genomic dna  (Roche)


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    Structured Review

    Roche genomic dna
    Analysis of adeno-associated virus (AAV) vector persistence and expression in mice. Muscles were harvested from animals (n = 5) injected intramuscularly with AAV8-NiV.G or AAV8-GFP and used for <t>DNA</t> and RNA extraction. A , AAV vector persistence was measured by <t>qRT-PCR,</t> using primers specific to AAV-NiV.G or AAV-GFP DNA. B , AAV-encoded transgene messenger RNA was measured by qRT-PCR, using primers specific to the Nipah virus (NiV) G or the green fluorescent protein complementary DNAs (cDNAs). Samples were normalized by quantifying the number of CD8 (DNA) or GAPDH (cDNA) copies. Results are expressed as the mean number ( ± standard error of the mean) of NiV G DNA and RNA copies per microgram of DNA and RNA, respectively (n = 5). * P
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    Images

    1) Product Images from "Protection Against Henipavirus Infection by Use of Recombinant Adeno-Associated Virus–Vector Vaccines"

    Article Title: Protection Against Henipavirus Infection by Use of Recombinant Adeno-Associated Virus–Vector Vaccines

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jis699

    Analysis of adeno-associated virus (AAV) vector persistence and expression in mice. Muscles were harvested from animals (n = 5) injected intramuscularly with AAV8-NiV.G or AAV8-GFP and used for DNA and RNA extraction. A , AAV vector persistence was measured by qRT-PCR, using primers specific to AAV-NiV.G or AAV-GFP DNA. B , AAV-encoded transgene messenger RNA was measured by qRT-PCR, using primers specific to the Nipah virus (NiV) G or the green fluorescent protein complementary DNAs (cDNAs). Samples were normalized by quantifying the number of CD8 (DNA) or GAPDH (cDNA) copies. Results are expressed as the mean number ( ± standard error of the mean) of NiV G DNA and RNA copies per microgram of DNA and RNA, respectively (n = 5). * P
    Figure Legend Snippet: Analysis of adeno-associated virus (AAV) vector persistence and expression in mice. Muscles were harvested from animals (n = 5) injected intramuscularly with AAV8-NiV.G or AAV8-GFP and used for DNA and RNA extraction. A , AAV vector persistence was measured by qRT-PCR, using primers specific to AAV-NiV.G or AAV-GFP DNA. B , AAV-encoded transgene messenger RNA was measured by qRT-PCR, using primers specific to the Nipah virus (NiV) G or the green fluorescent protein complementary DNAs (cDNAs). Samples were normalized by quantifying the number of CD8 (DNA) or GAPDH (cDNA) copies. Results are expressed as the mean number ( ± standard error of the mean) of NiV G DNA and RNA copies per microgram of DNA and RNA, respectively (n = 5). * P

    Techniques Used: Plasmid Preparation, Expressing, Mouse Assay, Injection, RNA Extraction, Quantitative RT-PCR

    2) Product Images from "Post-transcriptional modulation of the SigF regulon in Mycobacterium smegmatis by the PhoH2 toxin-antitoxin"

    Article Title: Post-transcriptional modulation of the SigF regulon in Mycobacterium smegmatis by the PhoH2 toxin-antitoxin

    Journal: bioRxiv

    doi: 10.1101/2020.01.15.907535

    PCR confirmation of phoH2 deletion from M. smegmatis mc 2 155. PCR using primers that flanked the deletion site were used with M. smegmatis mc 2 155 or mc 2 155 ΔphoH2 DNA as template to confirm the deletion of phoH2 . Expected band sizes: mc 2 155 – 2914 bp and mc 2 155 ΔphoH2 – 1622 bp.
    Figure Legend Snippet: PCR confirmation of phoH2 deletion from M. smegmatis mc 2 155. PCR using primers that flanked the deletion site were used with M. smegmatis mc 2 155 or mc 2 155 ΔphoH2 DNA as template to confirm the deletion of phoH2 . Expected band sizes: mc 2 155 – 2914 bp and mc 2 155 ΔphoH2 – 1622 bp.

    Techniques Used: Polymerase Chain Reaction

    3) Product Images from "New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing"

    Article Title: New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing

    Journal: bioRxiv

    doi: 10.1101/746826

    CRISPR- CLONInG : Replacement of partial cargo sequence (Cre-comp) with the desired donor sequence on AAV vector (Addgene #60229). (A) Schematic illustration of AAV vector with CRISPR cut sites (red scissors) at two ends of the Cre-comp segment. Guides (AAV-A and AAV-B) with high on-target scores were selected. (B) Cre-comp was cut out with ctRNP (Cas9-ctRNA) complex; donor for gene replacement (containing 15 bp AA replacement sequence, noted as ‘R’, sandwiched by HA) flanked with complementary Gibson overhangs of the adjacent AAV backbone was PCR-amplified from custom gene synthesized plasmid. (C) Assembled AAV-v1: donor template cloned into the customized AAV backbone via Gibson (HiFi) Assembly. (D) Excised AAV vector backbone (∼3.63 kb) and Cre-comp (∼2.73 kb) (left); PCR amplified donor template (∼0.8 kb) with Gibson overhangs (right). (E) After CRISPR- CLONInG , 15 out of 16 clones showed correct vector assembly, confirmed by BbsI RE(s) diagnosis (two DNA fragments; black arrow); 3 clones further validated by Sanger sequencing. Resolved on 0.9% agarose gel.
    Figure Legend Snippet: CRISPR- CLONInG : Replacement of partial cargo sequence (Cre-comp) with the desired donor sequence on AAV vector (Addgene #60229). (A) Schematic illustration of AAV vector with CRISPR cut sites (red scissors) at two ends of the Cre-comp segment. Guides (AAV-A and AAV-B) with high on-target scores were selected. (B) Cre-comp was cut out with ctRNP (Cas9-ctRNA) complex; donor for gene replacement (containing 15 bp AA replacement sequence, noted as ‘R’, sandwiched by HA) flanked with complementary Gibson overhangs of the adjacent AAV backbone was PCR-amplified from custom gene synthesized plasmid. (C) Assembled AAV-v1: donor template cloned into the customized AAV backbone via Gibson (HiFi) Assembly. (D) Excised AAV vector backbone (∼3.63 kb) and Cre-comp (∼2.73 kb) (left); PCR amplified donor template (∼0.8 kb) with Gibson overhangs (right). (E) After CRISPR- CLONInG , 15 out of 16 clones showed correct vector assembly, confirmed by BbsI RE(s) diagnosis (two DNA fragments; black arrow); 3 clones further validated by Sanger sequencing. Resolved on 0.9% agarose gel.

    Techniques Used: CRISPR, Clone Assay, Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Synthesized, Agarose Gel Electrophoresis

    CRISPR- CLIP : Procuration of lssDNA from dsDNA template Genotyping results of the CKO mice generated with the acquired lssDNA via Easi-CRISPR . (A) dsDNA template anchored in the default plasmid; the sense ssDNA (top strand) is the donor (lssDNA) of choice. (B) Cpf1 (with guide CLIP-B) was used to create a dsDNA incision on the plasmid at one end of the lssDNA cassette, while Cas9n (with guide CLIP-A) to create a ssDNA incision at the other end, specifically on the strand of interest (top strand in this case). (C) Upon DGLB treatment, the plasmid incised by Cpf1 and Cas9n was resolved into three stand-alone distinct-sized units (0.9% agarose gel electrophoresis): ∼4.9 kbases (donor+backbone) vs. ∼2.7 kbases (backbone) vs. ∼2.2 kbases (lssDNA donor). (D) Mice genotyping screened by RE HindIII (top) and EcoRV (bottom): a pair of external screening primers amplified 2.8 kb DNA fragment (black arrow); mice with the lssDNA donor integration should carry a floxed cassette with HindII/EcoRV site adjacent to LoxP. Upon RE digestion, mouse #4 showing the insertion of both LoxPs (∼0.8 kb vs. ∼2 kb; red asterisk), further confirmed by Sanger sequencing; mouse #6 showing only HindIII digest, indicating one LoxP insertion; mouse #7 was found to carry heterozygous 1.2 kb deletion between the two guides (used for creating CKO model), verified by Sanger sequencing, thus showing a band at ∼1.6 kb. Due to incomplete RE digest and usage of EtBr pre-stained gel, the smaller bands appeared in lighter intensity; purple asterisk: non-specific PCR band.
    Figure Legend Snippet: CRISPR- CLIP : Procuration of lssDNA from dsDNA template Genotyping results of the CKO mice generated with the acquired lssDNA via Easi-CRISPR . (A) dsDNA template anchored in the default plasmid; the sense ssDNA (top strand) is the donor (lssDNA) of choice. (B) Cpf1 (with guide CLIP-B) was used to create a dsDNA incision on the plasmid at one end of the lssDNA cassette, while Cas9n (with guide CLIP-A) to create a ssDNA incision at the other end, specifically on the strand of interest (top strand in this case). (C) Upon DGLB treatment, the plasmid incised by Cpf1 and Cas9n was resolved into three stand-alone distinct-sized units (0.9% agarose gel electrophoresis): ∼4.9 kbases (donor+backbone) vs. ∼2.7 kbases (backbone) vs. ∼2.2 kbases (lssDNA donor). (D) Mice genotyping screened by RE HindIII (top) and EcoRV (bottom): a pair of external screening primers amplified 2.8 kb DNA fragment (black arrow); mice with the lssDNA donor integration should carry a floxed cassette with HindII/EcoRV site adjacent to LoxP. Upon RE digestion, mouse #4 showing the insertion of both LoxPs (∼0.8 kb vs. ∼2 kb; red asterisk), further confirmed by Sanger sequencing; mouse #6 showing only HindIII digest, indicating one LoxP insertion; mouse #7 was found to carry heterozygous 1.2 kb deletion between the two guides (used for creating CKO model), verified by Sanger sequencing, thus showing a band at ∼1.6 kb. Due to incomplete RE digest and usage of EtBr pre-stained gel, the smaller bands appeared in lighter intensity; purple asterisk: non-specific PCR band.

    Techniques Used: CRISPR, Cross-linking Immunoprecipitation, Mouse Assay, Generated, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Sequencing, Staining, Polymerase Chain Reaction

    CRISPR- CLONInG : Replacement of Luciferase (Luc) on FLEx vector. (A) Schematic illustration of FLEx vector with CRISPR cut sites (red scissors) at the two junction sites flanking the undesired Luc fragment. Gray dot dashes: default backbone containing origin of replication and selection for propagation in bacterial host. (B) Luc was cut out with ctRNP (Cas9-ctRNA) complex; FRT-Neo-FRT and tdTomato were PCR-amplified from existing plasmids using primers carrying complementary Gibson overhangs from the adjacent DNA fragment and vector backbone. (C) Two new vector inserts were joined with the CRISPR-digested backbone via Gibson (HiFi) Cloning for final donor assembly. (D) Excised FLEx vector backbone (∼7.5 kb) and Luciferase (∼1.65 kb) (left); PCR amplified FRT-Neo-FRT (∼1.87 kb) and tdTomato (∼1.43 kb) (right). N.S.: non-specific bands. (E) After CRISPR- CLONInG , 14 out of 20 clones verified with PstI RE(s) diagnosis showed correct vector assembly (6 DNA fragments; black arrow); 3 clones validated for sequence integrity. Resolved on 0.9% agarose gel.
    Figure Legend Snippet: CRISPR- CLONInG : Replacement of Luciferase (Luc) on FLEx vector. (A) Schematic illustration of FLEx vector with CRISPR cut sites (red scissors) at the two junction sites flanking the undesired Luc fragment. Gray dot dashes: default backbone containing origin of replication and selection for propagation in bacterial host. (B) Luc was cut out with ctRNP (Cas9-ctRNA) complex; FRT-Neo-FRT and tdTomato were PCR-amplified from existing plasmids using primers carrying complementary Gibson overhangs from the adjacent DNA fragment and vector backbone. (C) Two new vector inserts were joined with the CRISPR-digested backbone via Gibson (HiFi) Cloning for final donor assembly. (D) Excised FLEx vector backbone (∼7.5 kb) and Luciferase (∼1.65 kb) (left); PCR amplified FRT-Neo-FRT (∼1.87 kb) and tdTomato (∼1.43 kb) (right). N.S.: non-specific bands. (E) After CRISPR- CLONInG , 14 out of 20 clones verified with PstI RE(s) diagnosis showed correct vector assembly (6 DNA fragments; black arrow); 3 clones validated for sequence integrity. Resolved on 0.9% agarose gel.

