hot firepol dna polymerase  (Solis BioDyne)


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    Solis BioDyne hot firepol dna polymerase
    The secondary structure of the vrn - A1 promoter may prevent successful amplification and sequencing. ( a ) Schematic representation of vrn - A1 promoter variants with 137 bp, 181 bp and 194 bp deletions found in winter cultivars and the position of the G-quadruplex (G4). ( b ) Sequence motif of G4 found in Triple Dirk C (TDC, MH347747). ( c ) <t>DNA</t> fold prediction of G4 found in TDC.
    Hot Firepol Dna Polymerase, supplied by Solis BioDyne, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hot firepol dna polymerase - by Bioz Stars, 2022-05
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    Images

    1) Product Images from "In-Depth Sequence Analysis of Bread Wheat VRN1 Genes"

    Article Title: In-Depth Sequence Analysis of Bread Wheat VRN1 Genes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms222212284

    The secondary structure of the vrn - A1 promoter may prevent successful amplification and sequencing. ( a ) Schematic representation of vrn - A1 promoter variants with 137 bp, 181 bp and 194 bp deletions found in winter cultivars and the position of the G-quadruplex (G4). ( b ) Sequence motif of G4 found in Triple Dirk C (TDC, MH347747). ( c ) DNA fold prediction of G4 found in TDC.
    Figure Legend Snippet: The secondary structure of the vrn - A1 promoter may prevent successful amplification and sequencing. ( a ) Schematic representation of vrn - A1 promoter variants with 137 bp, 181 bp and 194 bp deletions found in winter cultivars and the position of the G-quadruplex (G4). ( b ) Sequence motif of G4 found in Triple Dirk C (TDC, MH347747). ( c ) DNA fold prediction of G4 found in TDC.

    Techniques Used: Amplification, Sequencing

    2) Product Images from "TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo"

    Article Title: TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039141

    TYK2 K923E is enzymatically inactive and generation of Tyk2 K923E mice. A. The in vitro kinase activity assay was performed in a TYK2-deficient cell line transiently transfected with plasmids encoding GFP, wild-type TYK2 or kinase-inactive TYK2 K923E . TYK2 and TYK2 K923E proteins were immunoprecipitated from cell extracts and subjected to an in vitro kinase assay using GST-IFNAR cyt as an exogenous substrate (left panel). TYK2 was immunoprecipitated from whole cell extracts and Western Blot analysis performed to detect phosphorylated TYK2 (pTyk2, upper right panel) or TYK2 protein (lower right panel). B. Scheme of the murine Tyk2 locus from exons 9-24 (black boxes). The point mutations introduced in exon 20 resulting in the amino acid exchange K > E and the introduction of the BspTI restriction endonuclease site are depicted. The neomycin resistance cassette ( neo r , white box) flanked by loxP sites (black triangles) was inserted into the intron sequence between exons 21 and 22. The lower scheme shows the targeted locus with the restriction sites important for Southern blot analysis. Note that after germline transmission the neo r cassette was excised to leave a single loxP site in the mutated allele. C. Southern blot analysis using a non-radioactively labelled 471 bp neo r probe verified correct targeting and lack of heterologous integration in the ES cell clone 1, whereas two other clones (2 and 3) were not correctly targeted. D. DNA from WT (+/+), heterozygous (+/m) or homozygous Tyk2 K923E (m/m) mouse tails was used to amplify a 710 bp fragment with primers surrounding exon 20. The amplicons were digested with BspTI resulting in a 498 bp and a 212 bp fragment only in the Tyk2 K923E alleles. E. Conventional genotyping of mouse tails results in a 678 bp fragment corresponding to the WT and a 778 bp fragment specific for Tyk2 K923E .
    Figure Legend Snippet: TYK2 K923E is enzymatically inactive and generation of Tyk2 K923E mice. A. The in vitro kinase activity assay was performed in a TYK2-deficient cell line transiently transfected with plasmids encoding GFP, wild-type TYK2 or kinase-inactive TYK2 K923E . TYK2 and TYK2 K923E proteins were immunoprecipitated from cell extracts and subjected to an in vitro kinase assay using GST-IFNAR cyt as an exogenous substrate (left panel). TYK2 was immunoprecipitated from whole cell extracts and Western Blot analysis performed to detect phosphorylated TYK2 (pTyk2, upper right panel) or TYK2 protein (lower right panel). B. Scheme of the murine Tyk2 locus from exons 9-24 (black boxes). The point mutations introduced in exon 20 resulting in the amino acid exchange K > E and the introduction of the BspTI restriction endonuclease site are depicted. The neomycin resistance cassette ( neo r , white box) flanked by loxP sites (black triangles) was inserted into the intron sequence between exons 21 and 22. The lower scheme shows the targeted locus with the restriction sites important for Southern blot analysis. Note that after germline transmission the neo r cassette was excised to leave a single loxP site in the mutated allele. C. Southern blot analysis using a non-radioactively labelled 471 bp neo r probe verified correct targeting and lack of heterologous integration in the ES cell clone 1, whereas two other clones (2 and 3) were not correctly targeted. D. DNA from WT (+/+), heterozygous (+/m) or homozygous Tyk2 K923E (m/m) mouse tails was used to amplify a 710 bp fragment with primers surrounding exon 20. The amplicons were digested with BspTI resulting in a 498 bp and a 212 bp fragment only in the Tyk2 K923E alleles. E. Conventional genotyping of mouse tails results in a 678 bp fragment corresponding to the WT and a 778 bp fragment specific for Tyk2 K923E .

