pcr fragments  (Roche)


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    Structured Review

    Roche pcr fragments
    The example of <t>HRM</t> analysis in patients 385 and 419 (exon 39). 1 Detection of mutations by HRM. The green curve in the diagram corresponds to the <t>PCR</t> fragment carrying the nonsense mutation c.11172G > A (p.Trp3724X) in patient 385. The red curve refers to the PCR fragment carrying the likely pathogenic substitution c.11248C > G (p.Arg3750Gly) in patient 419. The rest of fragments with blue curves are wild type samples (patients without sequence changes). 2, 3 Confirmation of mutations by direct sequencing in patient 385 (number 2) and patient 419 (number 3). Both sequences are in reverse direction. The letter A corresponds to the mutated sequence; B is the wild type.
    Pcr Fragments, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Novel mutations of PKD genes in the Czech population with autosomal dominant polycystic kidney disease"

    Article Title: Novel mutations of PKD genes in the Czech population with autosomal dominant polycystic kidney disease

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-15-41

    The example of HRM analysis in patients 385 and 419 (exon 39). 1 Detection of mutations by HRM. The green curve in the diagram corresponds to the PCR fragment carrying the nonsense mutation c.11172G > A (p.Trp3724X) in patient 385. The red curve refers to the PCR fragment carrying the likely pathogenic substitution c.11248C > G (p.Arg3750Gly) in patient 419. The rest of fragments with blue curves are wild type samples (patients without sequence changes). 2, 3 Confirmation of mutations by direct sequencing in patient 385 (number 2) and patient 419 (number 3). Both sequences are in reverse direction. The letter A corresponds to the mutated sequence; B is the wild type.
    Figure Legend Snippet: The example of HRM analysis in patients 385 and 419 (exon 39). 1 Detection of mutations by HRM. The green curve in the diagram corresponds to the PCR fragment carrying the nonsense mutation c.11172G > A (p.Trp3724X) in patient 385. The red curve refers to the PCR fragment carrying the likely pathogenic substitution c.11248C > G (p.Arg3750Gly) in patient 419. The rest of fragments with blue curves are wild type samples (patients without sequence changes). 2, 3 Confirmation of mutations by direct sequencing in patient 385 (number 2) and patient 419 (number 3). Both sequences are in reverse direction. The letter A corresponds to the mutated sequence; B is the wild type.

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Sequencing

    2) Product Images from "The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating γ-glutamyltranspeptidase (GGT)"

    Article Title: The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating γ-glutamyltranspeptidase (GGT)

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.00567

    RacR binds to the ggt promoter region as shown by electrophoretic mobility shift assays (EMSA) . DIG-labeled PCR fragments (~50 fmol) containing the ggt, phoX , or Cj0200c promoter regions were incubated with RacR as indicated. (A) Influence of the phosphorylation of RacR on the binding to the ggt promoter. RacR was phosphorylated by RacScyto in the presence of ATP. (B) EMSA of the ggt, phoX , and Cj0200c promoter regions with phosphorylated RacR protein. The phoX and Cj0200c promoter regions were used as negative controls. RacR-P, phosphorylated RacR.
    Figure Legend Snippet: RacR binds to the ggt promoter region as shown by electrophoretic mobility shift assays (EMSA) . DIG-labeled PCR fragments (~50 fmol) containing the ggt, phoX , or Cj0200c promoter regions were incubated with RacR as indicated. (A) Influence of the phosphorylation of RacR on the binding to the ggt promoter. RacR was phosphorylated by RacScyto in the presence of ATP. (B) EMSA of the ggt, phoX , and Cj0200c promoter regions with phosphorylated RacR protein. The phoX and Cj0200c promoter regions were used as negative controls. RacR-P, phosphorylated RacR.

    Techniques Used: Electrophoretic Mobility Shift Assay, Labeling, Polymerase Chain Reaction, Incubation, Binding Assay

    RacR binds to a specific region on the ggt promoter. (A) Nucleotide sequence and features of the ggt promoter. The start codon ATG is indicated in bold face, the putative -10 and -16 regions and ribosomal binding site (RBS) are underlined. A palindromic sequence is indicated with a horizontal bar. The previously identified RacR binding consensus sequence ( van der Stel et al., 2015 ) is indicated above the predicted RacR binding site, vertical lines indicate matching nucleotides. Arrows indicate the 5′ termini and direction of the primers used to generate the ggt promoter elements for the luciferase reporter plasmids and EMSA bait DNA. The transcriptional start site of the ggt gene identified by ( Dugar et al., 2013 ) is indicated with a hooked arrow. (B) Luciferase activities using different lengths of the region upstream of the ggt gene are determined in wt and racRS ::Cm mutant bacteria. Cultures were grown until late-log phase at oxygen limiting conditions with the addition of nitrate. Data represents the mean and SE of three independent experiments. (C) EMSA experiments using the different ggt promoter elements. DIG-labeled PCR fragments (~50 fmol) were mixed with or without 50 pmol RacR and 25 pmol RacScyto in the presence of ATP. RacR-P phosphorylated RacR.
    Figure Legend Snippet: RacR binds to a specific region on the ggt promoter. (A) Nucleotide sequence and features of the ggt promoter. The start codon ATG is indicated in bold face, the putative -10 and -16 regions and ribosomal binding site (RBS) are underlined. A palindromic sequence is indicated with a horizontal bar. The previously identified RacR binding consensus sequence ( van der Stel et al., 2015 ) is indicated above the predicted RacR binding site, vertical lines indicate matching nucleotides. Arrows indicate the 5′ termini and direction of the primers used to generate the ggt promoter elements for the luciferase reporter plasmids and EMSA bait DNA. The transcriptional start site of the ggt gene identified by ( Dugar et al., 2013 ) is indicated with a hooked arrow. (B) Luciferase activities using different lengths of the region upstream of the ggt gene are determined in wt and racRS ::Cm mutant bacteria. Cultures were grown until late-log phase at oxygen limiting conditions with the addition of nitrate. Data represents the mean and SE of three independent experiments. (C) EMSA experiments using the different ggt promoter elements. DIG-labeled PCR fragments (~50 fmol) were mixed with or without 50 pmol RacR and 25 pmol RacScyto in the presence of ATP. RacR-P phosphorylated RacR.