    Techniques Used: CRISPR, Clone Assay, Luciferase, Plasmid Preparation, Selection, Polymerase Chain Reaction, Amplification, Sequencing, Agarose Gel Electrophoresis

    4) Product Images from "The importance of DNA methylation of exons on alternative splicing"

    Article Title: The importance of DNA methylation of exons on alternative splicing

    Journal: RNA

    doi: 10.1261/rna.064865.117

    dCas9-TET1 targeting of DHODH exon 5 affects alternative splicing. ( A ) Schematic illustration of genomic region of DHODH from exon 4 to exon 6. Gray boxes and blue lines indicate exons and introns, respectively. Brown and black arrows represent sgRNA binding sites and RT-PCR primer binding sites, respectively. Red dashed lines indicate the region analyzed for DNA methylation. ( B ) Gel electrophoresis of RT-PCR products of the DHODH exon 5 region after overexpression of dCas9-TET1 with or without sgRNAs targeting exon 5 of DHODH gene in HCT116 cells. RNA was extracted, reverse transcribed, and primers were used to amplify the included (370 bp) or the skipped (172 bp) isoforms. NTC is non-template control. ( C ) HCT116 cells were either transfected with dCas9-TET1 only (control) or co-transfected with dCas9-TET1 and sgRNAs targeting different regions: DHODH exon 5 itself (+ex5 gRNA), DHODH intron 4 (+int4 gRNA), or DHODH ). Plotted are means ± SEM, (*) indicates P -value
    Figure Legend Snippet: dCas9-TET1 targeting of DHODH exon 5 affects alternative splicing. ( A ) Schematic illustration of genomic region of DHODH from exon 4 to exon 6. Gray boxes and blue lines indicate exons and introns, respectively. Brown and black arrows represent sgRNA binding sites and RT-PCR primer binding sites, respectively. Red dashed lines indicate the region analyzed for DNA methylation. ( B ) Gel electrophoresis of RT-PCR products of the DHODH exon 5 region after overexpression of dCas9-TET1 with or without sgRNAs targeting exon 5 of DHODH gene in HCT116 cells. RNA was extracted, reverse transcribed, and primers were used to amplify the included (370 bp) or the skipped (172 bp) isoforms. NTC is non-template control. ( C ) HCT116 cells were either transfected with dCas9-TET1 only (control) or co-transfected with dCas9-TET1 and sgRNAs targeting different regions: DHODH exon 5 itself (+ex5 gRNA), DHODH intron 4 (+int4 gRNA), or DHODH ). Plotted are means ± SEM, (*) indicates P -value

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, DNA Methylation Assay, Nucleic Acid Electrophoresis, Over Expression, Transfection

    In vivo site-specific changes in DNA methylation induced by dCas9-DNMT3A-3L and dCas9-TET1. ( A ) Schematic illustration of EDI minigene. Gray and orange boxes indicate constitutive and alternative exons, respectively. Blue and black lines indicate introns and pFRT sequence, respectively. Black arrow represents the transcription start site (TSS). ( B ) Enlargement of EDI exon 5 region. Regions targeted by sgRNAs are indicated by brackets and letters above the gene schematic. Arrows below the gene schematic represent sgRNA binding sites. The arrow colors represent the different pools of sgRNAs used in panels C and D . Red dashed lines indicate the region analyzed for DNA methylation. ( C , D ) Methylation levels within exon 5 in ( C ) Met– cells transfected with dCas9-DNMT3A-3L or ( D ) Met+ cells transfected with dCas9-TET1 and a combination of three different sgRNAs (from the five or six sgRNAs designed to each region shown in panel B ). Different combinations of sgRNAs (marked #1, #2, or #3 below bars) were tested. “dCas9-DNMT3A-3L only” and “dCas9-TET1 only” are controls without sgRNAs and “untreated” indicates cells without transfection. (*) Indicates P -value
    Figure Legend Snippet: In vivo site-specific changes in DNA methylation induced by dCas9-DNMT3A-3L and dCas9-TET1. ( A ) Schematic illustration of EDI minigene. Gray and orange boxes indicate constitutive and alternative exons, respectively. Blue and black lines indicate introns and pFRT sequence, respectively. Black arrow represents the transcription start site (TSS). ( B ) Enlargement of EDI exon 5 region. Regions targeted by sgRNAs are indicated by brackets and letters above the gene schematic. Arrows below the gene schematic represent sgRNA binding sites. The arrow colors represent the different pools of sgRNAs used in panels C and D . Red dashed lines indicate the region analyzed for DNA methylation. ( C , D ) Methylation levels within exon 5 in ( C ) Met– cells transfected with dCas9-DNMT3A-3L or ( D ) Met+ cells transfected with dCas9-TET1 and a combination of three different sgRNAs (from the five or six sgRNAs designed to each region shown in panel B ). Different combinations of sgRNAs (marked #1, #2, or #3 below bars) were tested. “dCas9-DNMT3A-3L only” and “dCas9-TET1 only” are controls without sgRNAs and “untreated” indicates cells without transfection. (*) Indicates P -value

    Techniques Used: In Vivo, DNA Methylation Assay, Sequencing, Binding Assay, Methylation, Transfection

    Schematic illustration of region-specific DNA methylation manipulations using the CRISPR system. Cells are co-transfected with a plasmid for expression of dCas9-DNMT3A-3L and the sgRNA expression plasmids. The dCas9 fusion protein is guided to a specific region of the genome by the sgRNAs where it brings DNMT3A-3L close to the DNA, resulting in site-specific methylation. As the transfection is transient, after several cell divisions, the fusion protein expression is lost but the DNA methylation pattern is maintained by the endogenous DNMT1 during genomic DNA replication. A similar method involving the dCas9-TET1 fusion enabled site-specific demethylation of DNA. The yellow spheres indicate methylated CpG.
    Figure Legend Snippet: Schematic illustration of region-specific DNA methylation manipulations using the CRISPR system. Cells are co-transfected with a plasmid for expression of dCas9-DNMT3A-3L and the sgRNA expression plasmids. The dCas9 fusion protein is guided to a specific region of the genome by the sgRNAs where it brings DNMT3A-3L close to the DNA, resulting in site-specific methylation. As the transfection is transient, after several cell divisions, the fusion protein expression is lost but the DNA methylation pattern is maintained by the endogenous DNMT1 during genomic DNA replication. A similar method involving the dCas9-TET1 fusion enabled site-specific demethylation of DNA. The yellow spheres indicate methylated CpG.

    Techniques Used: DNA Methylation Assay, CRISPR, Transfection, Plasmid Preparation, Expressing, Methylation

    dCas9-TET1 induces DNA demethylation in a limited region. ( A ) Schematic illustration of EDI minigene exons 4 and 5. Exon 4 is alternatively spliced, whereas exon 5 is constitutively spliced. Blue and black lines indicate EDI introns and pFRT sequence, respectively. The six regions analyzed for methylation are indicated with letters. ( B ) The level of methylated CpGs (in percent) within the six regions denoted in panel A after transfection of dCas9-TET1 plasmid into Met+ cells with or without sgRNA expression (black and gray bars, respectively).
    Figure Legend Snippet: dCas9-TET1 induces DNA demethylation in a limited region. ( A ) Schematic illustration of EDI minigene exons 4 and 5. Exon 4 is alternatively spliced, whereas exon 5 is constitutively spliced. Blue and black lines indicate EDI introns and pFRT sequence, respectively. The six regions analyzed for methylation are indicated with letters. ( B ) The level of methylated CpGs (in percent) within the six regions denoted in panel A after transfection of dCas9-TET1 plasmid into Met+ cells with or without sgRNA expression (black and gray bars, respectively).

    Techniques Used: Sequencing, Methylation, Transfection, Plasmid Preparation, Expressing

    5) Product Images from "NF-kappa-B activation unveils the presence of inflammatory hotspots in human gut xenografts"

    Article Title: NF-kappa-B activation unveils the presence of inflammatory hotspots in human gut xenografts

    Journal: bioRxiv

    doi: 10.1101/2020.07.23.212621

    Foci of NF-κB activity are not associated with high copy number of luminescence reporter. Real-time PCR was used for analyses of Luciferase gene copy number in human gut xenografts. Amplification of the NFκB-Luciferase plasmid (A) and derived standard curve (B). The plasmid was serially diluted 1:5 starting from 6×10 4 vector DNA molecules and was amplified using the Luc2P primers. Plasmid DNA was diluted with genomic DNA to generate the standard curve (B) as described in Materials and methods. The amplification plot shows the change in fluorescence (Rn) as a function of the PCR cycle (A). Gut transplants were imaged ex-vivo for luminescence activity as described above (see Figure 2 ) to enable accurate sampling of hotspots (red arrows in C) and quiescent regions for extraction of genomic DNA from transduced and non-transduced transplants (top left panel in C) as negative controls. Copy number of reporter genes was not consistently higher in the luminescence foci compared with quiescent regions of NF-κB activity in transduced transplants (D-E). Each data point represents sampled transplant in luminescence focus (red hotspots in D) or quiescent area (blue in D). Paring of hotspot and quiescent copy number in individual transplants is presented in E.
    Figure Legend Snippet: Foci of NF-κB activity are not associated with high copy number of luminescence reporter. Real-time PCR was used for analyses of Luciferase gene copy number in human gut xenografts. Amplification of the NFκB-Luciferase plasmid (A) and derived standard curve (B). The plasmid was serially diluted 1:5 starting from 6×10 4 vector DNA molecules and was amplified using the Luc2P primers. Plasmid DNA was diluted with genomic DNA to generate the standard curve (B) as described in Materials and methods. The amplification plot shows the change in fluorescence (Rn) as a function of the PCR cycle (A). Gut transplants were imaged ex-vivo for luminescence activity as described above (see Figure 2 ) to enable accurate sampling of hotspots (red arrows in C) and quiescent regions for extraction of genomic DNA from transduced and non-transduced transplants (top left panel in C) as negative controls. Copy number of reporter genes was not consistently higher in the luminescence foci compared with quiescent regions of NF-κB activity in transduced transplants (D-E). Each data point represents sampled transplant in luminescence focus (red hotspots in D) or quiescent area (blue in D). Paring of hotspot and quiescent copy number in individual transplants is presented in E.

    Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction, Luciferase, Amplification, Plasmid Preparation, Derivative Assay, Fluorescence, Polymerase Chain Reaction, Ex Vivo, Sampling

    6) Product Images from "Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference"

    Article Title: Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz748

    Identities of cells that require RRF-1 for silencing by neuronal dsRNA vary from animal to animal. ( A ) GFP expression from the sur-5::gfp chimeric gene enables simultaneous visualization of most somatic nuclei in C. elegans . A depth coded (one color for ∼53 frames) projection of 5 Z-stacks that were stitched together from a single L4-staged animal is shown (also see Materials and Methods). Scale bar = 100 μm. ( B-D ) Expression of gfp -dsRNA in neurons causes silencing throughout the animal that is entirely dependent on SID-1 and partially dependent on RRF-1. Representative images of L4-staged sur-5::gfp animals that express Prgef-1::gfp -dsRNA (B) and additionally lack sid-1 (C) or rrf-1 (D) are shown. Maximum intensity projections of sections were stitched together to generate whole-worm images. Presence of gfp- dsRNA causes worms to twist because of the rol-6 co-injection marker. Cells that require RRF-1 for silencing (e.g. the excretory canal cell indicated by red arrows and some intestinal cells in red dashed lines) and cells that can silence in the absence of RRF-1 (e.g. some intestinal cells shown in blue dashed lines) are highlighted in Prgef-1::gfp- dsRNA ; rrf-1(-) and in Prgef-1::gfp -dsRNA; sid-1(-) animals. Scale bar = 100μm. ( E, F ) Silencing in rrf-1(- ) animals by neuronal dsRNA is associated with a decrease in sur-5::gfp mRNA levels. Single molecule FISH was used to detect sur-5::gfp mRNA in L4-staged wild-type animals (E, left ) or in sur-5::gfp animals that express Prgef-1::gfp- dsRNA (E, middle ) and that in addition lack sid-1 (E, right ) or rrf-1 (F). RNA from Prgef-1::gfp- dsRNA is prominently detected by gfp probes in neuronal nuclei (red arrows and bracket). A representative pair of intestinal nuclei is shown for each animal as an overlay of DNA (DAPI in blue), mRNA ( gfp in green) and protein (GFP in magenta). Cytoplasmic mRNA foci were counted (see Materials and Methods) between two nuclei in wild-type or in sid-1(-) backgrounds (E), and between two nuclei where GFP is silenced (off, blue) and where GFP is expressed (on, red) in rrf-1(-) animals. Errors indicate 95% confidence intervals, n = 3 in E and n = 4 in F. Top scale bar = 10 μm and bottom scale bar = 5 μm. ( G ) No intestinal cell requires RRF-1 for silencing in all animals. The E blastomere divides to generate 20 intestinal cells (EaLAAD to EpRPPP). Of the 20 cells, 10 undergo nuclear division without cell division (two grey circles per cell), 4 sometimes undergo similar nuclear division (one grey circle and one open circle per cell), and 6 do not undergo any division (one grey circle per cell). In each of 10 sur-5::gfp; rrf-1(-); Prgef-1::gfp -dsRNA L4-staged animals, GFP-positive nuclei (use only RRF-1, gray) and GFP-negative nuclei (use EGO-1 or RRF-1, white) were scored. Every binucleate cell had both nuclei with the same requirement. White boxes with a slash indicate absence of second nucleus because of lack of nuclear division ( 52 ). See Supplementary Figure S7 for images of additional animals.
    Figure Legend Snippet: Identities of cells that require RRF-1 for silencing by neuronal dsRNA vary from animal to animal. ( A ) GFP expression from the sur-5::gfp chimeric gene enables simultaneous visualization of most somatic nuclei in C. elegans . A depth coded (one color for ∼53 frames) projection of 5 Z-stacks that were stitched together from a single L4-staged animal is shown (also see Materials and Methods). Scale bar = 100 μm. ( B-D ) Expression of gfp -dsRNA in neurons causes silencing throughout the animal that is entirely dependent on SID-1 and partially dependent on RRF-1. Representative images of L4-staged sur-5::gfp animals that express Prgef-1::gfp -dsRNA (B) and additionally lack sid-1 (C) or rrf-1 (D) are shown. Maximum intensity projections of sections were stitched together to generate whole-worm images. Presence of gfp- dsRNA causes worms to twist because of the rol-6 co-injection marker. Cells that require RRF-1 for silencing (e.g. the excretory canal cell indicated by red arrows and some intestinal cells in red dashed lines) and cells that can silence in the absence of RRF-1 (e.g. some intestinal cells shown in blue dashed lines) are highlighted in Prgef-1::gfp- dsRNA ; rrf-1(-) and in Prgef-1::gfp -dsRNA; sid-1(-) animals. Scale bar = 100μm. ( E, F ) Silencing in rrf-1(- ) animals by neuronal dsRNA is associated with a decrease in sur-5::gfp mRNA levels. Single molecule FISH was used to detect sur-5::gfp mRNA in L4-staged wild-type animals (E, left ) or in sur-5::gfp animals that express Prgef-1::gfp- dsRNA (E, middle ) and that in addition lack sid-1 (E, right ) or rrf-1 (F). RNA from Prgef-1::gfp- dsRNA is prominently detected by gfp probes in neuronal nuclei (red arrows and bracket). A representative pair of intestinal nuclei is shown for each animal as an overlay of DNA (DAPI in blue), mRNA ( gfp in green) and protein (GFP in magenta). Cytoplasmic mRNA foci were counted (see Materials and Methods) between two nuclei in wild-type or in sid-1(-) backgrounds (E), and between two nuclei where GFP is silenced (off, blue) and where GFP is expressed (on, red) in rrf-1(-) animals. Errors indicate 95% confidence intervals, n = 3 in E and n = 4 in F. Top scale bar = 10 μm and bottom scale bar = 5 μm. ( G ) No intestinal cell requires RRF-1 for silencing in all animals. The E blastomere divides to generate 20 intestinal cells (EaLAAD to EpRPPP). Of the 20 cells, 10 undergo nuclear division without cell division (two grey circles per cell), 4 sometimes undergo similar nuclear division (one grey circle and one open circle per cell), and 6 do not undergo any division (one grey circle per cell). In each of 10 sur-5::gfp; rrf-1(-); Prgef-1::gfp -dsRNA L4-staged animals, GFP-positive nuclei (use only RRF-1, gray) and GFP-negative nuclei (use EGO-1 or RRF-1, white) were scored. Every binucleate cell had both nuclei with the same requirement. White boxes with a slash indicate absence of second nucleus because of lack of nuclear division ( 52 ). See Supplementary Figure S7 for images of additional animals.

    Techniques Used: Expressing, Injection, Marker, Fluorescence In Situ Hybridization

    7) Product Images from "Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase *"

    Article Title: Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.131235

    Co-purification of HSV-1 DNA polymerase and uracil DNA glycosylase. A , fractions eluting from the Resource Q column were resolved by 10% SDS-PAGE followed by silver staining. The positions of markers ( M L and M H , Bio-Rad low and high range standards, respectively), UL30 and UL42 are as indicated. X indicates the position of an ∼36-kDa polypeptide. B , the specified fractions were assayed for pol (●) and UDG (○) activities as described under “Experimental Procedures.”
    Figure Legend Snippet: Co-purification of HSV-1 DNA polymerase and uracil DNA glycosylase. A , fractions eluting from the Resource Q column were resolved by 10% SDS-PAGE followed by silver staining. The positions of markers ( M L and M H , Bio-Rad low and high range standards, respectively), UL30 and UL42 are as indicated. X indicates the position of an ∼36-kDa polypeptide. B , the specified fractions were assayed for pol (●) and UDG (○) activities as described under “Experimental Procedures.”

    Techniques Used: Copurification, SDS Page, Silver Staining

    8) Product Images from "Evidence That Runt Acts as a Counter-Repressor of Groucho During Drosophila melanogaster Primary Sex Determination"

    Article Title: Evidence That Runt Acts as a Counter-Repressor of Groucho During Drosophila melanogaster Primary Sex Determination

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.120.401384

    The initial run t expression pattern is recapitulated by the run t -VWRPY + transgene, but not the pair-rule expression pattern. Schematic of genomic DNA present in the run t -VWRPY + transgene. Boxed regions represent coding (solid blue) and non-coding (white) sequences of the run -RA transcript (Flybase). The C-terminal peptide VWRPY is indicated. (A) Early run t expression pattern. Embryos were stained following in situ hybridizations to detect run t mRNA. Top panels show wild-type embryos ( w 1118 ) at the indicated nuclear cycles. Lower panels show embryos containing one copy of run t -VWRPY + from the cross: w f run 3 /Binsin sc y females X w f run 3 /Yy + mal 108 (run + ) ; run t -VWRPY + males. Equal numbers of run 3 and run 3 /+ females and +/ Yy + mal 108 ( run + ) and run 3 / Yy + mal 108 (run + ) males, each bearing one copy of run t -VWRPY + , were expected. The run expression patterns could not be distinguished among the embryo types as all embryos appeared wt. (B) run t pair rule expression pattern. Wild type and run 3 mutant embryos at the indicated times during nuclear cycle 14 stained to detect run t mRNA following in situ hybridization. Embryos were staged by nuclear morphology and the degree of cellularization. Stripes are located as in wild type, but are more weakly expressed, particularly in dorsal regions. Embryos are oriented anterior to the left, dorsal to the top. Genetic crosses as in (A).
    Figure Legend Snippet: The initial run t expression pattern is recapitulated by the run t -VWRPY + transgene, but not the pair-rule expression pattern. Schematic of genomic DNA present in the run t -VWRPY + transgene. Boxed regions represent coding (solid blue) and non-coding (white) sequences of the run -RA transcript (Flybase). The C-terminal peptide VWRPY is indicated. (A) Early run t expression pattern. Embryos were stained following in situ hybridizations to detect run t mRNA. Top panels show wild-type embryos ( w 1118 ) at the indicated nuclear cycles. Lower panels show embryos containing one copy of run t -VWRPY + from the cross: w f run 3 /Binsin sc y females X w f run 3 /Yy + mal 108 (run + ) ; run t -VWRPY + males. Equal numbers of run 3 and run 3 /+ females and +/ Yy + mal 108 ( run + ) and run 3 / Yy + mal 108 (run + ) males, each bearing one copy of run t -VWRPY + , were expected. The run expression patterns could not be distinguished among the embryo types as all embryos appeared wt. (B) run t pair rule expression pattern. Wild type and run 3 mutant embryos at the indicated times during nuclear cycle 14 stained to detect run t mRNA following in situ hybridization. Embryos were staged by nuclear morphology and the degree of cellularization. Stripes are located as in wild type, but are more weakly expressed, particularly in dorsal regions. Embryos are oriented anterior to the left, dorsal to the top. Genetic crosses as in (A).