    Techniques Used: Mouse Assay, In Vitro, Kinase Assay, Transfection, Immunoprecipitation, Western Blot, Sequencing, Southern Blot, Transmission Assay, Clone Assay, Genotyping Assay

    3) Product Images from "Age-related profiling of DNA methylation in CD8+ T cells reveals changes in immune response and transcriptional regulator genes"

    Article Title: Age-related profiling of DNA methylation in CD8+ T cells reveals changes in immune response and transcriptional regulator genes

    Journal: Scientific Reports

    doi: 10.1038/srep13107

    Examples of genes with known functional role in CD8+ T cells that display inverse correlation between gene expression and DNA methylation. ( A ) LGALS1 , ( B ) IFNG , ( C ) CCR7 and ( D ) SATB1 gene. Each panel is composed of three sections. The top sections display the different transcripts of the gene, marked according to the expression level fold change between younger and older individuals. The middle sections show the associated CpG sites that are differentially methylated. The scatter plots in the bottom sections illustrate the correspondence between expression and methylation levels for each site separately. The scatter plots are displayed in the same order as the sites in the middle section; the red and blue dots display levels detected in the younger and older individuals, respectively.
    Figure Legend Snippet: Examples of genes with known functional role in CD8+ T cells that display inverse correlation between gene expression and DNA methylation. ( A ) LGALS1 , ( B ) IFNG , ( C ) CCR7 and ( D ) SATB1 gene. Each panel is composed of three sections. The top sections display the different transcripts of the gene, marked according to the expression level fold change between younger and older individuals. The middle sections show the associated CpG sites that are differentially methylated. The scatter plots in the bottom sections illustrate the correspondence between expression and methylation levels for each site separately. The scatter plots are displayed in the same order as the sites in the middle section; the red and blue dots display levels detected in the younger and older individuals, respectively.

    Techniques Used: Functional Assay, Expressing, DNA Methylation Assay, Methylation

    4) Product Images from "Pharmacological or TRIB3-Mediated Suppression of ATF4 Transcriptional Activity Promotes Hepatoma Cell Resistance to Proteasome Inhibitor Bortezomib"

    Article Title: Pharmacological or TRIB3-Mediated Suppression of ATF4 Transcriptional Activity Promotes Hepatoma Cell Resistance to Proteasome Inhibitor Bortezomib