    Techniques Used: Sequencing, Binding Assay, Luciferase, Mutagenesis, Labeling, Polymerase Chain Reaction

    3) Product Images from "circLMTK2 acts as a sponge of miR-150-5p and promotes proliferation and metastasis in gastric cancer"

    Article Title: circLMTK2 acts as a sponge of miR-150-5p and promotes proliferation and metastasis in gastric cancer

    Journal: Molecular Cancer

    doi: 10.1186/s12943-019-1081-4

    Characteristics of the circLMTK2 in GC cells. ( a ) The genomic loci of the LMTK2 gene and circLMTK2. The expression of circLMTK2 was detected by qRT-PCR and was validated by Sanger sequencing. The arrows represent divergent primers that bind to the genomic region of circLMTK2. ( b ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA after treatment with RNase R. ( c ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA after treatment with actinomycin D at the indicated time points. ( d ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA in either the cytoplasm or the nucleus. ( e ) RNA fluorescence in situ hybridization (FISH) for circLMTK2. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm
    Figure Legend Snippet: Characteristics of the circLMTK2 in GC cells. ( a ) The genomic loci of the LMTK2 gene and circLMTK2. The expression of circLMTK2 was detected by qRT-PCR and was validated by Sanger sequencing. The arrows represent divergent primers that bind to the genomic region of circLMTK2. ( b ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA after treatment with RNase R. ( c ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA after treatment with actinomycin D at the indicated time points. ( d ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA in either the cytoplasm or the nucleus. ( e ) RNA fluorescence in situ hybridization (FISH) for circLMTK2. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm

    Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Staining

    4) Product Images from "A PCR-Based Method for RNA Probes and Applications in Neuroscience"

    Article Title: A PCR-Based Method for RNA Probes and Applications in Neuroscience

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00266

    Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro -transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml proteinase K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.
    Figure Legend Snippet: Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro -transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml proteinase K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, In Vitro, Staining, Expressing, Permeability

    5) Product Images from "XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima"

    Article Title: XX/XY System of Sex Determination in the Geophilomorph Centipede Strigamia maritima

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150292

    Identification of the X chromosome in the Strigamia karyotype by FISH with a set of X-chromosome derived DNA probes. (A) The relative positions and sizes of the five PCR fragments, distributed on two different X-linked scaffolds, which were used to produce the X-probes. Two of the fragments are located on scf7180001248200 and three on scf7180001248049. The genomic distance between these scaffolds is not known. The black line depicts the scaffold. The numbers above the line indicate the number of bases, starting at 1 on the left hand side. Green boxes represent the length and relative position of the PCR products, numbered arbitrarily from 1 to 5. (B) A mitotic metaphase chromosome spread prepared from a single embryo. Hybridization signals of the X-probes identify a middle-sized element in the Strigamia karyotype as the X chromosome. As there are two chromosomes with the X-probe signals, we infer that this chromosome spread is derived from a female embryo (XX). (C) Two Strigamia karyotypes constructed from the mitotic metaphases of embryonic cells. They are derived from different embryos. Upper panel: karyotype derived from the female metaphase shown in (B). Lower panel: karyotype derived from an inverted image of a DAPI-stained metaphase of unknown sex. It is the same as that shown in Fig 1A . We infer that the pair of sex chromosomes represents the 4 th pair of chromosomes by size (asterisks). (D, E, F) Meiotic chromosome spreads, prepared from sub-adult male testes. (D) Late zygotene complement showing a clump of incompletely paired bivalents. The X-probes label the longer chromosome of a partially paired bivalent, as schematically illustrated in (D’). We thus infer that this is the X chromosome, and that the other shorter chromosome, without hybridization signals, is the Y chromosome. The X and Y chromosomes are only paired at the distal part of the X chromosome, with a large proximal part unpaired. (E) A particularly clear and well-spread XY bivalent at a similar stage to (D). It shows hybridization signals of X-probes on the unpaired proximal part of the X chromosome, while the Y chromosome is completely paired except for the DAPI-highlighted centromere (see schematic drawing below the XY bivalent). (F) Pachytene complement showing 8 bivalents, each with DAPI-highlighted centromeric chromatin. X-probe hybridization signals are visible on the unpaired segment of the longer chromosome, near the centromere (see schematic drawing on the right-hand side). The X and Y chromosomes now appear almost equal in length in the bivalent. Scale bar is equal to 5 μm in (B) and 10 μm in (D, E, F). Chromosomes were counterstained with DAPI (blue). Arrowheads indicate hybridization signals of the digoxigenin-labelled X-probes (green); arrows indicate a pair of the largest chromosomes (B) or the largest bivalent (F).
    Figure Legend Snippet: Identification of the X chromosome in the Strigamia karyotype by FISH with a set of X-chromosome derived DNA probes. (A) The relative positions and sizes of the five PCR fragments, distributed on two different X-linked scaffolds, which were used to produce the X-probes. Two of the fragments are located on scf7180001248200 and three on scf7180001248049. The genomic distance between these scaffolds is not known. The black line depicts the scaffold. The numbers above the line indicate the number of bases, starting at 1 on the left hand side. Green boxes represent the length and relative position of the PCR products, numbered arbitrarily from 1 to 5. (B) A mitotic metaphase chromosome spread prepared from a single embryo. Hybridization signals of the X-probes identify a middle-sized element in the Strigamia karyotype as the X chromosome. As there are two chromosomes with the X-probe signals, we infer that this chromosome spread is derived from a female embryo (XX). (C) Two Strigamia karyotypes constructed from the mitotic metaphases of embryonic cells. They are derived from different embryos. Upper panel: karyotype derived from the female metaphase shown in (B). Lower panel: karyotype derived from an inverted image of a DAPI-stained metaphase of unknown sex. It is the same as that shown in Fig 1A . We infer that the pair of sex chromosomes represents the 4 th pair of chromosomes by size (asterisks). (D, E, F) Meiotic chromosome spreads, prepared from sub-adult male testes. (D) Late zygotene complement showing a clump of incompletely paired bivalents. The X-probes label the longer chromosome of a partially paired bivalent, as schematically illustrated in (D’). We thus infer that this is the X chromosome, and that the other shorter chromosome, without hybridization signals, is the Y chromosome. The X and Y chromosomes are only paired at the distal part of the X chromosome, with a large proximal part unpaired. (E) A particularly clear and well-spread XY bivalent at a similar stage to (D). It shows hybridization signals of X-probes on the unpaired proximal part of the X chromosome, while the Y chromosome is completely paired except for the DAPI-highlighted centromere (see schematic drawing below the XY bivalent). (F) Pachytene complement showing 8 bivalents, each with DAPI-highlighted centromeric chromatin. X-probe hybridization signals are visible on the unpaired segment of the longer chromosome, near the centromere (see schematic drawing on the right-hand side). The X and Y chromosomes now appear almost equal in length in the bivalent. Scale bar is equal to 5 μm in (B) and 10 μm in (D, E, F). Chromosomes were counterstained with DAPI (blue). Arrowheads indicate hybridization signals of the digoxigenin-labelled X-probes (green); arrows indicate a pair of the largest chromosomes (B) or the largest bivalent (F).

