein–DNA complexes were analyzed by agarose gel electrophoresis, as described in ‘Materials and Methods’ section. The supercoiled att P DNAs were labeled with fluorescein donor dye (D) at position +18, plus or minus TAMRA acceptor dye (A) at position +63 (see inset of Figure 1 ). A tt B was radiolabeled with 32 P at its 5′ termini. Different size aliquots (7, 9.5, 12.5 and 16 μl for the HJ and 4, 7, 10, 13 and 16 μl for the recombinant) were loaded onto each lane (rising wedge). ( C ) The donor fluorescence and 32 P radioactivity of the Holliday junction and recombinant complexes were quantified using Multigauge software as described in ‘Materials and Methods’ section, and in the text . The values on the y -axis, ( ), are the donor fluorescence in the donor plus acceptor complexes multiplied by the ratio of 32 P radioactivity in the donor only versus the donor plus acceptor complexes (averaged as described in Supplementary Data ). The values for the x -axis are the fluorescence of the donor in the corresponding donor only complexes, . The slope of the line is , where is the energy of transfer, and in our experiments , as described in ‘Materials and Methods’ section, and the text.

Techniques Used: Recombinant, Agarose Gel Electrophoresis, Labeling, Fluorescence, Radioactivity, Software

35) Product Images from "Detection of Leishmania RNA Virus in Leishmania Parasites"

Article Title: Detection of Leishmania RNA Virus in Leishmania Parasites

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0002006

Screening for LRV in freshly-isolated human L. braziliensis . A. Dot blot analysis of two parasite samples obtained from separate lesion biopsies in an infected patient: Lb 2169 and Lb 2192. Live parasites (1 to 4 µg total proteins) were spotted on a nitrocellulose membrane for LRV dsRNA detection by dot blot (J2 antibody). Lg M4147 LRV high and LRV neg were used as positive and negative controls. Upper panel: dsRNA detection by dot blot (J2). Lower panel: verification of protein quantity by Ponceau staining. B. J2 anti-dsRNA analysis of Lb 2169 by fluorescence microscopy. Green: dsRNA (J2 Ab). Blue: DAPI. C. Isolation of viral genomic dsRNA from the Lb 2169 strain. Intact and DNase-digested total nucleic acids from Lb 2169 parasites and Lg M4147 LRV high as a control, were analyzed by gel electrophoresis (similarly to Figure 1A ). Note: with high resolution gels such as presented here (in contrast to Figure 1 ), the viral genome often appears as a doublet.
Figure Legend Snippet: Screening for LRV in freshly-isolated human L. braziliensis . A. Dot blot analysis of two parasite samples obtained from separate lesion biopsies in an infected patient: Lb 2169 and Lb 2192. Live parasites (1 to 4 µg total proteins) were spotted on a nitrocellulose membrane for LRV dsRNA detection by dot blot (J2 antibody). Lg M4147 LRV high and LRV neg were used as positive and negative controls. Upper panel: dsRNA detection by dot blot (J2). Lower panel: verification of protein quantity by Ponceau staining. B. J2 anti-dsRNA analysis of Lb 2169 by fluorescence microscopy. Green: dsRNA (J2 Ab). Blue: DAPI. C. Isolation of viral genomic dsRNA from the Lb 2169 strain. Intact and DNase-digested total nucleic acids from Lb 2169 parasites and Lg M4147 LRV high as a control, were analyzed by gel electrophoresis (similarly to Figure 1A ). Note: with high resolution gels such as presented here (in contrast to Figure 1 ), the viral genome often appears as a doublet.