    Journal: Cancers

    doi: 10.3390/cancers13102341

    TRIB3 and ATF4 binding near genes that are regulated in response to bortezomib (Btz), nelfinavir (Nelf) and ISRIB in HepG2 cells. ( A ) Gene ontology enrichment analysis for genes with a TRIB3 ChIP-Seq peak within 2 kb of the transcription start site (TSS). The GO Biological Process database was used. ( B ) RNA-Seq differential expression results overlap with genes harboring TRIB3 ChIP-Seq peaks (TSS ± 2 kb). ISRIB-downregulated genes were obtained comparing ISRIB and Btz co-treatment to Btz alone. ( C ) Btz-upregulated genes with TSS-proximal (TSS ± 2 kb) TRIB3 ChIP-Seq peaks. Genes are ranked by the TRIB3 Btz ChIP-Seq signal strength. ( D ) ChIP-Seq coverage plots for selected loci. The αFlag neg. cells track represents ChIP-Seq of unedited HepG2 cells with anti-Flag antibody. ( E ) ChIP-qPCR validation for the binding of ATF4, TRIB3 (anti-Flag M2) and non-targeting IgG at selected loci detected in ChIP-Seq. A negative control locus is also shown (-Ctrl). The means ± SD of 2–5 ChIP experiments from two biological replicates are shown. The DNA quantity is presented as a percentage of input chromatin.
    Figure Legend Snippet: TRIB3 and ATF4 binding near genes that are regulated in response to bortezomib (Btz), nelfinavir (Nelf) and ISRIB in HepG2 cells. ( A ) Gene ontology enrichment analysis for genes with a TRIB3 ChIP-Seq peak within 2 kb of the transcription start site (TSS). The GO Biological Process database was used. ( B ) RNA-Seq differential expression results overlap with genes harboring TRIB3 ChIP-Seq peaks (TSS ± 2 kb). ISRIB-downregulated genes were obtained comparing ISRIB and Btz co-treatment to Btz alone. ( C ) Btz-upregulated genes with TSS-proximal (TSS ± 2 kb) TRIB3 ChIP-Seq peaks. Genes are ranked by the TRIB3 Btz ChIP-Seq signal strength. ( D ) ChIP-Seq coverage plots for selected loci. The αFlag neg. cells track represents ChIP-Seq of unedited HepG2 cells with anti-Flag antibody. ( E ) ChIP-qPCR validation for the binding of ATF4, TRIB3 (anti-Flag M2) and non-targeting IgG at selected loci detected in ChIP-Seq. A negative control locus is also shown (-Ctrl). The means ± SD of 2–5 ChIP experiments from two biological replicates are shown. The DNA quantity is presented as a percentage of input chromatin.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, Negative Control

    5) Product Images from "Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays"

    Article Title: Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn357

    Generation of specific products using multiplex PCR. ( A ) Product quantity is visible as duplicates after phase 1 PCR, sampled in two cycle increments (cycles 16–24). Representative images include marker (M) and gel band intensities corresponding to multiplex PCR products. DNA was visualized using the Agilent DNA 1000 LabChip after Exonuclease I treatment to remove primers. ( B ) The product band after universal primer amplification and column purification.
    Figure Legend Snippet: Generation of specific products using multiplex PCR. ( A ) Product quantity is visible as duplicates after phase 1 PCR, sampled in two cycle increments (cycles 16–24). Representative images include marker (M) and gel band intensities corresponding to multiplex PCR products. DNA was visualized using the Agilent DNA 1000 LabChip after Exonuclease I treatment to remove primers. ( B ) The product band after universal primer amplification and column purification.

    Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Marker, Amplification, Purification

    6) Product Images from "Annexin A5 Promoter Haplotype M2 Is Not a Risk Factor for Recurrent Pregnancy Loss in Northern Europe"

    Article Title: Annexin A5 Promoter Haplotype M2 Is Not a Risk Factor for Recurrent Pregnancy Loss in Northern Europe