    Techniques Used: Fluorescence In Situ Hybridization, Derivative Assay, Polymerase Chain Reaction, Hybridization, Construct, Staining

    6) Product Images from "Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus"

    Article Title: Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54766-y

    Gdf11 -IRES-GFP expression is primarily detected within T and B lymphocytes of the peripheral blood. ( A ) Representative flow cytometry analysis of GFP expression within CD3 + T cells, CD19 + B cells, CD11b + /Ly6G − monocytes and CD11b + /Ly6G + neutrophils from peripheral blood. ( B ) Quantification of GFP+ T cells, B cells, monocytes and neutrophils in 2 month old mice from lines 1B, 11 and 12 and WT controls. N = 3–8 males and 3–8 females per genotype. Circles: males. Triangles: Females. Individual data points overlaid with mean ± SD. ( C ) Real time PCR analysis of Gdf11 levels in CD19+ and CD19- splenic cells from young (2-month old) and aged (24-month old) mice. Hprt was used as a housekeeping gene. ( D,E ) Quantification of ( D ), GFP + peripheral blood T cells and ( E ), GFP + peripheral blood B cells within heterozygous mice from line 1B during aging. ( F,G ) Quantification of GFP mean fluorescence intensity within F , peripheral blood T cells and ( G ), peripheral blood B cells in heterozygous mice from line 1B during aging. Mean fluorescence intensity (MFI) values normalized to wild type mice for each timepoint. ( H ) Quantification of total T cell frequency (red) and B cell frequency (blue) out of live peripheral blood cells during aging. N = 25 males and 19 females. Data points represent mean with error bars denoting SEM.
    Figure Legend Snippet: Gdf11 -IRES-GFP expression is primarily detected within T and B lymphocytes of the peripheral blood. ( A ) Representative flow cytometry analysis of GFP expression within CD3 + T cells, CD19 + B cells, CD11b + /Ly6G − monocytes and CD11b + /Ly6G + neutrophils from peripheral blood. ( B ) Quantification of GFP+ T cells, B cells, monocytes and neutrophils in 2 month old mice from lines 1B, 11 and 12 and WT controls. N = 3–8 males and 3–8 females per genotype. Circles: males. Triangles: Females. Individual data points overlaid with mean ± SD. ( C ) Real time PCR analysis of Gdf11 levels in CD19+ and CD19- splenic cells from young (2-month old) and aged (24-month old) mice. Hprt was used as a housekeeping gene. ( D,E ) Quantification of ( D ), GFP + peripheral blood T cells and ( E ), GFP + peripheral blood B cells within heterozygous mice from line 1B during aging. ( F,G ) Quantification of GFP mean fluorescence intensity within F , peripheral blood T cells and ( G ), peripheral blood B cells in heterozygous mice from line 1B during aging. Mean fluorescence intensity (MFI) values normalized to wild type mice for each timepoint. ( H ) Quantification of total T cell frequency (red) and B cell frequency (blue) out of live peripheral blood cells during aging. N = 25 males and 19 females. Data points represent mean with error bars denoting SEM.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Mouse Assay, Real-time Polymerase Chain Reaction, Fluorescence

    Generation of Gdf11 -IRES-GFP knock-in reporter mice using CRISPR/Cas9. ( A) Schematic of Gdf11 -IRES-GFP targeting to the Gdf11 locus. Blue underlined text indicates the protospacer adjacent motif (PAM) for sgRNA3. Red text indicates the target sequence for sgRNA3. Black arrowhead indicates the predicted cut site for sgRNA3. Primers used for PCR-based screening are designated as A, B, C, D, and E above each allele, and predicted amplicon sizes are listed beneath each allele. The location of NcoI restriction sites and Southern blot probe sequences are indicated in red and blue text, respectively. HA-L: Left homology arm. HA-R: Right homology arm. ( B ) PCR screening of 5 founder mice from Round #1 of injections using primer pair A–C. Expected size: WT = 3.1 kb; KI = 4.3 kb. Gel image is uncropped with the entirety of the captured image shown. ( C) Chromatogram illustrating sequence of boundaries between top: left homology arm (HA-L) and IRES-GFP, and bottom: IRES-GFP and right homology arm (HA-R). ( D) PCR screening of 36 founder mice from Rounds #3 and #4 of using primer pair B-C. Expected size: WT = 0.8 kb; KI = 2.1 kb; *Non-specific band. Green boxes indicate founder animals harboring the Gdf11 -IRES-GFP knock-in allele. Red boxes indicate founder animals harboring large deletions in Gdf11 . Gel image is uncropped. Positive and negative control reactions for PCR amplification were run on a separate gel, which is presented in Supplementary Fig. 12A .
    Figure Legend Snippet: Generation of Gdf11 -IRES-GFP knock-in reporter mice using CRISPR/Cas9. ( A) Schematic of Gdf11 -IRES-GFP targeting to the Gdf11 locus. Blue underlined text indicates the protospacer adjacent motif (PAM) for sgRNA3. Red text indicates the target sequence for sgRNA3. Black arrowhead indicates the predicted cut site for sgRNA3. Primers used for PCR-based screening are designated as A, B, C, D, and E above each allele, and predicted amplicon sizes are listed beneath each allele. The location of NcoI restriction sites and Southern blot probe sequences are indicated in red and blue text, respectively. HA-L: Left homology arm. HA-R: Right homology arm. ( B ) PCR screening of 5 founder mice from Round #1 of injections using primer pair A–C. Expected size: WT = 3.1 kb; KI = 4.3 kb. Gel image is uncropped with the entirety of the captured image shown. ( C) Chromatogram illustrating sequence of boundaries between top: left homology arm (HA-L) and IRES-GFP, and bottom: IRES-GFP and right homology arm (HA-R). ( D) PCR screening of 36 founder mice from Rounds #3 and #4 of using primer pair B-C. Expected size: WT = 0.8 kb; KI = 2.1 kb; *Non-specific band. Green boxes indicate founder animals harboring the Gdf11 -IRES-GFP knock-in allele. Red boxes indicate founder animals harboring large deletions in Gdf11 . Gel image is uncropped. Positive and negative control reactions for PCR amplification were run on a separate gel, which is presented in Supplementary Fig. 12A .