Techniques Used: Isolation, Dot Blot, Infection, Staining, Fluorescence, Microscopy, Nucleic Acid Electrophoresis

Detection of LRV in nucleic acid extracts. A and B. Visualization of viral genomic dsRNA by gel electrophoresis. A. Total nucleic acid from stationary phase promastigotes was treated with ssRNase then migrated in a 1% agarose gel. The sample was either kept intact (1 µg, upper panel) or digested with RQ-DNase (5 µg, lower panel). B. To quantify viral dsRNA in Lg 1398 relative to Lg M4147 LRV high , various concentrations of nucleic acid (2, 1 and 0.5 µg) were digested with RQ-DNase and migrated as above. C. Quantification of LRV transcript by qRT-PCR. Total parasitic and viral cDNA was prepared for qRT-PCR and amplified using primers specific for LRV (SetA and SetB, see material and methods for sequences). Viral transcript was quantified as normalized to the parasitic housekeeping gene kmp11 then adjusted relative to Lg M4147 LRV high .
Figure Legend Snippet: Detection of LRV in nucleic acid extracts. A and B. Visualization of viral genomic dsRNA by gel electrophoresis. A. Total nucleic acid from stationary phase promastigotes was treated with ssRNase then migrated in a 1% agarose gel. The sample was either kept intact (1 µg, upper panel) or digested with RQ-DNase (5 µg, lower panel). B. To quantify viral dsRNA in Lg 1398 relative to Lg M4147 LRV high , various concentrations of nucleic acid (2, 1 and 0.5 µg) were digested with RQ-DNase and migrated as above. C. Quantification of LRV transcript by qRT-PCR. Total parasitic and viral cDNA was prepared for qRT-PCR and amplified using primers specific for LRV (SetA and SetB, see material and methods for sequences). Viral transcript was quantified as normalized to the parasitic housekeeping gene kmp11 then adjusted relative to Lg M4147 LRV high .

Techniques Used: Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Quantitative RT-PCR, Amplification

36) Product Images from "Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products"

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsn004

ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.
Figure Legend Snippet: ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.

Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, Binding Assay, Expressing

37) Product Images from "Fast Hepatitis C Virus RNA Elimination and NS5A Redistribution by NS5A Inhibitors Studied by a Multiplex Assay Approach"

Article Title: Fast Hepatitis C Virus RNA Elimination and NS5A Redistribution by NS5A Inhibitors Studied by a Multiplex Assay Approach

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.00223-15

Assessment of HCV inhibition by DAAs from 3 classes using a Gaussia luciferase reporter system. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. Culture supernatants were analyzed for luciferase activity at the indicated time points as described in Materials and Methods. Data were normalized to data for DMSO controls. Each data point represents the average value from 3 individual experiments. Error bars represent SEM.
Figure Legend Snippet: Assessment of HCV inhibition by DAAs from 3 classes using a Gaussia luciferase reporter system. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. Culture supernatants were analyzed for luciferase activity at the indicated time points as described in Materials and Methods. Data were normalized to data for DMSO controls. Each data point represents the average value from 3 individual experiments. Error bars represent SEM.

Techniques Used: Inhibition, Luciferase, Infection, Activity Assay

Assessment of HCV inhibition by DAAs from 3 classes using the infectivity assay. Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or 100× EC 50 of the HCV inhibitors. Extracellular virus was precipitated by PEG and analyzed for viral titer by a TCID 50 assay, and extracellular viral RNA was detected by RT-qPCR at 8 hpt as described in Materials and Methods. Data were normalized to data for DMSO controls. Each data point represents the average value from two individual experiments. Error bars represent SEM. The statistical analysis was performed by using Student's t test (* P
Figure Legend Snippet: Assessment of HCV inhibition by DAAs from 3 classes using the infectivity assay. Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or 100× EC 50 of the HCV inhibitors. Extracellular virus was precipitated by PEG and analyzed for viral titer by a TCID 50 assay, and extracellular viral RNA was detected by RT-qPCR at 8 hpt as described in Materials and Methods. Data were normalized to data for DMSO controls. Each data point represents the average value from two individual experiments. Error bars represent SEM. The statistical analysis was performed by using Student's t test (* P

Techniques Used: Inhibition, Infection, Quantitative RT-PCR

Assessment of HCV inhibition by DAAs from 3 classes using the image profiling assay. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. Cells were fixed and stained with DRAQ5 and 9E10 anti-NS5A antibodies as described in Materials and Methods. Images were acquired at ×40 magnification and analyzed for NS5A Gabor texture changes at the indicated time points. Data were normalized to data for DMSO controls. Each data point represents the average value from 2 individual experiments. Error bars represent SEM.
Figure Legend Snippet: Assessment of HCV inhibition by DAAs from 3 classes using the image profiling assay. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. Cells were fixed and stained with DRAQ5 and 9E10 anti-NS5A antibodies as described in Materials and Methods. Images were acquired at ×40 magnification and analyzed for NS5A Gabor texture changes at the indicated time points. Data were normalized to data for DMSO controls. Each data point represents the average value from 2 individual experiments. Error bars represent SEM.