    Techniques Used: Expressing, Staining, In Situ, Mutagenesis, In Situ Hybridization

    9) Product Images from "Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference"

    Article Title: Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference

    Journal: bioRxiv

    doi: 10.1101/393074

    Identities of cells that require RRF-1 for silencing by neuronal dsRNA vary from animal to animal. ( A ) GFP expression from the sur-5::gfp chimeric gene enables simultaneous visualization of most somatic nuclei in C. elegans . A depth coded (one color for ∼53 frames) projection of 5 Z-stacks that were stitched together from a single L4-staged animal is shown (also see Materials and Methods). Scale bar = 100 µm. ( B-D ) Expression of gfp -dsRNA in neurons causes silencing throughout the animal that is entirely dependent on SID-1 and partially dependent on RRF-1. Representative images of L4-staged sur-5::gfp animals that express Prgef-1::gfp -dsRNA (B) and additionally lack sid-1 (C) or rrf-1 (D) are shown. Maximum intensity projections of sections were stitched together to generate whole-worm images. Presence of gfp- dsRNA causes worms to twist because of the rol-6 co-injection marker. Cells that require RRF-1 for silencing (e.g. the excretory canal cell indicated by red arrows and some intestinal cells in red dashed lines) and cells that can silence in the absence of RRF-1 (e.g. some intestinal cells shown in blue dashed lines) are highlighted in Prgef-1::gfp- dsRNA ; rrf-1(-) and in Prgef-1::gfp -dsRNA; sid-1(-) animals. Scale bar = 100µm. (E-F) Silencing in rrf-1(- ) animals by neuronal dsRNA is associated with a decrease in sur-5::gfp mRNA levels. Single molecule FISH was used to detect sur-5::gfp mRNA in L4-staged wild-type animals (E, left ) or in sur-5::gfp animals that express Prgef-1::gfp- dsRNA (E, middle ) and that in addition lack sid-1 (E, right ) or rrf-1 (F). RNA from Prgef-1::gfp- dsRNA is prominently detected by gfp probes in neuronal nuclei (red arrows and bracket). A representative pair of intestinal nuclei is shown for each animal as an overlay of DNA (DAPI in blue), mRNA ( gfp in green) and protein (GFP in magenta). Cytoplasmic mRNA foci were counted (see Materials and Methods) between two nuclei in wild-type or in sid-1(-) backgrounds (E), and between two nuclei where GFP is silenced (off, blue) and where GFP is expressed (on, red) in rrf-1(-) animals. Errors indicate 95% confidence intervals, n=3 in E and n=4 in F. Top scale bar = 10 µm and bottom scale bar = 5 µm. ( G ) Identities of intestinal cells that require RRF-1 for silencing vary from animal to animal. The E blastomere divides to generate 20 intestinal cells (EaLAAD to EpRPPP). Of the 20 cells, 10 undergo nuclear division without cell division (two grey circles per cell), 4 sometimes undergo similar nuclear division (one grey circle and one open circle per cell), and 6 do not undergo any division (one grey circle per cell). In each of 10 sur-5::gfp; rrf-1(-); Prgef-1::gfp -dsRNA L4-staged animals, GFP-positive nuclei (use only RRF-1, grey) and GFP-negative nuclei (use EGO-1 or RRF-1, white) were scored. Every binucleate cell had both nuclei of the same class. White boxes with a slash indicate absence of second nucleus because of lack of nuclear division ( 47 ). See Supplementary Figure S7 for images of additional animals.
    Figure Legend Snippet: Identities of cells that require RRF-1 for silencing by neuronal dsRNA vary from animal to animal. ( A ) GFP expression from the sur-5::gfp chimeric gene enables simultaneous visualization of most somatic nuclei in C. elegans . A depth coded (one color for ∼53 frames) projection of 5 Z-stacks that were stitched together from a single L4-staged animal is shown (also see Materials and Methods). Scale bar = 100 µm. ( B-D ) Expression of gfp -dsRNA in neurons causes silencing throughout the animal that is entirely dependent on SID-1 and partially dependent on RRF-1. Representative images of L4-staged sur-5::gfp animals that express Prgef-1::gfp -dsRNA (B) and additionally lack sid-1 (C) or rrf-1 (D) are shown. Maximum intensity projections of sections were stitched together to generate whole-worm images. Presence of gfp- dsRNA causes worms to twist because of the rol-6 co-injection marker. Cells that require RRF-1 for silencing (e.g. the excretory canal cell indicated by red arrows and some intestinal cells in red dashed lines) and cells that can silence in the absence of RRF-1 (e.g. some intestinal cells shown in blue dashed lines) are highlighted in Prgef-1::gfp- dsRNA ; rrf-1(-) and in Prgef-1::gfp -dsRNA; sid-1(-) animals. Scale bar = 100µm. (E-F) Silencing in rrf-1(- ) animals by neuronal dsRNA is associated with a decrease in sur-5::gfp mRNA levels. Single molecule FISH was used to detect sur-5::gfp mRNA in L4-staged wild-type animals (E, left ) or in sur-5::gfp animals that express Prgef-1::gfp- dsRNA (E, middle ) and that in addition lack sid-1 (E, right ) or rrf-1 (F). RNA from Prgef-1::gfp- dsRNA is prominently detected by gfp probes in neuronal nuclei (red arrows and bracket). A representative pair of intestinal nuclei is shown for each animal as an overlay of DNA (DAPI in blue), mRNA ( gfp in green) and protein (GFP in magenta). Cytoplasmic mRNA foci were counted (see Materials and Methods) between two nuclei in wild-type or in sid-1(-) backgrounds (E), and between two nuclei where GFP is silenced (off, blue) and where GFP is expressed (on, red) in rrf-1(-) animals. Errors indicate 95% confidence intervals, n=3 in E and n=4 in F. Top scale bar = 10 µm and bottom scale bar = 5 µm. ( G ) Identities of intestinal cells that require RRF-1 for silencing vary from animal to animal. The E blastomere divides to generate 20 intestinal cells (EaLAAD to EpRPPP). Of the 20 cells, 10 undergo nuclear division without cell division (two grey circles per cell), 4 sometimes undergo similar nuclear division (one grey circle and one open circle per cell), and 6 do not undergo any division (one grey circle per cell). In each of 10 sur-5::gfp; rrf-1(-); Prgef-1::gfp -dsRNA L4-staged animals, GFP-positive nuclei (use only RRF-1, grey) and GFP-negative nuclei (use EGO-1 or RRF-1, white) were scored. Every binucleate cell had both nuclei of the same class. White boxes with a slash indicate absence of second nucleus because of lack of nuclear division ( 47 ). See Supplementary Figure S7 for images of additional animals.

    Techniques Used: Expressing, Injection, Marker, Fluorescence In Situ Hybridization

    10) Product Images from "New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing"

    Article Title: New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing

    Journal: The CRISPR Journal

    doi: 10.1089/crispr.2019.0062

    CRISPR- CLONInG : Replacement of partial cargo sequence (Cre-comp) with the desired donor sequence on adeno-associated virus (AAV) vector (#60229; Addgene). (A) Schematic illustration of AAV vector with CRISPR cut sites (red scissors) at two ends of the Cre-comp segment. Guides (AAV-A and AAV-B) with high on-target scores were selected. (B) Cre-comp was cut out with ctRNP (Cas9-ctRNA) complex; donor for gene replacement (containing 15 bp AA replacement sequence, noted as “R,” sandwiched by HA) flanked with complementary Gibson overhangs of the adjacent AAV backbone was PCR-amplified from custom gene synthesized plasmid. (C) Assembled AAV-v1: donor template cloned into the customized AAV backbone via Gibson (HiFi) Assembly. (D) Excised AAV vector backbone (∼3.63 kb) and Cre-comp (∼2.73 kb; left); PCR amplified donor template (∼0.8 kb) with Gibson overhangs (right). (E) After CRISPR- CLONInG , 15/16 clones showed correct vector assembly, confirmed by BbsI RE(s) diagnosis (two DNA fragments; black arrow); three clones further validated by Sanger sequencing. Resolved on 0.9% agarose gel.
    Figure Legend Snippet: CRISPR- CLONInG : Replacement of partial cargo sequence (Cre-comp) with the desired donor sequence on adeno-associated virus (AAV) vector (#60229; Addgene). (A) Schematic illustration of AAV vector with CRISPR cut sites (red scissors) at two ends of the Cre-comp segment. Guides (AAV-A and AAV-B) with high on-target scores were selected. (B) Cre-comp was cut out with ctRNP (Cas9-ctRNA) complex; donor for gene replacement (containing 15 bp AA replacement sequence, noted as “R,” sandwiched by HA) flanked with complementary Gibson overhangs of the adjacent AAV backbone was PCR-amplified from custom gene synthesized plasmid. (C) Assembled AAV-v1: donor template cloned into the customized AAV backbone via Gibson (HiFi) Assembly. (D) Excised AAV vector backbone (∼3.63 kb) and Cre-comp (∼2.73 kb; left); PCR amplified donor template (∼0.8 kb) with Gibson overhangs (right). (E) After CRISPR- CLONInG , 15/16 clones showed correct vector assembly, confirmed by BbsI RE(s) diagnosis (two DNA fragments; black arrow); three clones further validated by Sanger sequencing. Resolved on 0.9% agarose gel.

    Techniques Used: CRISPR, Clone Assay, Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Synthesized, Agarose Gel Electrophoresis

    CRISPR- CLONInG : Replacement of Luciferase (Luc) on FLEx vector. (A) Schematic illustration of FLEx vector with CRISPR cut sites (red scissors) at the two junction sites flanking the undesired Luc fragment. Gray dot dashes: default backbone containing origin of replication and selection for propagation in bacterial host. (B) Luc was cut out with ctRNP (Cas9-ctRNA) complex; FRT-Neo-FRT and tdTomato were polymerase chain reaction (PCR)-amplified from existing plasmids using primers carrying complementary Gibson overhangs from the adjacent DNA fragment and vector backbone. (C) Two new vector inserts were joined with the CRISPR-digested backbone via Gibson (HiFi) Cloning for final donor assembly. (D) Excised FLEx vector backbone (∼7.5 kb) and Luciferase (∼1.65 kb; left); PCR amplified FRT-Neo-FRT (∼1.87 kb) and tdTomato (∼1.43 kb; right). N.S., nonspecific bands. (E) After CRISPR- CLONInG , 14/20 clones verified with PstI RE(s) diagnosis showed correct vector assembly (six DNA fragments; black arrow); three clones validated for sequence integrity. Resolved on 0.9% agarose gel.
    Figure Legend Snippet: CRISPR- CLONInG : Replacement of Luciferase (Luc) on FLEx vector. (A) Schematic illustration of FLEx vector with CRISPR cut sites (red scissors) at the two junction sites flanking the undesired Luc fragment. Gray dot dashes: default backbone containing origin of replication and selection for propagation in bacterial host. (B) Luc was cut out with ctRNP (Cas9-ctRNA) complex; FRT-Neo-FRT and tdTomato were polymerase chain reaction (PCR)-amplified from existing plasmids using primers carrying complementary Gibson overhangs from the adjacent DNA fragment and vector backbone. (C) Two new vector inserts were joined with the CRISPR-digested backbone via Gibson (HiFi) Cloning for final donor assembly. (D) Excised FLEx vector backbone (∼7.5 kb) and Luciferase (∼1.65 kb; left); PCR amplified FRT-Neo-FRT (∼1.87 kb) and tdTomato (∼1.43 kb; right). N.S., nonspecific bands. (E) After CRISPR- CLONInG , 14/20 clones verified with PstI RE(s) diagnosis showed correct vector assembly (six DNA fragments; black arrow); three clones validated for sequence integrity. Resolved on 0.9% agarose gel.