    Techniques Used: Knock-In, Mouse Assay, CRISPR, Sequencing, Polymerase Chain Reaction, Amplification, Southern Blot, Negative Control

    Mice homozygous for Gdf11 deletion allele 7 ( Gdf11 WE ) lack skeletal phenotypes seen in Gdf11 loss-of-function mutants and are viable with no substantial alterations in body parameters. ( A ) Images of E18.5 embryos from line 7. Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE embryos all exhibit an external tail. ( B ) Skeletal analysis of Gdf11 WE / + and Gdf11 WE/WE embryos stained with Alcian Blue (to mark cartilage) and Alizarin Red (to mark bone). Numbers indicate thoracic vertebrae. hl: hindlimb. Scale bar: 1 mm. ( C ) Skeletal preparations of skulls from Gdf11 WE/+ and Gdf11 WE/WE embryos stained with Alcian Blue and Alizarin Red. oc: otic capsule. White arrow denotes oc. Inset depicts isolated oc. Scale bar: 1 mm. ( D ) Representative images of lumbar vertebrae within Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE embryos. Arrows denote L5 and L6/S1 vertebrae. ( E ) Quantification of the number of cervical, thoracic and lumbar vertebrae among the genotypes indicated. ( F ) PCR analysis of Gdf11 locus in Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE weanlings using primer pair B-C. NTC: no template control. Gel image was cropped to focus on the amplicons. Full length gel is presented in Supplementary Fig. 12D . ( G – J ) Quantification of ( G ), Body weight, ( H ), Normalized heart weight (relative to tibia length), I , Raw spleen weight, and ( J ), Normalized muscle weight (relative to tibia length) in 5–8 month old mice. N = 5–8 males (blue) and N = 7–14 females (red) per genotype. ( K,L ) Quantification of ( K ), raw and ( L ), normalized grip strength measurements (relative to body weight) in 5–8 month old mice. N = 5–8 males (blue) and N = 7–14 females (red) per genotype. Each data point represents the average of two technical replicates. ( M,N ) Quantification of ( M ) GDF11 serum levels and N , GDF8 serum levels in 6–8-week old mice. N = 8–11 males (blue) and N = 8–14 females (red) per genotype. Individual data points overlaid with mean ± SD.
    Figure Legend Snippet: Mice homozygous for Gdf11 deletion allele 7 ( Gdf11 WE ) lack skeletal phenotypes seen in Gdf11 loss-of-function mutants and are viable with no substantial alterations in body parameters. ( A ) Images of E18.5 embryos from line 7. Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE embryos all exhibit an external tail. ( B ) Skeletal analysis of Gdf11 WE / + and Gdf11 WE/WE embryos stained with Alcian Blue (to mark cartilage) and Alizarin Red (to mark bone). Numbers indicate thoracic vertebrae. hl: hindlimb. Scale bar: 1 mm. ( C ) Skeletal preparations of skulls from Gdf11 WE/+ and Gdf11 WE/WE embryos stained with Alcian Blue and Alizarin Red. oc: otic capsule. White arrow denotes oc. Inset depicts isolated oc. Scale bar: 1 mm. ( D ) Representative images of lumbar vertebrae within Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE embryos. Arrows denote L5 and L6/S1 vertebrae. ( E ) Quantification of the number of cervical, thoracic and lumbar vertebrae among the genotypes indicated. ( F ) PCR analysis of Gdf11 locus in Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE weanlings using primer pair B-C. NTC: no template control. Gel image was cropped to focus on the amplicons. Full length gel is presented in Supplementary Fig. 12D . ( G – J ) Quantification of ( G ), Body weight, ( H ), Normalized heart weight (relative to tibia length), I , Raw spleen weight, and ( J ), Normalized muscle weight (relative to tibia length) in 5–8 month old mice. N = 5–8 males (blue) and N = 7–14 females (red) per genotype. ( K,L ) Quantification of ( K ), raw and ( L ), normalized grip strength measurements (relative to body weight) in 5–8 month old mice. N = 5–8 males (blue) and N = 7–14 females (red) per genotype. Each data point represents the average of two technical replicates. ( M,N ) Quantification of ( M ) GDF11 serum levels and N , GDF8 serum levels in 6–8-week old mice. N = 8–11 males (blue) and N = 8–14 females (red) per genotype. Individual data points overlaid with mean ± SD.

    Techniques Used: Mouse Assay, Staining, Isolation, Polymerase Chain Reaction

    Validation of Gdf11 -IRES-GFP knock-in reporter mouse lines. ( A , B ) Southern blot analysis of ( A ), Gdf11- IRES-GFP targeted founder mice and ( B ), Gdf11- IRES-GFP F1 progeny. Nco1-digested genomic DNA was hybridized with the internal probe. Expected fragment size: WT = n/a; T (targeted) = 3.5 kb. AI: Additional integration. Blot images were cropped to focus on the target bands. Uncropped blots are presented in Supplementary Fig. 12B,C . ( C ) TLA sequencing coverage and analysis plots from line 1B using outward facing primers residing in the GFP transgene. ( D ) Flow cytometry analysis of GFP expression in live (7AAD − ) peripheral blood cells in left : mice exhibiting correct targeting (lines 1B, 11 and 12) and right: mice exhibiting incorrect targeting (lines 1A and 13). ( E,F ) Real time PCR analysis of Gdf11 levels in FACS-purified GFP high and GFP low splenocytes from line 1B using ( E ), primers spanning exons 1-2 and ( F ), primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in GFP low splenocytes. N = 4 males (blue), 4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( G,H ) Real time PCR analysis of Gdf11 levels in whole spleen from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. Relative Gdf11 expression levels were assayed using ( G ), primers spanning exons 1–2 and ( H) , primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in C57BL/6J mice. N = 3–4 males (blue), 3–4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( I,J ), Quantification of ( I ), GDF11 protein levels, and ( J) , GDF8 protein levels, in serum from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. ( K,L ) Whole mount in situ hybridization for Gdf11 (top) and Gfp (bottom) in E10.5 Gdf11 +/+ and Gdf11 KI/+ embryos from line 1B. For each embryo, the right-most images show the dissected forelimb. mb: midbrain, fb: forebrain, psm: pre-somitic mesoderm, fl: forelimb, hl: hindlimb, s: somite. Scale bar: 0.5 mm.
    Figure Legend Snippet: Validation of Gdf11 -IRES-GFP knock-in reporter mouse lines. ( A , B ) Southern blot analysis of ( A ), Gdf11- IRES-GFP targeted founder mice and ( B ), Gdf11- IRES-GFP F1 progeny. Nco1-digested genomic DNA was hybridized with the internal probe. Expected fragment size: WT = n/a; T (targeted) = 3.5 kb. AI: Additional integration. Blot images were cropped to focus on the target bands. Uncropped blots are presented in Supplementary Fig. 12B,C . ( C ) TLA sequencing coverage and analysis plots from line 1B using outward facing primers residing in the GFP transgene. ( D ) Flow cytometry analysis of GFP expression in live (7AAD − ) peripheral blood cells in left : mice exhibiting correct targeting (lines 1B, 11 and 12) and right: mice exhibiting incorrect targeting (lines 1A and 13). ( E,F ) Real time PCR analysis of Gdf11 levels in FACS-purified GFP high and GFP low splenocytes from line 1B using ( E ), primers spanning exons 1-2 and ( F ), primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in GFP low splenocytes. N = 4 males (blue), 4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( G,H ) Real time PCR analysis of Gdf11 levels in whole spleen from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. Relative Gdf11 expression levels were assayed using ( G ), primers spanning exons 1–2 and ( H) , primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in C57BL/6J mice. N = 3–4 males (blue), 3–4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( I,J ), Quantification of ( I ), GDF11 protein levels, and ( J) , GDF8 protein levels, in serum from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. ( K,L ) Whole mount in situ hybridization for Gdf11 (top) and Gfp (bottom) in E10.5 Gdf11 +/+ and Gdf11 KI/+ embryos from line 1B. For each embryo, the right-most images show the dissected forelimb. mb: midbrain, fb: forebrain, psm: pre-somitic mesoderm, fl: forelimb, hl: hindlimb, s: somite. Scale bar: 0.5 mm.