Techniques Used: Inhibition, Infection, Staining

Assessment of HCV inhibition by DAAs from 3 classes using Western blot analysis. Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or the HCV inhibitors at concentrations of 100× EC 50 (DCV, 3.2 nM; LDV, 3 μM; DNV, 0.32 μM; SOF, 20 μM). Cell lysates were harvested at 8 hpt (C and D) or at 24 hpt (A and B) and blotted for NS5A (A and C) or core (B and D). Data were normalized to GAPDH and quantified as relative fold change with respect to DMSO. Each data point represents the average value from 3 individual experiments. Error bars represent SEM. Statistical analysis was performed by using Student's t test (* P
Figure Legend Snippet: Assessment of HCV inhibition by DAAs from 3 classes using Western blot analysis. Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or the HCV inhibitors at concentrations of 100× EC 50 (DCV, 3.2 nM; LDV, 3 μM; DNV, 0.32 μM; SOF, 20 μM). Cell lysates were harvested at 8 hpt (C and D) or at 24 hpt (A and B) and blotted for NS5A (A and C) or core (B and D). Data were normalized to GAPDH and quantified as relative fold change with respect to DMSO. Each data point represents the average value from 3 individual experiments. Error bars represent SEM. Statistical analysis was performed by using Student's t test (* P

Techniques Used: Inhibition, Western Blot, Infection

HCV gt2a viral genome organization and experimental design of HCV infection and DAA treatment. (A) Schematic diagrams of the HCV gt2a virus expressing an NS5A-Ypet fusion protein as a marker for viral replication and the HCV gt2a virus carrying a Gaussia luciferase reporter gene at the p7-NS2 junction as a secretable marker for viral replication. (B) Huh-7.5.1 cells were seeded into wells followed by infection with either Jc1/Ypet virus or Jc1/Gluc2A virus for 48 h and then treated with compounds from 3 different classes of HCV DAAs: NS5A inhibitors (DCV and LDV), NS3/4A protease inhibitor (DNV), or NS5B polymerase inhibitor (SOF). IRES, internal ribosome entry site.
Figure Legend Snippet: HCV gt2a viral genome organization and experimental design of HCV infection and DAA treatment. (A) Schematic diagrams of the HCV gt2a virus expressing an NS5A-Ypet fusion protein as a marker for viral replication and the HCV gt2a virus carrying a Gaussia luciferase reporter gene at the p7-NS2 junction as a secretable marker for viral replication. (B) Huh-7.5.1 cells were seeded into wells followed by infection with either Jc1/Ypet virus or Jc1/Gluc2A virus for 48 h and then treated with compounds from 3 different classes of HCV DAAs: NS5A inhibitors (DCV and LDV), NS3/4A protease inhibitor (DNV), or NS5B polymerase inhibitor (SOF). IRES, internal ribosome entry site.

Techniques Used: Infection, Expressing, Marker, Luciferase, Protease Inhibitor

Assessment of HCV inhibition by DAAs from 3 classes using the RT-qPCR assay. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. The total cellular RNA was extracted by TRIzol reagent and analyzed for HCV 5′ UTR RNA by RT-qPCR at the indicated time points as described in Materials and Methods. Cycle threshold ( C T ) values were converted to copy numbers based on the standard curve of pure Jc1/Gluc2A genome-encoded plasmid. Data were normalized to data for DMSO controls. Each data point represents the average value from 2 individual experiments. Error bars represent SEM.
Figure Legend Snippet: Assessment of HCV inhibition by DAAs from 3 classes using the RT-qPCR assay. Dose-response curves are shown for DCV (A), LDV (B), DNV (C), and SOF (D). Jc1/Gluc2A virus-infected Huh-7.5.1 cells were treated with DMSO or different concentrations of the HCV inhibitors. The total cellular RNA was extracted by TRIzol reagent and analyzed for HCV 5′ UTR RNA by RT-qPCR at the indicated time points as described in Materials and Methods. Cycle threshold ( C T ) values were converted to copy numbers based on the standard curve of pure Jc1/Gluc2A genome-encoded plasmid. Data were normalized to data for DMSO controls. Each data point represents the average value from 2 individual experiments. Error bars represent SEM.