    Techniques Used: CRISPR, Clone Assay, Luciferase, Plasmid Preparation, Selection, Polymerase Chain Reaction, Amplification, Sequencing, Agarose Gel Electrophoresis

    CRISPR- CLIP : Procuration of lssDNA from dsDNA template and genotyping results of the CKO mice generated with the acquired lssDNA via Easi-CRISPR . (A) dsDNA template anchored in the default plasmid; the sense ssDNA (top strand) is the donor (lssDNA) of choice. (B) Cpf1 (with guide CLIP-B) was used to create a dsDNA incision on the plasmid at one end of the lssDNA cassette, while Cas9n (with guide CLIP-A) was used to create a ssDNA incision at the other end, specifically on the strand of interest (top strand in this case). (C) Upon denaturing gel-loading buffer (DGLB) treatment, the plasmid incised by Cpf1 and Cas9n was resolved into three stand-alone distinct-sized units (0.9% agarose gel electrophoresis): ∼4.9 kbase (donor + backbone) vs. ∼2.7 kbase (backbone) vs. ∼2.2 kbase (lssDNA donor). (D) Mice genotyping screened by RE HindIII (top) and EcoRV (bottom): a pair of external screening primers amplified 2.8 kb DNA fragment (black arrow); mice with the lssDNA donor integration should carry a floxed cassette with HindIII/EcoRV site adjacent to LoxP. Upon RE digestion, mouse #4 showing the insertion of both LoxPs (∼0.8 kb vs. ∼2 kb; red asterisk), further confirmed by Sanger sequencing; mouse #6 showing only HindIII digest, indicating one LoxP insertion; mouse #7 was found to carry a heterozygous 1.2 kb deletion between the two guides (used for creating CKO model), verified by Sanger sequencing, thus showing a band at ∼1.6 kb. Due to incomplete RE digest and usage of EtBr pre-stained gel, the smaller bands appeared in lighter intensity; purple asterisk: non-specific PCR band.
    Figure Legend Snippet: CRISPR- CLIP : Procuration of lssDNA from dsDNA template and genotyping results of the CKO mice generated with the acquired lssDNA via Easi-CRISPR . (A) dsDNA template anchored in the default plasmid; the sense ssDNA (top strand) is the donor (lssDNA) of choice. (B) Cpf1 (with guide CLIP-B) was used to create a dsDNA incision on the plasmid at one end of the lssDNA cassette, while Cas9n (with guide CLIP-A) was used to create a ssDNA incision at the other end, specifically on the strand of interest (top strand in this case). (C) Upon denaturing gel-loading buffer (DGLB) treatment, the plasmid incised by Cpf1 and Cas9n was resolved into three stand-alone distinct-sized units (0.9% agarose gel electrophoresis): ∼4.9 kbase (donor + backbone) vs. ∼2.7 kbase (backbone) vs. ∼2.2 kbase (lssDNA donor). (D) Mice genotyping screened by RE HindIII (top) and EcoRV (bottom): a pair of external screening primers amplified 2.8 kb DNA fragment (black arrow); mice with the lssDNA donor integration should carry a floxed cassette with HindIII/EcoRV site adjacent to LoxP. Upon RE digestion, mouse #4 showing the insertion of both LoxPs (∼0.8 kb vs. ∼2 kb; red asterisk), further confirmed by Sanger sequencing; mouse #6 showing only HindIII digest, indicating one LoxP insertion; mouse #7 was found to carry a heterozygous 1.2 kb deletion between the two guides (used for creating CKO model), verified by Sanger sequencing, thus showing a band at ∼1.6 kb. Due to incomplete RE digest and usage of EtBr pre-stained gel, the smaller bands appeared in lighter intensity; purple asterisk: non-specific PCR band.

    Techniques Used: CRISPR, Cross-linking Immunoprecipitation, Mouse Assay, Generated, Plasmid Preparation, Agarose Gel Electrophoresis, Amplification, Sequencing, Staining, Polymerase Chain Reaction

    11) Product Images from "The importance of DNA methylation of exons on alternative splicing"

    Article Title: The importance of DNA methylation of exons on alternative splicing

    Journal: RNA

    doi: 10.1261/rna.064865.117

    dCas9-TET1 targeting of DHODH exon 5 affects alternative splicing. ( A ) Schematic illustration of genomic region of DHODH from exon 4 to exon 6. Gray boxes and blue lines indicate exons and introns, respectively. Brown and black arrows represent sgRNA binding sites and RT-PCR primer binding sites, respectively. Red dashed lines indicate the region analyzed for DNA methylation. ( B ) Gel electrophoresis of RT-PCR products of the DHODH exon 5 region after overexpression of dCas9-TET1 with or without sgRNAs targeting exon 5 of DHODH gene in HCT116 cells. RNA was extracted, reverse transcribed, and primers were used to amplify the included (370 bp) or the skipped (172 bp) isoforms. NTC is non-template control. ( C ) HCT116 cells were either transfected with dCas9-TET1 only (control) or co-transfected with dCas9-TET1 and sgRNAs targeting different regions: DHODH exon 5 itself (+ex5 gRNA), DHODH intron 4 (+int4 gRNA), or DHODH ). Plotted are means ± SEM, (*) indicates P -value
    Figure Legend Snippet: dCas9-TET1 targeting of DHODH exon 5 affects alternative splicing. ( A ) Schematic illustration of genomic region of DHODH from exon 4 to exon 6. Gray boxes and blue lines indicate exons and introns, respectively. Brown and black arrows represent sgRNA binding sites and RT-PCR primer binding sites, respectively. Red dashed lines indicate the region analyzed for DNA methylation. ( B ) Gel electrophoresis of RT-PCR products of the DHODH exon 5 region after overexpression of dCas9-TET1 with or without sgRNAs targeting exon 5 of DHODH gene in HCT116 cells. RNA was extracted, reverse transcribed, and primers were used to amplify the included (370 bp) or the skipped (172 bp) isoforms. NTC is non-template control. ( C ) HCT116 cells were either transfected with dCas9-TET1 only (control) or co-transfected with dCas9-TET1 and sgRNAs targeting different regions: DHODH exon 5 itself (+ex5 gRNA), DHODH intron 4 (+int4 gRNA), or DHODH ). Plotted are means ± SEM, (*) indicates P -value

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, DNA Methylation Assay, Nucleic Acid Electrophoresis, Over Expression, Transfection

    In vivo site-specific changes in DNA methylation induced by dCas9-DNMT3A-3L and dCas9-TET1. ( A ) Schematic illustration of EDI minigene. Gray and orange boxes indicate constitutive and alternative exons, respectively. Blue and black lines indicate introns and pFRT sequence, respectively. Black arrow represents the transcription start site (TSS). ( B ) Enlargement of EDI exon 5 region. Regions targeted by sgRNAs are indicated by brackets and letters above the gene schematic. Arrows below the gene schematic represent sgRNA binding sites. The arrow colors represent the different pools of sgRNAs used in panels C and D . Red dashed lines indicate the region analyzed for DNA methylation. ( C , D ) Methylation levels within exon 5 in ( C ) Met– cells transfected with dCas9-DNMT3A-3L or ( D ) Met+ cells transfected with dCas9-TET1 and a combination of three different sgRNAs (from the five or six sgRNAs designed to each region shown in panel B ). Different combinations of sgRNAs (marked #1, #2, or #3 below bars) were tested. “dCas9-DNMT3A-3L only” and “dCas9-TET1 only” are controls without sgRNAs and “untreated” indicates cells without transfection. (*) Indicates P -value
    Figure Legend Snippet: In vivo site-specific changes in DNA methylation induced by dCas9-DNMT3A-3L and dCas9-TET1. ( A ) Schematic illustration of EDI minigene. Gray and orange boxes indicate constitutive and alternative exons, respectively. Blue and black lines indicate introns and pFRT sequence, respectively. Black arrow represents the transcription start site (TSS). ( B ) Enlargement of EDI exon 5 region. Regions targeted by sgRNAs are indicated by brackets and letters above the gene schematic. Arrows below the gene schematic represent sgRNA binding sites. The arrow colors represent the different pools of sgRNAs used in panels C and D . Red dashed lines indicate the region analyzed for DNA methylation. ( C , D ) Methylation levels within exon 5 in ( C ) Met– cells transfected with dCas9-DNMT3A-3L or ( D ) Met+ cells transfected with dCas9-TET1 and a combination of three different sgRNAs (from the five or six sgRNAs designed to each region shown in panel B ). Different combinations of sgRNAs (marked #1, #2, or #3 below bars) were tested. “dCas9-DNMT3A-3L only” and “dCas9-TET1 only” are controls without sgRNAs and “untreated” indicates cells without transfection. (*) Indicates P -value

    Techniques Used: In Vivo, DNA Methylation Assay, Sequencing, Binding Assay, Methylation, Transfection

    Schematic illustration of region-specific DNA methylation manipulations using the CRISPR system. Cells are co-transfected with a plasmid for expression of dCas9-DNMT3A-3L and the sgRNA expression plasmids. The dCas9 fusion protein is guided to a specific region of the genome by the sgRNAs where it brings DNMT3A-3L close to the DNA, resulting in site-specific methylation. As the transfection is transient, after several cell divisions, the fusion protein expression is lost but the DNA methylation pattern is maintained by the endogenous DNMT1 during genomic DNA replication. A similar method involving the dCas9-TET1 fusion enabled site-specific demethylation of DNA. The yellow spheres indicate methylated CpG.
    Figure Legend Snippet: Schematic illustration of region-specific DNA methylation manipulations using the CRISPR system. Cells are co-transfected with a plasmid for expression of dCas9-DNMT3A-3L and the sgRNA expression plasmids. The dCas9 fusion protein is guided to a specific region of the genome by the sgRNAs where it brings DNMT3A-3L close to the DNA, resulting in site-specific methylation. As the transfection is transient, after several cell divisions, the fusion protein expression is lost but the DNA methylation pattern is maintained by the endogenous DNMT1 during genomic DNA replication. A similar method involving the dCas9-TET1 fusion enabled site-specific demethylation of DNA. The yellow spheres indicate methylated CpG.

    Techniques Used: DNA Methylation Assay, CRISPR, Transfection, Plasmid Preparation, Expressing, Methylation

    dCas9-TET1 induces DNA demethylation in a limited region. ( A ) Schematic illustration of EDI minigene exons 4 and 5. Exon 4 is alternatively spliced, whereas exon 5 is constitutively spliced. Blue and black lines indicate EDI introns and pFRT sequence, respectively. The six regions analyzed for methylation are indicated with letters. ( B ) The level of methylated CpGs (in percent) within the six regions denoted in panel A after transfection of dCas9-TET1 plasmid into Met+ cells with or without sgRNA expression (black and gray bars, respectively).
    Figure Legend Snippet: dCas9-TET1 induces DNA demethylation in a limited region. ( A ) Schematic illustration of EDI minigene exons 4 and 5. Exon 4 is alternatively spliced, whereas exon 5 is constitutively spliced. Blue and black lines indicate EDI introns and pFRT sequence, respectively. The six regions analyzed for methylation are indicated with letters. ( B ) The level of methylated CpGs (in percent) within the six regions denoted in panel A after transfection of dCas9-TET1 plasmid into Met+ cells with or without sgRNA expression (black and gray bars, respectively).