    Techniques Used: Knock-In, Southern Blot, Mouse Assay, Sequencing, Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction, FACS, Purification, In Situ Hybridization

    7) Product Images from "Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos"

    Article Title: Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068374

    Pertubation of HDAC activity disrupts the cell cycle in HeLa cells. ( A ) The efficiency of Sirt1 and Sirt2 depletion in HeLa cells following treatment with siRNAs targeting Sirt1 and/or Sirt2 was evaluated by semi-quantitative PCR. Luciferase siRNA was used as a negative control. Tubulin was used as a loading control. ( B ) Western blot analysis of H3K56ac in total histone extracts, chromatin, and non-chromatin fractions of HeLa cells after the indicated treatments. The purity of histone extracts and protein composition of chromatin and non-chromatin fractions were evaluated by Coomassie-stained gels. Histone H3 was used as a loading control for the total histone extract and chromatin fractions. Tubulin was used as a loading control for the non-chromatin fractions. ( C ) The bar graphs show the relative percentage of cells in G1, S, and G2/M phase analysed by flow cytometry after various treatments. ( D ) Western blotting of total HeLa cell extracts following the indicated treatments. Tubulin was used as a loading control. In all cases, the means of three independent biological replicates are shown; error bars represent S.E.M Significant differences were evaluated using a Student’s t -test. ** indicates P
    Figure Legend Snippet: Pertubation of HDAC activity disrupts the cell cycle in HeLa cells. ( A ) The efficiency of Sirt1 and Sirt2 depletion in HeLa cells following treatment with siRNAs targeting Sirt1 and/or Sirt2 was evaluated by semi-quantitative PCR. Luciferase siRNA was used as a negative control. Tubulin was used as a loading control. ( B ) Western blot analysis of H3K56ac in total histone extracts, chromatin, and non-chromatin fractions of HeLa cells after the indicated treatments. The purity of histone extracts and protein composition of chromatin and non-chromatin fractions were evaluated by Coomassie-stained gels. Histone H3 was used as a loading control for the total histone extract and chromatin fractions. Tubulin was used as a loading control for the non-chromatin fractions. ( C ) The bar graphs show the relative percentage of cells in G1, S, and G2/M phase analysed by flow cytometry after various treatments. ( D ) Western blotting of total HeLa cell extracts following the indicated treatments. Tubulin was used as a loading control. In all cases, the means of three independent biological replicates are shown; error bars represent S.E.M Significant differences were evaluated using a Student’s t -test. ** indicates P

    Techniques Used: Activity Assay, Real-time Polymerase Chain Reaction, Luciferase, Negative Control, Western Blot, Staining, Flow Cytometry, Cytometry

    8) Product Images from "Cas9 fusions for precision in vivo editing"

    Article Title: Cas9 fusions for precision in vivo editing

    Journal: bioRxiv

    doi: 10.1101/2020.07.15.199620

    CtIP fusion and HMEJ donor yield high-performance knockin. (A) Schematic outlining FACS isolation of distinct cell populations following gene editing with Cas9 WT -CtIP and HMEJ donor, genotyping PCR of the target genomic DNA (gDNA) locus, and Sanger sequencing of the amplified fragment pool. (B) Alignment of Sanger sequencing reads for wild-type, knockout, and knockin populations to the reference template (top). (C) Editing outcomes (indels, knockin frequency) were quantified by decomposition of Sanger sequencing reads using the ICE algorithm and plotted in histograms binned by indel size.
    Figure Legend Snippet: CtIP fusion and HMEJ donor yield high-performance knockin. (A) Schematic outlining FACS isolation of distinct cell populations following gene editing with Cas9 WT -CtIP and HMEJ donor, genotyping PCR of the target genomic DNA (gDNA) locus, and Sanger sequencing of the amplified fragment pool. (B) Alignment of Sanger sequencing reads for wild-type, knockout, and knockin populations to the reference template (top). (C) Editing outcomes (indels, knockin frequency) were quantified by decomposition of Sanger sequencing reads using the ICE algorithm and plotted in histograms binned by indel size.

    Techniques Used: Knock-In, FACS, Isolation, Polymerase Chain Reaction, Sequencing, Amplification, Knock-Out

    9) Product Images from "A PCR-Based Method for RNA Probes and Applications in Neuroscience"

    Article Title: A PCR-Based Method for RNA Probes and Applications in Neuroscience

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00266

    Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro -transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml proteinase K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.
    Figure Legend Snippet: Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro -transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml proteinase K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, In Vitro, Staining, Expressing, Permeability

    10) Product Images from "Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs"

    Article Title: Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs

    Journal: Nature Communications

    doi: 10.1038/ncomms11215

    Characterization of circHIPK3 RNA in human cells. ( a ) The genomic loci of five circRNAs in HIPK3 gene. The supported unique reads were presented. The expression of circHIPK3 was validated by RT–PCR followed by sanger sequencing. Arrows represent divergent primers binding to the genome region of circHIPK3. ( b ) Absolute quantification for circHIPK3 and HIPK3 mRNA in six human normal tissues. ( c ) qRT–PCR for the abundance of circHIPK3 and HIPK3 mRNA in HeLa cells treated with Actinomycin D at the indicated time points. ( d ) qRT–PCR for the abundance of circHIPK3 and HIPK3 mRNA in HeLa cells treated with RNase R. The amount of circHIPK3 and HIPK3 mRNA were normalized to the value measured in the mock treatment. ( e ) qRT–PCR data indicating the abundance of circHIPK3 and HIPK3 mRNA in either the cytoplasm or nucleus of HeLa cells. The amounts of circHIPK3 and HIPK3 mRNA were normalized to the value measured in the cytoplasm. Data in ( c – e ) are the means±s.e.m. of three experiments. ( f ) RNA fluorescence in situ hybridization for circHIPK3. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm.
    Figure Legend Snippet: Characterization of circHIPK3 RNA in human cells. ( a ) The genomic loci of five circRNAs in HIPK3 gene. The supported unique reads were presented. The expression of circHIPK3 was validated by RT–PCR followed by sanger sequencing. Arrows represent divergent primers binding to the genome region of circHIPK3. ( b ) Absolute quantification for circHIPK3 and HIPK3 mRNA in six human normal tissues. ( c ) qRT–PCR for the abundance of circHIPK3 and HIPK3 mRNA in HeLa cells treated with Actinomycin D at the indicated time points. ( d ) qRT–PCR for the abundance of circHIPK3 and HIPK3 mRNA in HeLa cells treated with RNase R. The amount of circHIPK3 and HIPK3 mRNA were normalized to the value measured in the mock treatment. ( e ) qRT–PCR data indicating the abundance of circHIPK3 and HIPK3 mRNA in either the cytoplasm or nucleus of HeLa cells. The amounts of circHIPK3 and HIPK3 mRNA were normalized to the value measured in the cytoplasm. Data in ( c – e ) are the means±s.e.m. of three experiments. ( f ) RNA fluorescence in situ hybridization for circHIPK3. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Binding Assay, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Staining