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131606

    Detection of SNP profile and haplotype distribution within the ANXA5 promoter region in Estonia and Denmark. (A) Genomic context of the ANXA5 gene. Chromosomal positions are based on hg19. (B) Location of identified single nucleotide polymorphisms (SNPs) within the resequenced region of ANXA5 core promoter. SNP positions are given according to the initially reported [ 10 ] first transcription start site (arrow) of the non-conserved untranslated first exon (blue box). Common SNPs are denoted with black and rare singleton SNPs with white triangles. SNP nomenclature according to dbSNP database ( http://www.ncbi.nlm.nih.gov/SNP/ ) is the following: -19G/A, rs112782763; 1A/C, rs28717001; 27T/C, rs28651243; 76G/A, rs113588187. In the current human genome assembly ( http://www.ensembl.org/ ), the determined transcription start site is shifted for all annotated ANXA5 transcripts and only rs113588187 is located within the transcribed region. (C) Linkage disequilibrium (r 2 ) between pairs of SNPs within the resequenced region of the ANXA5 promoter. The order of SNPs is given according to the direction of transcription. Black box indicates complete LD between a pair of SNPs. (D) Distribution of haplotypes identified within the core promoter of Estonian subjects based on haplotype reconstruction analysis with all common SNPs (n = 5) or four common SNPs within the LD block. Haplotype phasing of either five or four SNPs yielded identical results. (E) Detection and observed prevalence of M2 haplotype by Restriction Fragment Length Polymorphism (RFLP) analysis targeting the M2 tag-SNP 76G/A (rs113588187). In case of a GG homozygote at position 76, RFLP analysis results in two fragments (188 bp and 106 bp), three fragments are detected in subjects heterozygous for the M2 haplotype (294 bp, 188 bp and 106 bp), whereas one uncut fragment is observed in homozygous carriers of M2 haplotype (294 bp). Minor allele of the 76G/A tag-SNP defining the M2 haplotype is denoted in red. M, molecular weight marker, 100 bp DNA Ladder (Solid Biodyne). PCR, uncut PCR product not subjected to RFLP analysis. N, number of subjects. *Fisher’s exact P -value.
    Figure Legend Snippet: Detection of SNP profile and haplotype distribution within the ANXA5 promoter region in Estonia and Denmark. (A) Genomic context of the ANXA5 gene. Chromosomal positions are based on hg19. (B) Location of identified single nucleotide polymorphisms (SNPs) within the resequenced region of ANXA5 core promoter. SNP positions are given according to the initially reported [ 10 ] first transcription start site (arrow) of the non-conserved untranslated first exon (blue box). Common SNPs are denoted with black and rare singleton SNPs with white triangles. SNP nomenclature according to dbSNP database ( http://www.ncbi.nlm.nih.gov/SNP/ ) is the following: -19G/A, rs112782763; 1A/C, rs28717001; 27T/C, rs28651243; 76G/A, rs113588187. In the current human genome assembly ( http://www.ensembl.org/ ), the determined transcription start site is shifted for all annotated ANXA5 transcripts and only rs113588187 is located within the transcribed region. (C) Linkage disequilibrium (r 2 ) between pairs of SNPs within the resequenced region of the ANXA5 promoter. The order of SNPs is given according to the direction of transcription. Black box indicates complete LD between a pair of SNPs. (D) Distribution of haplotypes identified within the core promoter of Estonian subjects based on haplotype reconstruction analysis with all common SNPs (n = 5) or four common SNPs within the LD block. Haplotype phasing of either five or four SNPs yielded identical results. (E) Detection and observed prevalence of M2 haplotype by Restriction Fragment Length Polymorphism (RFLP) analysis targeting the M2 tag-SNP 76G/A (rs113588187). In case of a GG homozygote at position 76, RFLP analysis results in two fragments (188 bp and 106 bp), three fragments are detected in subjects heterozygous for the M2 haplotype (294 bp, 188 bp and 106 bp), whereas one uncut fragment is observed in homozygous carriers of M2 haplotype (294 bp). Minor allele of the 76G/A tag-SNP defining the M2 haplotype is denoted in red. M, molecular weight marker, 100 bp DNA Ladder (Solid Biodyne). PCR, uncut PCR product not subjected to RFLP analysis. N, number of subjects. *Fisher’s exact P -value.

    Techniques Used: Blocking Assay, Molecular Weight, Marker, Polymerase Chain Reaction

    7) Product Images from "Wnt pathway antagonists, SFRP1, SFRP2, SOX17, and PPP2R2B, are methylated in gliomas and SFRP1 methylation predicts shorter survival"

    Article Title: Wnt pathway antagonists, SFRP1, SFRP2, SOX17, and PPP2R2B, are methylated in gliomas and SFRP1 methylation predicts shorter survival

    Journal: Journal of Applied Genetics

    doi: 10.1007/s13353-015-0312-7

    The visualization of the most important results and representative electropherograms presenting the method of the analysis. a Mean methylation index in different age groups with 95 % confidence intervals. b Representative methylation-specific polymerase chain reaction (MSP) electropherograms of SFRP1 and SFRP2 promoter methylation analysis. For each gene, the upper part represents the reaction with primers specific for methylated sequence, whereas the lower part represents the reaction with primers binding to unmethylated sequence. M completely methylated human genomic DNA used as positive control; WBC white blood cells used as negative control; Bl blank control; T tumor. c The frequency of Wnt antagonists’ promoter methylation. d The relationship between promoter methylation of SFRP1 and patients’ age
    Figure Legend Snippet: The visualization of the most important results and representative electropherograms presenting the method of the analysis. a Mean methylation index in different age groups with 95 % confidence intervals. b Representative methylation-specific polymerase chain reaction (MSP) electropherograms of SFRP1 and SFRP2 promoter methylation analysis. For each gene, the upper part represents the reaction with primers specific for methylated sequence, whereas the lower part represents the reaction with primers binding to unmethylated sequence. M completely methylated human genomic DNA used as positive control; WBC white blood cells used as negative control; Bl blank control; T tumor. c The frequency of Wnt antagonists’ promoter methylation. d The relationship between promoter methylation of SFRP1 and patients’ age