    Techniques Used: Sequencing, Methylation, Transfection, Plasmid Preparation, Expressing

    12) Product Images from "The Immunophilin Ligands Cyclosporin A and FK506 Suppress Prostate Cancer Cell Growth by Androgen Receptor-Dependent and -Independent Mechanisms"

    Article Title: The Immunophilin Ligands Cyclosporin A and FK506 Suppress Prostate Cancer Cell Growth by Androgen Receptor-Dependent and -Independent Mechanisms

    Journal:

    doi: 10.1210/en.2007-0145

    CsA and FK506 do not induce apoptosis. LNCaP cells were cultured in the presence and absence of CsA (5.0 μM) or FK506 (10.0 μM) for 7 days with a change of media and ligands on days 2, 4, and 6. DNA was isolated, subjected to agarose gel
    Figure Legend Snippet: CsA and FK506 do not induce apoptosis. LNCaP cells were cultured in the presence and absence of CsA (5.0 μM) or FK506 (10.0 μM) for 7 days with a change of media and ligands on days 2, 4, and 6. DNA was isolated, subjected to agarose gel

    Techniques Used: Cell Culture, Isolation, Agarose Gel Electrophoresis

    13) Product Images from "Comparative analyses of DNA repeats and identification of a novel Fesreba centromeric element in fescues and ryegrasses"

    Article Title: Comparative analyses of DNA repeats and identification of a novel Fesreba centromeric element in fescues and ryegrasses

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-020-02495-0

    Graphical layouts of selected DNA repeats and their validation by Southern blotting. Graphical layouts were obtained after clustering analyses done by RepeatExplorer. a Cluster CL1. b Cluster CL38 containing the Ty3/gypsy Athila element. c Cluster CL20 containing Ty3/gypsy Ogre/Tat elements. Sequencing reads from Festuca species are in pink, whereas sequencing reads from Lolium species are in yellow. d – f Validation of clustering results by Southern hybridization with sequences derived from clusters CL1, CL38, and CL20. Lanes contained genomic DNA digested by Hae III restriction endonuclease. Lane 1: F. pratensis cv. Fure (2n = 2x = 14); lane 2: F. pratensis cv. Westa (2n = 4x = 28); lane 3: F. arundinacea subsp. arundinacea (2n = 6x = 42); lane 4: F. gigantea (2n = 6x = 42); lane 5: F. glaucescens (2n = 4x = 28); lane 6: F. mairei (2n = 4x = 28); lane 7: L. multiflorum cv. Mitos (2n = 4x = 28); lane 8: L. multiflorum cv. Kuri1 (2n = 2x = 14); lane 9: L. perenne cv. Neptun (2n = 4x = 28); lane 10: L. perenne GR 3320 (2n = 2x = 14)
    Figure Legend Snippet: Graphical layouts of selected DNA repeats and their validation by Southern blotting. Graphical layouts were obtained after clustering analyses done by RepeatExplorer. a Cluster CL1. b Cluster CL38 containing the Ty3/gypsy Athila element. c Cluster CL20 containing Ty3/gypsy Ogre/Tat elements. Sequencing reads from Festuca species are in pink, whereas sequencing reads from Lolium species are in yellow. d – f Validation of clustering results by Southern hybridization with sequences derived from clusters CL1, CL38, and CL20. Lanes contained genomic DNA digested by Hae III restriction endonuclease. Lane 1: F. pratensis cv. Fure (2n = 2x = 14); lane 2: F. pratensis cv. Westa (2n = 4x = 28); lane 3: F. arundinacea subsp. arundinacea (2n = 6x = 42); lane 4: F. gigantea (2n = 6x = 42); lane 5: F. glaucescens (2n = 4x = 28); lane 6: F. mairei (2n = 4x = 28); lane 7: L. multiflorum cv. Mitos (2n = 4x = 28); lane 8: L. multiflorum cv. Kuri1 (2n = 2x = 14); lane 9: L. perenne cv. Neptun (2n = 4x = 28); lane 10: L. perenne GR 3320 (2n = 2x = 14)

    Techniques Used: Southern Blot, Sequencing, Hybridization, Derivative Assay

    Genome proportion of the most abundant DNA repeats. The genome proportion of individual repeat types was obtained as a ratio of reads specific to individual repeat types to all reads used for clustering analyses by the RepeatExplorer pipeline. Diploid Festuca pratensis cv. Fure (FPF); tetraploid F. pratensis cv. Westa (FPW); hexaploid F. arundinacea subsp. arundinacea (FAR); hexaploid F. gigantea (FGI); tetraploid F. glaucescens (FGL); tetraploid F. mairei (FMA); diploid cv. Kuri1 of L. multiflorum (LM2); tetraploid cv. Mitos of L. multiflorum (LMM); diploid L. perenne (LP2); tetraploid cv. Neptun of L. perenne (LPN)
    Figure Legend Snippet: Genome proportion of the most abundant DNA repeats. The genome proportion of individual repeat types was obtained as a ratio of reads specific to individual repeat types to all reads used for clustering analyses by the RepeatExplorer pipeline. Diploid Festuca pratensis cv. Fure (FPF); tetraploid F. pratensis cv. Westa (FPW); hexaploid F. arundinacea subsp. arundinacea (FAR); hexaploid F. gigantea (FGI); tetraploid F. glaucescens (FGL); tetraploid F. mairei (FMA); diploid cv. Kuri1 of L. multiflorum (LM2); tetraploid cv. Mitos of L. multiflorum (LMM); diploid L. perenne (LP2); tetraploid cv. Neptun of L. perenne (LPN)

    Techniques Used:

    14) Product Images from "Characteristics of Age-Related Changes in Cultured Human Vocal Fold Fibroblasts"

    Article Title: Characteristics of Age-Related Changes in Cultured Human Vocal Fold Fibroblasts

    Journal:

    doi: 10.1097/MLG.0b013e31817aec6c

    Terminal restriction fragment (telomere) length in cultures human vocal fold fibroblasts (21T, 59T and 79T) during passage 7 to 18 by Southern blot analysis. Genomic DNA was isolated from hVFF of passage 7, 13 and 18, and hybridized with a telomeric probe
    Figure Legend Snippet: Terminal restriction fragment (telomere) length in cultures human vocal fold fibroblasts (21T, 59T and 79T) during passage 7 to 18 by Southern blot analysis. Genomic DNA was isolated from hVFF of passage 7, 13 and 18, and hybridized with a telomeric probe

    Techniques Used: Southern Blot, Isolation

    Related Articles

    SYBR Green Assay:

    Article Title: Protection Against Henipavirus Infection by Use of Recombinant Adeno-Associated Virus–Vector Vaccines
    Article Snippet: .. The qRT-PCR reaction was conducted on 10 ng of complementary DNA or genomic DNA, using the FastStart Universal SYBR Green Master reagent (Roche Diagnostics). qRT-PCR was run on the Step One Plus Real Time PCR system (Applied Biosystems). .. All samples were run in duplicate, and results were analyzed using ABI StepOne software v2.1.

    Transfection:

    Article Title: The importance of DNA methylation of exons on alternative splicing
    Article Snippet: .. Following transfection, cells were incubated for 4 d and then genomic DNA was extracted using High Pure PCR Template Preparation Kit (Roche) or using the following protocol: Cells from each well were pelleted by centrifugation and washed with PBS. .. Pellets were resuspended in DNA Lysis Buffer (50 mM Tris-HCl, 60 mM NaCl, 0.65% SDS, 0.8 mg/mL Proteinase K [NEB]) and incubated at 37°C overnight or at 55°C for 3 h. Samples were treated with RNase A. DNA was extracted using Phase Lock Gel tubes (5Prime) and phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 100 mM Tris, pH 8.0 (Sigma) according to the protocol provided by 5Prime.

    Amplification:

    Article Title: Post-transcriptional modulation of the SigF regulon in Mycobacterium smegmatis by the PhoH2 toxin-antitoxin
    Article Snippet: .. Biological target assays Target DNA sequences, sigF, rsbW-sigF and chaB-rsbW-sigF were amplified from M. smegmatis mc 2 155 genomic DNA using SF Fwd and Rev, RS Fwd and SF Rev, and UCRS Fwd and SF Rev respectively ( ) in PCR reactions with either KAPA HiFi DNA polymerase with high GC buffers (KAPA Biosystems) or Hot FIREPol Blend Master mix (Solis BioDyne). .. These products were used as DNA template for a second round of PCR using T7+SF Fwd, T7+RS Fwd and T7+UCRS Fwd in place of the original forward primer to introduce the T7 promoter sequence to the 5’ end of the PCR product.

    Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
    Article Snippet: .. The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States). .. All the PCR primers are listed in .

    Article Title: ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells
    Article Snippet: .. A total of 50 µl of washed culture was mixed with 1 µg of either gDNA derived from V. cholerae strain A1552-lacZ-Kan , commercially available phage lambda DNA (Roche) or a 10.3 kb fragment amplified through PCR. .. After 5 min of incubation with the DNA the bacteria were mounted on agarose pads and imaged.

    Mutagenesis:

    Article Title: New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing
    Article Snippet: .. Genomic DNA was extracted using High Pure PCR Template Preparation Kit (Roche, ref#11 796 828 001), and Herculase II PCR system was used to amplify 1,352bp of exon 10 mutant sequence along with neighboring sequence using TD PCR condition of 95 ℃ for 3 min; 95 ℃ for 15 sec, 61 ℃ for 20 sec (−0.5/cycle), 72 ℃ for 1.5 min, for 10 cycles; 95 ℃ for 15 sec, 56 ℃ for 20 sec, 72 ℃ for 1.5 min, for 25 cycles with primer pair (PSEN1-F & PSEN1-R, Table 2), followed by Sanger sequencing read with PSEN1-seq primer (Table 2). .. We thank Jing Gao and Chiayun Han for assistance with mESCs culture and handling; Transgenic and Reproductive Technology Center for zygote microinjection and mouse husbandary; Gali Umschweif Nevo for the gift of Neuro-2a/Cas9-Rosa26-Neo cell line; Daniel Weinberg for providing the original FLEx vector; Flow Cytometry Resource Center for clonal isolation of the N2A cell; Betty Shih of Enlightagen Consulting for help with the manuscript.

    Lambda DNA Preparation:

    Article Title: ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells
    Article Snippet: .. A total of 50 µl of washed culture was mixed with 1 µg of either gDNA derived from V. cholerae strain A1552-lacZ-Kan , commercially available phage lambda DNA (Roche) or a 10.3 kb fragment amplified through PCR. .. After 5 min of incubation with the DNA the bacteria were mounted on agarose pads and imaged.

    Nick Translation:

    Article Title: Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping
    Article Snippet: .. The gDNA and all repeat DNA probes were labeled with either biotin-dUTP or digoxigenin-dUTP (Roche, http://www.roche-applied-science.com ) by standard nick translation reaction. .. Fluorescence in situ hybridization FISH was carried out essentially according to published procedures [ ].

    Centrifugation:

    Article Title: The importance of DNA methylation of exons on alternative splicing
    Article Snippet: .. Following transfection, cells were incubated for 4 d and then genomic DNA was extracted using High Pure PCR Template Preparation Kit (Roche) or using the following protocol: Cells from each well were pelleted by centrifugation and washed with PBS. .. Pellets were resuspended in DNA Lysis Buffer (50 mM Tris-HCl, 60 mM NaCl, 0.65% SDS, 0.8 mg/mL Proteinase K [NEB]) and incubated at 37°C overnight or at 55°C for 3 h. Samples were treated with RNase A. DNA was extracted using Phase Lock Gel tubes (5Prime) and phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 100 mM Tris, pH 8.0 (Sigma) according to the protocol provided by 5Prime.