    Silencing of circHIPK3 RNA inhibits human cell proliferation. ( a ) Schematic illustration showing three targeted siRNAs. Si-HIPK3 targets the HIPK3 linear transcript, si-circHIPK3 targets the back-splice junction of circHIPK3, and si-both targets both the linear and circular species. ( b ) qRT–PCR for circHIPK3 and HIPK3 mRNA in HEK-293 T cells treated with three siRNAs as described above. Data are the means±s.e.m. of three experiments. ( c – e ) Proliferation of HuH-7, HCT-116 and HeLa cells transfected with the above three siRNAs assessed using a CCK-8 kit at the indicated days. Data in c – e are the means±s.e.m. of three experiments. ( f ) DNA synthesis assessed using an EdU (5-ethynyl-2'-deoxyuridine) assay in HuH-7 cells transfected with the above three siRNAs for 48 h. Cells were fluorescently stained with EdU (red). Nuclei were stained with DAPI (blue). Micrographs represent at least three experiments. Scale bar, 200 μm. ( g ) Quantitative EdU assay data from f and Supplementary Fig. 7 . Data in d – g are the means±s.e.m. of three experiments. * P
    Figure Legend Snippet: Silencing of circHIPK3 RNA inhibits human cell proliferation. ( a ) Schematic illustration showing three targeted siRNAs. Si-HIPK3 targets the HIPK3 linear transcript, si-circHIPK3 targets the back-splice junction of circHIPK3, and si-both targets both the linear and circular species. ( b ) qRT–PCR for circHIPK3 and HIPK3 mRNA in HEK-293 T cells treated with three siRNAs as described above. Data are the means±s.e.m. of three experiments. ( c – e ) Proliferation of HuH-7, HCT-116 and HeLa cells transfected with the above three siRNAs assessed using a CCK-8 kit at the indicated days. Data in c – e are the means±s.e.m. of three experiments. ( f ) DNA synthesis assessed using an EdU (5-ethynyl-2'-deoxyuridine) assay in HuH-7 cells transfected with the above three siRNAs for 48 h. Cells were fluorescently stained with EdU (red). Nuclei were stained with DAPI (blue). Micrographs represent at least three experiments. Scale bar, 200 μm. ( g ) Quantitative EdU assay data from f and Supplementary Fig. 7 . Data in d – g are the means±s.e.m. of three experiments. * P

    Techniques Used: Quantitative RT-PCR, Transfection, CCK-8 Assay, DNA Synthesis, Staining, EdU Assay

    circHIPK3 sponges with miR-124 and inhibits its activity. ( a ) Proliferation assessed using a CCK-8 kit in HEK-293 T cells transfected with nine miRNA mimics or control RNA (20 nM). ( b ) qRT–PCR analysis of circHIPK3 level in the streptavidin captured fractions from the HEK-293 T cell lysates after transfection with 3′-end biotinylated miR-124 or control RNA (NC). ( c ) Co-localization between miR-124 and circHIPK3 was observed (arrowheads) by RNA in situ hybridization in HeLa cells after co-transfection with circHIPK3 and miR-124 expressing vectors. Nuclei were stained with DAPI. Scale bar, 5μm. ( d ) qRT–PCR analysis of IL6R and DLX2 expression in HEK-293 T cells after transfected with si-cHIPK3, miR-124 mimics or miR-124 with circHIPK3 expressing vector (p-cHIPK3). ( e ) Proliferation assessed using a CCK-8 kit in cells transfected with circHIPK3 or miR-124 (10 nM) as indicated. Data in a , b , d are the means±s.e.m. of three experiments. ( f ) qRT–PCR for the abundance of circHIPK3 relative to ACTB and miR-124 relative RNU6B in six human normal tissues. The correlation between circHIPK3 and miR-124 is also shown. * P
    Figure Legend Snippet: circHIPK3 sponges with miR-124 and inhibits its activity. ( a ) Proliferation assessed using a CCK-8 kit in HEK-293 T cells transfected with nine miRNA mimics or control RNA (20 nM). ( b ) qRT–PCR analysis of circHIPK3 level in the streptavidin captured fractions from the HEK-293 T cell lysates after transfection with 3′-end biotinylated miR-124 or control RNA (NC). ( c ) Co-localization between miR-124 and circHIPK3 was observed (arrowheads) by RNA in situ hybridization in HeLa cells after co-transfection with circHIPK3 and miR-124 expressing vectors. Nuclei were stained with DAPI. Scale bar, 5μm. ( d ) qRT–PCR analysis of IL6R and DLX2 expression in HEK-293 T cells after transfected with si-cHIPK3, miR-124 mimics or miR-124 with circHIPK3 expressing vector (p-cHIPK3). ( e ) Proliferation assessed using a CCK-8 kit in cells transfected with circHIPK3 or miR-124 (10 nM) as indicated. Data in a , b , d are the means±s.e.m. of three experiments. ( f ) qRT–PCR for the abundance of circHIPK3 relative to ACTB and miR-124 relative RNU6B in six human normal tissues. The correlation between circHIPK3 and miR-124 is also shown. * P

    Techniques Used: Activity Assay, CCK-8 Assay, Transfection, Quantitative RT-PCR, RNA In Situ Hybridization, Cotransfection, Expressing, Staining, Plasmid Preparation

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    Amplification:

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    In Vitro:

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    Article Snippet: .. For in vitro transcription, the reaction solution with a final volume of 10 μl was prepared with the following components: 2–5 ng purified PCR fragments (template DNA), 1 μl DIG-RNA-labeling mixture (#11175025910, Roche, Germany), 0.25 μl RNase inhibitor (EO0382, Thermo Scientific, USA), 1 μl 0.1 M DTT (#1305658, Invitrogen, USA), and 1 μl T7 RNA polymerase (P2075, Promega, USA). .. The reaction was incubated at 37°C for 2 h. Extra template DNA was removed by digesting the products with 2 U RNAse-free DNAse I (M6101, Promega, USA) at 37°C for 30 min.