    Techniques Used: Methylation, Polymerase Chain Reaction, Sequencing, Binding Assay, Positive Control, Negative Control

    8) Product Images from "A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR"

    Article Title: A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-02535-1

    ( a ) Schematic illustration of the fundamental principle of plasmonic photothermal light-to-heat conversion for droplet-based photonic PCR (dpPCR): when a light is turned on, fast heating of sample droplets can be achieved by fast heat diffusion of hot electrons throughout the AuNF. When a light is turned off, the heat dissipation through the AuNF can occur, leading to the cooling of the heated droplets. After thermal cycling, the success of the amplification can be identified by analyzing the fluorescence intensity of droplets. ( b ) Circuit diagram of the plasmonic photothermal cycler. ( c ) An enlarged section of ( b ), indicating the position of the LED, heatsink, chips, and thermocouple.
    Figure Legend Snippet: ( a ) Schematic illustration of the fundamental principle of plasmonic photothermal light-to-heat conversion for droplet-based photonic PCR (dpPCR): when a light is turned on, fast heating of sample droplets can be achieved by fast heat diffusion of hot electrons throughout the AuNF. When a light is turned off, the heat dissipation through the AuNF can occur, leading to the cooling of the heated droplets. After thermal cycling, the success of the amplification can be identified by analyzing the fluorescence intensity of droplets. ( b ) Circuit diagram of the plasmonic photothermal cycler. ( c ) An enlarged section of ( b ), indicating the position of the LED, heatsink, chips, and thermocouple.

    Techniques Used: Polymerase Chain Reaction, Diffusion-based Assay, Amplification, Fluorescence

    9) Product Images from "Annexin A5 Promoter Haplotype M2 Is Not a Risk Factor for Recurrent Pregnancy Loss in Northern Europe"

    Article Title: Annexin A5 Promoter Haplotype M2 Is Not a Risk Factor for Recurrent Pregnancy Loss in Northern Europe