    Quantitative RT-PCR:

    Article Title: Protection Against Henipavirus Infection by Use of Recombinant Adeno-Associated Virus–Vector Vaccines
    Article Snippet: .. The qRT-PCR reaction was conducted on 10 ng of complementary DNA or genomic DNA, using the FastStart Universal SYBR Green Master reagent (Roche Diagnostics). qRT-PCR was run on the Step One Plus Real Time PCR system (Applied Biosystems). .. All samples were run in duplicate, and results were analyzed using ABI StepOne software v2.1.

    Purification:

    Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
    Article Snippet: .. The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States). .. All the PCR primers are listed in .

    Real-time Polymerase Chain Reaction:

    Article Title: Protection Against Henipavirus Infection by Use of Recombinant Adeno-Associated Virus–Vector Vaccines
    Article Snippet: .. The qRT-PCR reaction was conducted on 10 ng of complementary DNA or genomic DNA, using the FastStart Universal SYBR Green Master reagent (Roche Diagnostics). qRT-PCR was run on the Step One Plus Real Time PCR system (Applied Biosystems). .. All samples were run in duplicate, and results were analyzed using ABI StepOne software v2.1.

    Polymerase Chain Reaction:

    Article Title: Post-transcriptional modulation of the SigF regulon in Mycobacterium smegmatis by the PhoH2 toxin-antitoxin
    Article Snippet: .. Biological target assays Target DNA sequences, sigF, rsbW-sigF and chaB-rsbW-sigF were amplified from M. smegmatis mc 2 155 genomic DNA using SF Fwd and Rev, RS Fwd and SF Rev, and UCRS Fwd and SF Rev respectively ( ) in PCR reactions with either KAPA HiFi DNA polymerase with high GC buffers (KAPA Biosystems) or Hot FIREPol Blend Master mix (Solis BioDyne). .. These products were used as DNA template for a second round of PCR using T7+SF Fwd, T7+RS Fwd and T7+UCRS Fwd in place of the original forward primer to introduce the T7 promoter sequence to the 5’ end of the PCR product.

    Article Title: New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing
    Article Snippet: .. Genomic DNA was extracted using High Pure PCR Template Preparation Kit (Roche, ref#11 796 828 001), and Herculase II PCR system was used to amplify 1,352bp of exon 10 mutant sequence along with neighboring sequence using TD PCR condition of 95 ℃ for 3 min; 95 ℃ for 15 sec, 61 ℃ for 20 sec (−0.5/cycle), 72 ℃ for 1.5 min, for 10 cycles; 95 ℃ for 15 sec, 56 ℃ for 20 sec, 72 ℃ for 1.5 min, for 25 cycles with primer pair (PSEN1-F & PSEN1-R, Table 2), followed by Sanger sequencing read with PSEN1-seq primer (Table 2). .. We thank Jing Gao and Chiayun Han for assistance with mESCs culture and handling; Transgenic and Reproductive Technology Center for zygote microinjection and mouse husbandary; Gali Umschweif Nevo for the gift of Neuro-2a/Cas9-Rosa26-Neo cell line; Daniel Weinberg for providing the original FLEx vector; Flow Cytometry Resource Center for clonal isolation of the N2A cell; Betty Shih of Enlightagen Consulting for help with the manuscript.

    Article Title: Conversion of a recA-Mediated Non-toxigenic Vibrio cholerae O1 Strain to a Toxigenic Strain Using Chitin-Induced Transformation
    Article Snippet: .. The upstream and downstream fragments of orfU gene were amplified from gDNA of E7946 strain; gDNA of E7946 was extracted and purified using High Pure PCR Template Preparation Kit (Roche Life Science, Indianapolis, IN, United States). .. All the PCR primers are listed in .

    Article Title: The importance of DNA methylation of exons on alternative splicing
    Article Snippet: .. Following transfection, cells were incubated for 4 d and then genomic DNA was extracted using High Pure PCR Template Preparation Kit (Roche) or using the following protocol: Cells from each well were pelleted by centrifugation and washed with PBS. .. Pellets were resuspended in DNA Lysis Buffer (50 mM Tris-HCl, 60 mM NaCl, 0.65% SDS, 0.8 mg/mL Proteinase K [NEB]) and incubated at 37°C overnight or at 55°C for 3 h. Samples were treated with RNase A. DNA was extracted using Phase Lock Gel tubes (5Prime) and phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 100 mM Tris, pH 8.0 (Sigma) according to the protocol provided by 5Prime.

    Article Title: ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells
    Article Snippet: .. A total of 50 µl of washed culture was mixed with 1 µg of either gDNA derived from V. cholerae strain A1552-lacZ-Kan , commercially available phage lambda DNA (Roche) or a 10.3 kb fragment amplified through PCR. .. After 5 min of incubation with the DNA the bacteria were mounted on agarose pads and imaged.

    Incubation:

    Article Title: The importance of DNA methylation of exons on alternative splicing
    Article Snippet: .. Following transfection, cells were incubated for 4 d and then genomic DNA was extracted using High Pure PCR Template Preparation Kit (Roche) or using the following protocol: Cells from each well were pelleted by centrifugation and washed with PBS. .. Pellets were resuspended in DNA Lysis Buffer (50 mM Tris-HCl, 60 mM NaCl, 0.65% SDS, 0.8 mg/mL Proteinase K [NEB]) and incubated at 37°C overnight or at 55°C for 3 h. Samples were treated with RNase A. DNA was extracted using Phase Lock Gel tubes (5Prime) and phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 100 mM Tris, pH 8.0 (Sigma) according to the protocol provided by 5Prime.

    Labeling:

    Article Title: Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping
    Article Snippet: .. The gDNA and all repeat DNA probes were labeled with either biotin-dUTP or digoxigenin-dUTP (Roche, http://www.roche-applied-science.com ) by standard nick translation reaction. .. Fluorescence in situ hybridization FISH was carried out essentially according to published procedures [ ].

    Article Title: Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization
    Article Snippet: .. Rsa I-digested B. anthracis, B. cereus , and B. thuringiensis genomic DNAs were labeled with 32 P (Random-Primer Labeling System, Roche Molecular Biochemicals, Indianapolis, IN) and used to probe the two sets of colony filters for each SSH library. .. Plasmid DNA preparation and insert fragment isolation Plasmid DNA was isolated from 1–5 ml LB or TB/kanamycin cultures by either the boiling miniprep method [ ] or the Plasmid Mini Kit (Qiagen, Valencia, CA).

    Sequencing:

    Article Title: New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing
    Article Snippet: .. Genomic DNA was extracted using High Pure PCR Template Preparation Kit (Roche, ref#11 796 828 001), and Herculase II PCR system was used to amplify 1,352bp of exon 10 mutant sequence along with neighboring sequence using TD PCR condition of 95 ℃ for 3 min; 95 ℃ for 15 sec, 61 ℃ for 20 sec (−0.5/cycle), 72 ℃ for 1.5 min, for 10 cycles; 95 ℃ for 15 sec, 56 ℃ for 20 sec, 72 ℃ for 1.5 min, for 25 cycles with primer pair (PSEN1-F & PSEN1-R, Table 2), followed by Sanger sequencing read with PSEN1-seq primer (Table 2). .. We thank Jing Gao and Chiayun Han for assistance with mESCs culture and handling; Transgenic and Reproductive Technology Center for zygote microinjection and mouse husbandary; Gali Umschweif Nevo for the gift of Neuro-2a/Cas9-Rosa26-Neo cell line; Daniel Weinberg for providing the original FLEx vector; Flow Cytometry Resource Center for clonal isolation of the N2A cell; Betty Shih of Enlightagen Consulting for help with the manuscript.

    Derivative Assay:

    Article Title: ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells
    Article Snippet: .. A total of 50 µl of washed culture was mixed with 1 µg of either gDNA derived from V. cholerae strain A1552-lacZ-Kan , commercially available phage lambda DNA (Roche) or a 10.3 kb fragment amplified through PCR. .. After 5 min of incubation with the DNA the bacteria were mounted on agarose pads and imaged.

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  • 85
    Roche total aegilops tauschii genomic dna
    Substitution (1D/1A) line, 2 n = 42 chromosomes. ( a ) FISH with 5S rDNA (red) and 35S rDNA (green); ( b ) FISH with pAs1 (red) and pSc 119.2 (green). The chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue); ( c ) mc GISH with total genomic <t>DNA</t> from rye—R genome (red), total genomic DNA from Triticum monococcum —A genome (green) and total genomic DNA from Aegilops <t>tauschii</t> —D genome (yellow) with blocking genomic DNA of Aegilops speltoides —B genome (DAPI, blue).
    Total Aegilops Tauschii Genomic Dna, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gdna  (Roche)
    94
    Roche gdna
    ComEA binds to DNA in vivo . (A) Plasmid-encoded gfp (tat-GFP) or comEA-gfp (tat-ComEA-GFP), both preceded by a tat-signal sequence, were expressed in E. coli . The images shown correspond to the GFP channel, DAPI channel (to visualize DAPI-stained DNA), merged fluorescent images (merge), and phase contrast (Ph). The cells are outlined with dashed lines for tat-ComEA-GFP. Heat-maps showing the fluorescence intensities of the GFP and DAPI signals are depicted for the tat-comEA-gfp expressing cells below the images. (B) ComEA-mCherry aggregation and foci formation after the addition of external DNA. Competence-induced cells without (no DNA) or with external DNA were imaged in the red (mCherry; upper row) or the phase contrast channel (lower row) to visualize ComEA-mCherry localization. The DNA fragments differed in lengths (PCR fragment, 10.3 kb; λDNA, 48.5 kb; <t>gDNA,</t> various lengths). Transforming DNA did not lead to foci formation of periplasmic mCherry alone (preceded by the ComEA signal sequence; ss[ComEA]-mCherry). (C) Colocalization (merged image) of ComEA-mCherry (red channel) and YoYo-1-stained transforming DNA (green channel). The outline of the cells is shown in the phase contrast image (Ph). Scale bars in all images, 2 µm. (D) DNA uptake requires ComEA. DNA uptake of competent V. <t>cholerae</t> cells was tested using a whole-cell duplex PCR assay. All mutant strains were tested in a comEC positive (+) and negative (−) background. The lower PCR fragments indicate acceptor strain DNA (gDNA, acceptor); the upper band indicates internalized transforming DNA (tDNA). L, ladder.
    Gdna, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche bisulfite pyrosequencing genomic dna
    Lentiviral-mediated Kaiso knockdown (Kaiso KD ) is associated with a reduction of ICR1 methylation and decreased endogenous IGF2 and H19 transcription in human primary. As controls, transfections with shRNA against a scrambled locus ( S.L. ) were performed. a Knockdown efficiency was quantitated by RT-qPCR. The expression values are normalized to PDH and B2M transcripts and are represented as mean ± SEM from two independent experiments each. p values for the transcriptional comparisons are indicated. b ICR1 methylation was determined by <t>pyrosequencing</t> of the three established differentially methylated ICR1 CpGs in bisulfite-treated <t>DNA.</t> The location of the analysed CpGs is schematically depicted on top . Mean methylation values of three replicates are presented ± SEM for each CG position. The knockdown of Kaiso is associated with reduced methylation of endogenous ICR1
    Bisulfite Pyrosequencing Genomic Dna, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Roche negative genomic dnas
    Cq difference between positive and negative genomic <t>DNAs</t> obtained with different PCR mixes and machines. a Cq values of 20 primer-selective PCR SNP assays for gDNA samples that should be positive ( green dots ) or negative ( red squares ). b Individual delta-Cq values for 20 primer-selective PCR SNP assays between positive and negative gDNA samples for <t>ABI</t> and Roche PCR mixes on two different PCR machines. The flags indicate means ± SD
    Negative Genomic Dnas, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Substitution (1D/1A) line, 2 n = 42 chromosomes. ( a ) FISH with 5S rDNA (red) and 35S rDNA (green); ( b ) FISH with pAs1 (red) and pSc 119.2 (green). The chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue); ( c ) mc GISH with total genomic DNA from rye—R genome (red), total genomic DNA from Triticum monococcum —A genome (green) and total genomic DNA from Aegilops tauschii —D genome (yellow) with blocking genomic DNA of Aegilops speltoides —B genome (DAPI, blue).