Techniques Used: Inhibition, Quantitative RT-PCR, Infection, Plasmid Preparation

Schematic diagram of the image profiling assay. Cells were seeded in 96-well plates and infected with Jc1/Gluc2A virus followed by DAA treatment as described in Materials and Methods. (A) Nine images for every condition were obtained by an automated autofocus Zeiss LSM 510 Meta confocal microscope. Data were processed using the LSM Toolbox functions in the ImageJ program and converted to a format compatible with the CellProfiler software. CellProfiler was used to identify cells and define their cytoplasm and nuclei. Morphological changes in the cytoplasm were analyzed using image profiling methods. (B) Representative analyses based on changes of the Gabor texture are shown for DCV (left panel) and DNV (right panel). Dose-dependent NS5A texture changes were seen for DCV treatment but not for DNV treatment. Nuclei were stained with DRAQ5 (blue channel) and HCV NS5A with green fluorescent protein (GFP) (green channel).
Figure Legend Snippet: Schematic diagram of the image profiling assay. Cells were seeded in 96-well plates and infected with Jc1/Gluc2A virus followed by DAA treatment as described in Materials and Methods. (A) Nine images for every condition were obtained by an automated autofocus Zeiss LSM 510 Meta confocal microscope. Data were processed using the LSM Toolbox functions in the ImageJ program and converted to a format compatible with the CellProfiler software. CellProfiler was used to identify cells and define their cytoplasm and nuclei. Morphological changes in the cytoplasm were analyzed using image profiling methods. (B) Representative analyses based on changes of the Gabor texture are shown for DCV (left panel) and DNV (right panel). Dose-dependent NS5A texture changes were seen for DCV treatment but not for DNV treatment. Nuclei were stained with DRAQ5 (blue channel) and HCV NS5A with green fluorescent protein (GFP) (green channel).

Techniques Used: Infection, Microscopy, Software, Staining

38) Product Images from "Transient Shifts in Bacterial Communities Associated with the Temperate Gorgonian Paramuricea clavata in the Northwestern Mediterranean Sea"

Article Title: Transient Shifts in Bacterial Communities Associated with the Temperate Gorgonian Paramuricea clavata in the Northwestern Mediterranean Sea

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057385

MDS ordination plot of P. clavata bacterial communities. 2-D scatter plots of T-RFLP profiles from P. clavata colonies sampled at 3 sites in winter and summer from 2007 to 2010 are based on the Dice similarity matrix for the T-RFLP data retrieved from the amplified bacterial 16S rDNA digested with Cfo I (A) and Msp I (B). Each symbol represents the bacterial community of an individual sample from Riou (square), Medes (triangle) or Scandola (circle), and the colors correspond to different sampling seasons. Sample clusters are based on coordinates determined with the k-means method. Dotted ellipses contain 50% (grey dots) or 95% (dark dots) of the points that contribute to the cluster. The centers of the confidence ellipses are identified with a cross.
Figure Legend Snippet: MDS ordination plot of P. clavata bacterial communities. 2-D scatter plots of T-RFLP profiles from P. clavata colonies sampled at 3 sites in winter and summer from 2007 to 2010 are based on the Dice similarity matrix for the T-RFLP data retrieved from the amplified bacterial 16S rDNA digested with Cfo I (A) and Msp I (B). Each symbol represents the bacterial community of an individual sample from Riou (square), Medes (triangle) or Scandola (circle), and the colors correspond to different sampling seasons. Sample clusters are based on coordinates determined with the k-means method. Dotted ellipses contain 50% (grey dots) or 95% (dark dots) of the points that contribute to the cluster. The centers of the confidence ellipses are identified with a cross.