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131606

    Detection of SNP profile and haplotype distribution within the ANXA5 promoter region in Estonia and Denmark. (A) Genomic context of the ANXA5 gene. Chromosomal positions are based on hg19. (B) Location of identified single nucleotide polymorphisms (SNPs) within the resequenced region of ANXA5 core promoter. SNP positions are given according to the initially reported [ 10 ] first transcription start site (arrow) of the non-conserved untranslated first exon (blue box). Common SNPs are denoted with black and rare singleton SNPs with white triangles. SNP nomenclature according to dbSNP database ( http://www.ncbi.nlm.nih.gov/SNP/ ) is the following: -19G/A, rs112782763; 1A/C, rs28717001; 27T/C, rs28651243; 76G/A, rs113588187. In the current human genome assembly ( http://www.ensembl.org/ ), the determined transcription start site is shifted for all annotated ANXA5 transcripts and only rs113588187 is located within the transcribed region. (C) Linkage disequilibrium (r 2 ) between pairs of SNPs within the resequenced region of the ANXA5 promoter. The order of SNPs is given according to the direction of transcription. Black box indicates complete LD between a pair of SNPs. (D) Distribution of haplotypes identified within the core promoter of Estonian subjects based on haplotype reconstruction analysis with all common SNPs (n = 5) or four common SNPs within the LD block. Haplotype phasing of either five or four SNPs yielded identical results. (E) Detection and observed prevalence of M2 haplotype by Restriction Fragment Length Polymorphism (RFLP) analysis targeting the M2 tag-SNP 76G/A (rs113588187). In case of a GG homozygote at position 76, RFLP analysis results in two fragments (188 bp and 106 bp), three fragments are detected in subjects heterozygous for the M2 haplotype (294 bp, 188 bp and 106 bp), whereas one uncut fragment is observed in homozygous carriers of M2 haplotype (294 bp). Minor allele of the 76G/A tag-SNP defining the M2 haplotype is denoted in red. M, molecular weight marker, 100 bp DNA Ladder (Solid Biodyne). PCR, uncut PCR product not subjected to RFLP analysis. N, number of subjects. *Fisher’s exact P -value.
    Figure Legend Snippet: Detection of SNP profile and haplotype distribution within the ANXA5 promoter region in Estonia and Denmark. (A) Genomic context of the ANXA5 gene. Chromosomal positions are based on hg19. (B) Location of identified single nucleotide polymorphisms (SNPs) within the resequenced region of ANXA5 core promoter. SNP positions are given according to the initially reported [ 10 ] first transcription start site (arrow) of the non-conserved untranslated first exon (blue box). Common SNPs are denoted with black and rare singleton SNPs with white triangles. SNP nomenclature according to dbSNP database ( http://www.ncbi.nlm.nih.gov/SNP/ ) is the following: -19G/A, rs112782763; 1A/C, rs28717001; 27T/C, rs28651243; 76G/A, rs113588187. In the current human genome assembly ( http://www.ensembl.org/ ), the determined transcription start site is shifted for all annotated ANXA5 transcripts and only rs113588187 is located within the transcribed region. (C) Linkage disequilibrium (r 2 ) between pairs of SNPs within the resequenced region of the ANXA5 promoter. The order of SNPs is given according to the direction of transcription. Black box indicates complete LD between a pair of SNPs. (D) Distribution of haplotypes identified within the core promoter of Estonian subjects based on haplotype reconstruction analysis with all common SNPs (n = 5) or four common SNPs within the LD block. Haplotype phasing of either five or four SNPs yielded identical results. (E) Detection and observed prevalence of M2 haplotype by Restriction Fragment Length Polymorphism (RFLP) analysis targeting the M2 tag-SNP 76G/A (rs113588187). In case of a GG homozygote at position 76, RFLP analysis results in two fragments (188 bp and 106 bp), three fragments are detected in subjects heterozygous for the M2 haplotype (294 bp, 188 bp and 106 bp), whereas one uncut fragment is observed in homozygous carriers of M2 haplotype (294 bp). Minor allele of the 76G/A tag-SNP defining the M2 haplotype is denoted in red. M, molecular weight marker, 100 bp DNA Ladder (Solid Biodyne). PCR, uncut PCR product not subjected to RFLP analysis. N, number of subjects. *Fisher’s exact P -value.

    Techniques Used: Blocking Assay, Molecular Weight, Marker, Polymerase Chain Reaction

    10) Product Images from "Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect"

    Article Title: Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

    Journal: Bioorganic & Medicinal Chemistry

    doi: 10.1016/j.bmc.2014.02.004

    Gene knockdown of endogenous bcl-2. (A) After lipofectamine 2000-mediated transfection, (B) after unassisted application. The effect of PEG-siRNA conjugates (siRNA-PEG (S), 1 / 6 ; siRNA-PEG2 (S/AS), 5 / 6 ) was evaluated by qPCR-based quantification of bcl-2 mRNA levels in MCF-7 cells. The indicated siRNAs were either transfected into the cells with lipofectamine 2000 (A), or applied without any uptake-enhancing agent (B). Cells were lysed after 24 h, total RNA was extracted, transcribed into cDNA, and quantified with qPCR. Bcl-2 levels were normalized to the reference gene RNA polymerase II, which was shown not to be influenced by siRNA or the transfection procedure. After lipofectamine-mediated delivery, efficient gene silencing occurred with both modified and unmodified siRNAs ( p
    Figure Legend Snippet: Gene knockdown of endogenous bcl-2. (A) After lipofectamine 2000-mediated transfection, (B) after unassisted application. The effect of PEG-siRNA conjugates (siRNA-PEG (S), 1 / 6 ; siRNA-PEG2 (S/AS), 5 / 6 ) was evaluated by qPCR-based quantification of bcl-2 mRNA levels in MCF-7 cells. The indicated siRNAs were either transfected into the cells with lipofectamine 2000 (A), or applied without any uptake-enhancing agent (B). Cells were lysed after 24 h, total RNA was extracted, transcribed into cDNA, and quantified with qPCR. Bcl-2 levels were normalized to the reference gene RNA polymerase II, which was shown not to be influenced by siRNA or the transfection procedure. After lipofectamine-mediated delivery, efficient gene silencing occurred with both modified and unmodified siRNAs ( p

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Modification

    11) Product Images from "Equine Parvovirus-Hepatitis Screening in Horses and Donkeys with Histopathologic Liver Abnormalities"