    Journal: International Journal of Molecular Sciences

    Article Title: Molecular, Physicochemical and Rheological Characteristics of Introgressive Triticale/Triticum monococcum ssp. monococcum Lines with Wheat 1D/1A Chromosome Substitution

    doi: 10.3390/ijms140815595

    Figure Lengend Snippet: Substitution (1D/1A) line, 2 n = 42 chromosomes. ( a ) FISH with 5S rDNA (red) and 35S rDNA (green); ( b ) FISH with pAs1 (red) and pSc 119.2 (green). The chromosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue); ( c ) mc GISH with total genomic DNA from rye—R genome (red), total genomic DNA from Triticum monococcum —A genome (green) and total genomic DNA from Aegilops tauschii —D genome (yellow) with blocking genomic DNA of Aegilops speltoides —B genome (DAPI, blue).

    Article Snippet: Total Aegilops tauschii genomic DNA was used as a probe; it was labeled by nicktranslation with digoxigenin-11-dUTP for the screening, or digoxigenin-11-dUTP and tetramethyl-rhodamine-5-dUTP (Roche) (1:1 ratio) for the identification process.

    Techniques: Fluorescence In Situ Hybridization, Blocking Assay

    ComEA binds to DNA in vivo . (A) Plasmid-encoded gfp (tat-GFP) or comEA-gfp (tat-ComEA-GFP), both preceded by a tat-signal sequence, were expressed in E. coli . The images shown correspond to the GFP channel, DAPI channel (to visualize DAPI-stained DNA), merged fluorescent images (merge), and phase contrast (Ph). The cells are outlined with dashed lines for tat-ComEA-GFP. Heat-maps showing the fluorescence intensities of the GFP and DAPI signals are depicted for the tat-comEA-gfp expressing cells below the images. (B) ComEA-mCherry aggregation and foci formation after the addition of external DNA. Competence-induced cells without (no DNA) or with external DNA were imaged in the red (mCherry; upper row) or the phase contrast channel (lower row) to visualize ComEA-mCherry localization. The DNA fragments differed in lengths (PCR fragment, 10.3 kb; λDNA, 48.5 kb; gDNA, various lengths). Transforming DNA did not lead to foci formation of periplasmic mCherry alone (preceded by the ComEA signal sequence; ss[ComEA]-mCherry). (C) Colocalization (merged image) of ComEA-mCherry (red channel) and YoYo-1-stained transforming DNA (green channel). The outline of the cells is shown in the phase contrast image (Ph). Scale bars in all images, 2 µm. (D) DNA uptake requires ComEA. DNA uptake of competent V. cholerae cells was tested using a whole-cell duplex PCR assay. All mutant strains were tested in a comEC positive (+) and negative (−) background. The lower PCR fragments indicate acceptor strain DNA (gDNA, acceptor); the upper band indicates internalized transforming DNA (tDNA). L, ladder.

    Journal: PLoS Genetics

    Article Title: ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

    doi: 10.1371/journal.pgen.1004066

    Figure Lengend Snippet: ComEA binds to DNA in vivo . (A) Plasmid-encoded gfp (tat-GFP) or comEA-gfp (tat-ComEA-GFP), both preceded by a tat-signal sequence, were expressed in E. coli . The images shown correspond to the GFP channel, DAPI channel (to visualize DAPI-stained DNA), merged fluorescent images (merge), and phase contrast (Ph). The cells are outlined with dashed lines for tat-ComEA-GFP. Heat-maps showing the fluorescence intensities of the GFP and DAPI signals are depicted for the tat-comEA-gfp expressing cells below the images. (B) ComEA-mCherry aggregation and foci formation after the addition of external DNA. Competence-induced cells without (no DNA) or with external DNA were imaged in the red (mCherry; upper row) or the phase contrast channel (lower row) to visualize ComEA-mCherry localization. The DNA fragments differed in lengths (PCR fragment, 10.3 kb; λDNA, 48.5 kb; gDNA, various lengths). Transforming DNA did not lead to foci formation of periplasmic mCherry alone (preceded by the ComEA signal sequence; ss[ComEA]-mCherry). (C) Colocalization (merged image) of ComEA-mCherry (red channel) and YoYo-1-stained transforming DNA (green channel). The outline of the cells is shown in the phase contrast image (Ph). Scale bars in all images, 2 µm. (D) DNA uptake requires ComEA. DNA uptake of competent V. cholerae cells was tested using a whole-cell duplex PCR assay. All mutant strains were tested in a comEC positive (+) and negative (−) background. The lower PCR fragments indicate acceptor strain DNA (gDNA, acceptor); the upper band indicates internalized transforming DNA (tDNA). L, ladder.

    Article Snippet: A total of 50 µl of washed culture was mixed with 1 µg of either gDNA derived from V. cholerae strain A1552-lacZ-Kan , commercially available phage lambda DNA (Roche) or a 10.3 kb fragment amplified through PCR.

    Techniques: In Vivo, Plasmid Preparation, Sequencing, Staining, Fluorescence, Expressing, Polymerase Chain Reaction, Mutagenesis

    Localization of the ComEA protein in naturally competent V. cholerae cells. (A) Expression and distribution of ComEA-mCherry (upper row) or signal sequence[ComEA] (amino acids 1–25)-mCherry fusion proteins (lower row) within competent V. cholerae cells. Fluorescent signals for mCherry or DAPI-stained genomic DNA were visualized and compared with each other (merge) and the corresponding phase contrast image (Ph). (B) Representative fluorescence loss in photobleaching (FLIP) experiment to demonstrate the degree of mobility of ComEA-mCherry in live bacteria. Bleaching of the region-of-interest (ROI) 1 (indicated as 1 in the images on the right) was initiated after the acquisition of 20 frames and repeated after every frame. The fluorescence intensities of ROIs 1–3 were measured for a total of 20 sec and normalized to the average fluorescence intensity of the first 10 frames. The moving averages (period n = 5) are indicated with black lines. The average fluorescence intensity projections before (pre-bleach) and after bleaching (post-bleach) are shown on the right. Scale bars, 2 µm.

    Journal: PLoS Genetics

    Article Title: ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

    doi: 10.1371/journal.pgen.1004066

    Figure Lengend Snippet: Localization of the ComEA protein in naturally competent V. cholerae cells. (A) Expression and distribution of ComEA-mCherry (upper row) or signal sequence[ComEA] (amino acids 1–25)-mCherry fusion proteins (lower row) within competent V. cholerae cells. Fluorescent signals for mCherry or DAPI-stained genomic DNA were visualized and compared with each other (merge) and the corresponding phase contrast image (Ph). (B) Representative fluorescence loss in photobleaching (FLIP) experiment to demonstrate the degree of mobility of ComEA-mCherry in live bacteria. Bleaching of the region-of-interest (ROI) 1 (indicated as 1 in the images on the right) was initiated after the acquisition of 20 frames and repeated after every frame. The fluorescence intensities of ROIs 1–3 were measured for a total of 20 sec and normalized to the average fluorescence intensity of the first 10 frames. The moving averages (period n = 5) are indicated with black lines. The average fluorescence intensity projections before (pre-bleach) and after bleaching (post-bleach) are shown on the right. Scale bars, 2 µm.

    Article Snippet: A total of 50 µl of washed culture was mixed with 1 µg of either gDNA derived from V. cholerae strain A1552-lacZ-Kan , commercially available phage lambda DNA (Roche) or a 10.3 kb fragment amplified through PCR.

    Techniques: Expressing, Sequencing, Staining, Fluorescence, Size-exclusion Chromatography

    Lentiviral-mediated Kaiso knockdown (Kaiso KD ) is associated with a reduction of ICR1 methylation and decreased endogenous IGF2 and H19 transcription in human primary. As controls, transfections with shRNA against a scrambled locus ( S.L. ) were performed. a Knockdown efficiency was quantitated by RT-qPCR. The expression values are normalized to PDH and B2M transcripts and are represented as mean ± SEM from two independent experiments each. p values for the transcriptional comparisons are indicated. b ICR1 methylation was determined by pyrosequencing of the three established differentially methylated ICR1 CpGs in bisulfite-treated DNA. The location of the analysed CpGs is schematically depicted on top . Mean methylation values of three replicates are presented ± SEM for each CG position. The knockdown of Kaiso is associated with reduced methylation of endogenous ICR1

    Journal: Clinical Epigenetics

    Article Title: Kaiso mediates human ICR1 methylation maintenance and H19 transcriptional fine regulation

    doi: 10.1186/s13148-016-0215-4

    Figure Lengend Snippet: Lentiviral-mediated Kaiso knockdown (Kaiso KD ) is associated with a reduction of ICR1 methylation and decreased endogenous IGF2 and H19 transcription in human primary. As controls, transfections with shRNA against a scrambled locus ( S.L. ) were performed. a Knockdown efficiency was quantitated by RT-qPCR. The expression values are normalized to PDH and B2M transcripts and are represented as mean ± SEM from two independent experiments each. p values for the transcriptional comparisons are indicated. b ICR1 methylation was determined by pyrosequencing of the three established differentially methylated ICR1 CpGs in bisulfite-treated DNA. The location of the analysed CpGs is schematically depicted on top . Mean methylation values of three replicates are presented ± SEM for each CG position. The knockdown of Kaiso is associated with reduced methylation of endogenous ICR1

    Article Snippet: Bisulfite pyrosequencing Genomic DNA of primary fibroblasts was extracted with the High Pure PCR Template preparation Kit (Roche) according to the manufacturer’s instructions.

    Techniques: Methylation, Transfection, shRNA, Quantitative RT-PCR, Expressing

    Cq difference between positive and negative genomic DNAs obtained with different PCR mixes and machines. a Cq values of 20 primer-selective PCR SNP assays for gDNA samples that should be positive ( green dots ) or negative ( red squares ). b Individual delta-Cq values for 20 primer-selective PCR SNP assays between positive and negative gDNA samples for ABI and Roche PCR mixes on two different PCR machines. The flags indicate means ± SD

    Journal: BMC Research Notes

    Article Title: The source of SYBR green master mix determines outcome of nucleic acid amplification reactions

    doi: 10.1186/s13104-016-2093-4

    Figure Lengend Snippet: Cq difference between positive and negative genomic DNAs obtained with different PCR mixes and machines. a Cq values of 20 primer-selective PCR SNP assays for gDNA samples that should be positive ( green dots ) or negative ( red squares ). b Individual delta-Cq values for 20 primer-selective PCR SNP assays between positive and negative gDNA samples for ABI and Roche PCR mixes on two different PCR machines. The flags indicate means ± SD

    Article Snippet: The mean ΔCq for the 20 assays between positive and negative genomic DNAs was higher with the ABI mix than with the Roche mix (Fig. b), but this difference was not significant.

    Techniques: Polymerase Chain Reaction