Techniques Used: Amplification, Sampling

Relative abundances of Msp I TRFs in P. clavata samples analyzed by T-RFLP. As described in Figure 3 , with the restriction enzyme Msp I.
Figure Legend Snippet: Relative abundances of Msp I TRFs in P. clavata samples analyzed by T-RFLP. As described in Figure 3 , with the restriction enzyme Msp I.

Techniques Used:

39) Product Images from "Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans"

Article Title: Targeting Fungal Genes by Diced siRNAs: A Rapid Tool to Decipher Gene Function in Aspergillus nidulans

Journal: PLoS ONE

doi: 10.1371/journal.pone.0075443

Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.
Figure Legend Snippet: Strategies for generation of dsRNAs and d-siRNAs. (A) PCR template strategy was utilized for obtaining s GFP , An rasA and An ras B dsRNA in a single T7 transcription reaction. T7 promoter sequence was added to both forward and reverse gene specific primers of s GFP , An rasA and An rasB , and then PCR amplification was performed in order to generate templates for dsRNA synthesis. (B) For generation of template DNA for unrelated MiAchE , PCR amplification was performed on pGEM T- MiAchE vector using M13 primers. This amplification includes the T7 promoter to one end and SP6 promoter to another end of MiAchE . (C) PCR templates for transcription of respective target genes. M. ladder; B. blank; s GFP , An rasA , An rasB and MiAchE target gene templates. (D) Synthesis of dsRNAs with T7 or SP6 in vitro transcription reactions. M. Ladder; s GFP , An rasA , An rasB and MiAchE dsRNAs. (E) 20% PAGE analysis of purified diced siRNAs. M-Ladder, s GFP , An rasA , An rasB and MiAchE d-siRNAs. The d-siRNAs of all target genes were generated by cleaving respective dsRNAs with RNase III at 37°C for 30 mins, followed by subsequent purifications and finally dissolved in nuclease free water.

Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Plasmid Preparation, In Vitro, Polyacrylamide Gel Electrophoresis, Purification, Generated

40) Product Images from "Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography"

Article Title: Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography

Journal: Nucleic Acids Research

doi:

PCR amplification of CpG islands. ( A ) 294 bp human hMLH1 promoter from colon cancer cell line RKO; ( B ) 605 bp human Cox-2 promoter from gastric cancer cell line MKN45. M1, puC18 DNA/ Hea III marker; M2, 1 kb DNA ladder (Gibco no. 15615-016); lanes 1 and 4, untreated DNA; lanes 2 and 3, bisulfite-modified template.
Figure Legend Snippet: PCR amplification of CpG islands. ( A ) 294 bp human hMLH1 promoter from colon cancer cell line RKO; ( B ) 605 bp human Cox-2 promoter from gastric cancer cell line MKN45. M1, puC18 DNA/ Hea III marker; M2, 1 kb DNA ladder (Gibco no. 15615-016); lanes 1 and 4, untreated DNA; lanes 2 and 3, bisulfite-modified template.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Modification

Detection of methylation in the hMLH1 promoter by Bst UI COBRA assay in the RKO and PACM82 cell lines. DNA without or with bisulfite treatment was amplified by PCR or ssPCR. The amplicon mixture was digested with Bst UI at 60°C for 3 h. M1, PCR and ssPCR as in Figure 1.
Figure Legend Snippet: Detection of methylation in the hMLH1 promoter by Bst UI COBRA assay in the RKO and PACM82 cell lines. DNA without or with bisulfite treatment was amplified by PCR or ssPCR. The amplicon mixture was digested with Bst UI at 60°C for 3 h. M1, PCR and ssPCR as in Figure 1.

Techniques Used: Methylation, Combined Bisulfite Restriction Analysis Assay, Amplification, Polymerase Chain Reaction

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 system
    Screening and generation of Mp erf13 mutants (A) The flow chart of the screen for mutants defective in oil body formation from <t>T-DNA-insertion</t> 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) <t>PCR-based</t> 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 .
    Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3060 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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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    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 .

    Journal: bioRxiv

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

    doi: 10.1101/2020.03.02.956961

    Figure Lengend 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 .

    Article Snippet: After agarose gel electrophoresis of the final TAIL-PCR products, DNA bands were excised and purified using the Wizard SV Gel and PCR Clean-Up System (Promega).

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

    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

    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