    Nick Translation:

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    Article Snippet: .. PCR fragments were labelled by digoxigenin-11-dUTP (Roche) using the Nick Translation Kit (Abbott Molecular). .. The 25 μL labelling reaction contained 500 ng of purified PCR product, 1x nick translation buffer, 250 μM dATP, dCTP, and dGTP, 90 μM dTTP, 160 μM digoxigenin-11-dUTP and 5 μL of nick translation enzyme mix.

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    Article Title: Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos
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    Polymerase Chain Reaction:

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    Roche hbv dna pcr fragment
    ( a ) Schematic representation of the intramolecular ligation technique to obtain the preCore and YMDD Pol motif close to each other. ( 1 ) The circle in a continuous line represents the complete <t>HBV</t> genome (3200 bp). The semicircles in discontinuous lines represent the first (first <t>PCR,</t> 2700 bp) and the nested (2194 bp for genotype A2 sequences and 2155 bp for genotype D sequences) PCR products. ( 2 ) The product obtained after intramolecular ligation and primers used to sequence the circular molecule ( Table 2 ) are indicated in the figure. ( b ) Agarose gel obtained after intramolecular digestion. Two markers (M-I and M-II) were loaded in the gel; band lengths are shown at the left side of the image. Two samples (S1 and S2) were loaded and four bands were detected; band lengths are indicated at the right side of the image. A band with an apparent size of 1700 bp was the circular molecule, with the preCore and YMDD Pol motif close to each other. Band B was the nested PCR product without ligation, and band D were dimers of nested PCR products without ligation. Band C corresponded to the ligated dimers. B , C and D bands were not used, because the main interest was to analyze regions from the same genome. ( c ) Sequence of the circular construct including the preCore region, YMDD polymerase motif (in box), and HindIII sequence. PreCore (1, 2, 14, 15, 28 and 29) and Core ( 1 ) codons analyzed in the text are also enclosed in boxes.
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    Roche pcr fragments
    The example of <t>HRM</t> analysis in patients 385 and 419 (exon 39). 1 Detection of mutations by HRM. The green curve in the diagram corresponds to the <t>PCR</t> fragment carrying the nonsense mutation c.11172G > A (p.Trp3724X) in patient 385. The red curve refers to the PCR fragment carrying the likely pathogenic substitution c.11248C > G (p.Arg3750Gly) in patient 419. The rest of fragments with blue curves are wild type samples (patients without sequence changes). 2, 3 Confirmation of mutations by direct sequencing in patient 385 (number 2) and patient 419 (number 3). Both sequences are in reverse direction. The letter A corresponds to the mutated sequence; B is the wild type.
    Pcr Fragments, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( a ) Schematic representation of the intramolecular ligation technique to obtain the preCore and YMDD Pol motif close to each other. ( 1 ) The circle in a continuous line represents the complete HBV genome (3200 bp). The semicircles in discontinuous lines represent the first (first PCR, 2700 bp) and the nested (2194 bp for genotype A2 sequences and 2155 bp for genotype D sequences) PCR products. ( 2 ) The product obtained after intramolecular ligation and primers used to sequence the circular molecule ( Table 2 ) are indicated in the figure. ( b ) Agarose gel obtained after intramolecular digestion. Two markers (M-I and M-II) were loaded in the gel; band lengths are shown at the left side of the image. Two samples (S1 and S2) were loaded and four bands were detected; band lengths are indicated at the right side of the image. A band with an apparent size of 1700 bp was the circular molecule, with the preCore and YMDD Pol motif close to each other. Band B was the nested PCR product without ligation, and band D were dimers of nested PCR products without ligation. Band C corresponded to the ligated dimers. B , C and D bands were not used, because the main interest was to analyze regions from the same genome. ( c ) Sequence of the circular construct including the preCore region, YMDD polymerase motif (in box), and HindIII sequence. PreCore (1, 2, 14, 15, 28 and 29) and Core ( 1 ) codons analyzed in the text are also enclosed in boxes.

    Journal: Nucleic Acids Research

    Article Title: Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome

    doi: 10.1093/nar/gkr451

    Figure Lengend Snippet: ( a ) Schematic representation of the intramolecular ligation technique to obtain the preCore and YMDD Pol motif close to each other. ( 1 ) The circle in a continuous line represents the complete HBV genome (3200 bp). The semicircles in discontinuous lines represent the first (first PCR, 2700 bp) and the nested (2194 bp for genotype A2 sequences and 2155 bp for genotype D sequences) PCR products. ( 2 ) The product obtained after intramolecular ligation and primers used to sequence the circular molecule ( Table 2 ) are indicated in the figure. ( b ) Agarose gel obtained after intramolecular digestion. Two markers (M-I and M-II) were loaded in the gel; band lengths are shown at the left side of the image. Two samples (S1 and S2) were loaded and four bands were detected; band lengths are indicated at the right side of the image. A band with an apparent size of 1700 bp was the circular molecule, with the preCore and YMDD Pol motif close to each other. Band B was the nested PCR product without ligation, and band D were dimers of nested PCR products without ligation. Band C corresponded to the ligated dimers. B , C and D bands were not used, because the main interest was to analyze regions from the same genome. ( c ) Sequence of the circular construct including the preCore region, YMDD polymerase motif (in box), and HindIII sequence. PreCore (1, 2, 14, 15, 28 and 29) and Core ( 1 ) codons analyzed in the text are also enclosed in boxes.

    Article Snippet: Optimal results were obtained with 0.5 ng/µ1 of the HBV DNA-PCR fragment, and 1 U of T4 DNA ligase (Roche Diagnostics GmbH).

    Techniques: Ligation, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Nested PCR, Construct

    The example of HRM analysis in patients 385 and 419 (exon 39). 1 Detection of mutations by HRM. The green curve in the diagram corresponds to the PCR fragment carrying the nonsense mutation c.11172G > A (p.Trp3724X) in patient 385. The red curve refers to the PCR fragment carrying the likely pathogenic substitution c.11248C > G (p.Arg3750Gly) in patient 419. The rest of fragments with blue curves are wild type samples (patients without sequence changes). 2, 3 Confirmation of mutations by direct sequencing in patient 385 (number 2) and patient 419 (number 3). Both sequences are in reverse direction. The letter A corresponds to the mutated sequence; B is the wild type.

    Journal: BMC Medical Genetics

    Article Title: Novel mutations of PKD genes in the Czech population with autosomal dominant polycystic kidney disease

    doi: 10.1186/1471-2350-15-41

    Figure Lengend Snippet: The example of HRM analysis in patients 385 and 419 (exon 39). 1 Detection of mutations by HRM. The green curve in the diagram corresponds to the PCR fragment carrying the nonsense mutation c.11172G > A (p.Trp3724X) in patient 385. The red curve refers to the PCR fragment carrying the likely pathogenic substitution c.11248C > G (p.Arg3750Gly) in patient 419. The rest of fragments with blue curves are wild type samples (patients without sequence changes). 2, 3 Confirmation of mutations by direct sequencing in patient 385 (number 2) and patient 419 (number 3). Both sequences are in reverse direction. The letter A corresponds to the mutated sequence; B is the wild type.

    Article Snippet: PCR fragments were produced and analyzed by HRM using the LightCycler® 480 (Roche Applied Science).

    Techniques: Polymerase Chain Reaction, Mutagenesis, Sequencing

    RacR binds to the ggt promoter region as shown by electrophoretic mobility shift assays (EMSA) . DIG-labeled PCR fragments (~50 fmol) containing the ggt, phoX , or Cj0200c promoter regions were incubated with RacR as indicated. (A) Influence of the phosphorylation of RacR on the binding to the ggt promoter. RacR was phosphorylated by RacScyto in the presence of ATP. (B) EMSA of the ggt, phoX , and Cj0200c promoter regions with phosphorylated RacR protein. The phoX and Cj0200c promoter regions were used as negative controls. RacR-P, phosphorylated RacR.

    Journal: Frontiers in Microbiology

    Article Title: The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating γ-glutamyltranspeptidase (GGT)

    doi: 10.3389/fmicb.2015.00567

    Figure Lengend Snippet: RacR binds to the ggt promoter region as shown by electrophoretic mobility shift assays (EMSA) . DIG-labeled PCR fragments (~50 fmol) containing the ggt, phoX , or Cj0200c promoter regions were incubated with RacR as indicated. (A) Influence of the phosphorylation of RacR on the binding to the ggt promoter. RacR was phosphorylated by RacScyto in the presence of ATP. (B) EMSA of the ggt, phoX , and Cj0200c promoter regions with phosphorylated RacR protein. The phoX and Cj0200c promoter regions were used as negative controls. RacR-P, phosphorylated RacR.

    Article Snippet: After electrophoresis the DNA was blotted on a hybond-N+ membrane (Amersham) and PCR fragments were visualized using α-DIG-AP, Fab fragments, and CSPD substrate (Roche).

    Techniques: Electrophoretic Mobility Shift Assay, Labeling, Polymerase Chain Reaction, Incubation, Binding Assay

    RacR binds to a specific region on the ggt promoter. (A) Nucleotide sequence and features of the ggt promoter. The start codon ATG is indicated in bold face, the putative -10 and -16 regions and ribosomal binding site (RBS) are underlined. A palindromic sequence is indicated with a horizontal bar. The previously identified RacR binding consensus sequence ( van der Stel et al., 2015 ) is indicated above the predicted RacR binding site, vertical lines indicate matching nucleotides. Arrows indicate the 5′ termini and direction of the primers used to generate the ggt promoter elements for the luciferase reporter plasmids and EMSA bait DNA. The transcriptional start site of the ggt gene identified by ( Dugar et al., 2013 ) is indicated with a hooked arrow. (B) Luciferase activities using different lengths of the region upstream of the ggt gene are determined in wt and racRS ::Cm mutant bacteria. Cultures were grown until late-log phase at oxygen limiting conditions with the addition of nitrate. Data represents the mean and SE of three independent experiments. (C) EMSA experiments using the different ggt promoter elements. DIG-labeled PCR fragments (~50 fmol) were mixed with or without 50 pmol RacR and 25 pmol RacScyto in the presence of ATP. RacR-P phosphorylated RacR.

    Journal: Frontiers in Microbiology

    Article Title: The Campylobacter jejuni RacRS two-component system activates the glutamate synthesis by directly upregulating γ-glutamyltranspeptidase (GGT)

    doi: 10.3389/fmicb.2015.00567

    Figure Lengend Snippet: RacR binds to a specific region on the ggt promoter. (A) Nucleotide sequence and features of the ggt promoter. The start codon ATG is indicated in bold face, the putative -10 and -16 regions and ribosomal binding site (RBS) are underlined. A palindromic sequence is indicated with a horizontal bar. The previously identified RacR binding consensus sequence ( van der Stel et al., 2015 ) is indicated above the predicted RacR binding site, vertical lines indicate matching nucleotides. Arrows indicate the 5′ termini and direction of the primers used to generate the ggt promoter elements for the luciferase reporter plasmids and EMSA bait DNA. The transcriptional start site of the ggt gene identified by ( Dugar et al., 2013 ) is indicated with a hooked arrow. (B) Luciferase activities using different lengths of the region upstream of the ggt gene are determined in wt and racRS ::Cm mutant bacteria. Cultures were grown until late-log phase at oxygen limiting conditions with the addition of nitrate. Data represents the mean and SE of three independent experiments. (C) EMSA experiments using the different ggt promoter elements. DIG-labeled PCR fragments (~50 fmol) were mixed with or without 50 pmol RacR and 25 pmol RacScyto in the presence of ATP. RacR-P phosphorylated RacR.

    Article Snippet: After electrophoresis the DNA was blotted on a hybond-N+ membrane (Amersham) and PCR fragments were visualized using α-DIG-AP, Fab fragments, and CSPD substrate (Roche).

    Techniques: Sequencing, Binding Assay, Luciferase, Mutagenesis, Labeling, Polymerase Chain Reaction

    Characteristics of the circLMTK2 in GC cells. ( a ) The genomic loci of the LMTK2 gene and circLMTK2. The expression of circLMTK2 was detected by qRT-PCR and was validated by Sanger sequencing. The arrows represent divergent primers that bind to the genomic region of circLMTK2. ( b ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA after treatment with RNase R. ( c ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA after treatment with actinomycin D at the indicated time points. ( d ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA in either the cytoplasm or the nucleus. ( e ) RNA fluorescence in situ hybridization (FISH) for circLMTK2. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm

    Journal: Molecular Cancer

    Article Title: circLMTK2 acts as a sponge of miR-150-5p and promotes proliferation and metastasis in gastric cancer

    doi: 10.1186/s12943-019-1081-4

    Figure Lengend Snippet: Characteristics of the circLMTK2 in GC cells. ( a ) The genomic loci of the LMTK2 gene and circLMTK2. The expression of circLMTK2 was detected by qRT-PCR and was validated by Sanger sequencing. The arrows represent divergent primers that bind to the genomic region of circLMTK2. ( b ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA after treatment with RNase R. ( c ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA after treatment with actinomycin D at the indicated time points. ( d ) qRT-PCR analysis of circLMTK2 and LMTK2 mRNA in either the cytoplasm or the nucleus. ( e ) RNA fluorescence in situ hybridization (FISH) for circLMTK2. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm

    Article Snippet: Digoxin or biotin-labelled RNA probes were transcribed from PCR fragments using the DIG or biotin RNA labelling mix and T7 RNA polymerase (Roche) according to the manufacturers’ instructions.

    Techniques: Expressing, Quantitative RT-PCR, Sequencing, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Staining