    Article Title: Equine Parvovirus-Hepatitis Screening in Horses and Donkeys with Histopathologic Liver Abnormalities

    Journal: Viruses

    doi: 10.3390/v13081599

    Detection of EqPV-H ( A ) and the cellular calibrator gene TTC17 ( B ) by dPCR in liver samples #1 and #2. Each panel represents a separate experimental chamber on a chip where the reaction mix containing DNA from liver samples (1:20 dilution for TTC17 ) was segregated into individual droplets and assessed for the presence of EqPV-H and TTC17 . • : positive samples; •: negative signals, respectively. The blue line designates the arbitrary fluorescence threshold separating the signals from background.
    Figure Legend Snippet: Detection of EqPV-H ( A ) and the cellular calibrator gene TTC17 ( B ) by dPCR in liver samples #1 and #2. Each panel represents a separate experimental chamber on a chip where the reaction mix containing DNA from liver samples (1:20 dilution for TTC17 ) was segregated into individual droplets and assessed for the presence of EqPV-H and TTC17 . • : positive samples; •: negative signals, respectively. The blue line designates the arbitrary fluorescence threshold separating the signals from background.

    Techniques Used: Digital PCR, Chromatin Immunoprecipitation, Fluorescence

    12) Product Images from "DNA-based identification of Calendula officinalis (Asteraceae) 1"

    Article Title: DNA-based identification of Calendula officinalis (Asteraceae) 1

    Journal: Applications in Plant Sciences

    doi: 10.3732/apps.1500069

    Analysis of artificial admixtures of Calendula officinalis in saffron. All properly amplified admixture samples showed an equivalent HRM curve like the C. officinalis references. (A) HRM analysis with the primer pair Cal_trnK_2F R. (B) Amplification plot of the qPCR corresponding to A. (C) HRM analysis with the primer pair Cal_trnL-F_1F R. (D) Amplification plot of the qPCR corresponding to B. %-Values mean proportion of C. officinalis DNA in saffron DNA of each sample. NTC = no template control.
    Figure Legend Snippet: Analysis of artificial admixtures of Calendula officinalis in saffron. All properly amplified admixture samples showed an equivalent HRM curve like the C. officinalis references. (A) HRM analysis with the primer pair Cal_trnK_2F R. (B) Amplification plot of the qPCR corresponding to A. (C) HRM analysis with the primer pair Cal_trnL-F_1F R. (D) Amplification plot of the qPCR corresponding to B. %-Values mean proportion of C. officinalis DNA in saffron DNA of each sample. NTC = no template control.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction

    13) Product Images from "Two are better than one: HPoxBS - hairpin oxidative bisulfite sequencing"

    Article Title: Two are better than one: HPoxBS - hairpin oxidative bisulfite sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky422

    HPoxBS pipeline. Individual steps of HPoxBS starting from DNA quality assessment to 5hmC prediction and enzyme efficiency estimation.
    Figure Legend Snippet: HPoxBS pipeline. Individual steps of HPoxBS starting from DNA quality assessment to 5hmC prediction and enzyme efficiency estimation.

    Techniques Used:

    Hairpin Methylation Pattern Maps. Methylation patterns for the single copy genes Afp, Ttc25 and Zim3, as well as the retrotransposable elements IAP, L1mdT, L1mdA, mSat and MuERVL for BS and oxBS of ECS cultivated under Serum/LIF (d0) and 2i medium (d1 = 24 h 2i, d3 = 72 h 2i, d6 = 144 h 2i). Each column represents one CpG dyad, each row one sequenced chromosome. The very left column gives the mean methylation pattern over all analysed CpGs. Red = CpG dyad is modified on both DNA strands (BS = 5mC or 5hmC; oxBS = 5mC only); Dark green = CpG dyad is only modified on the plus strand (BS = 5mC or 5hmC; oxBS = 5mC only); Light green = CpG dyad is only modified on the lower strand (BS = 5mC or 5hmC; oxBS = 5mC only); Blue = CpG dyad is unmodified on both strands (BS = C only; oxBS = C or 5hmC); White = CpG dyad was not analysable.
    Figure Legend Snippet: Hairpin Methylation Pattern Maps. Methylation patterns for the single copy genes Afp, Ttc25 and Zim3, as well as the retrotransposable elements IAP, L1mdT, L1mdA, mSat and MuERVL for BS and oxBS of ECS cultivated under Serum/LIF (d0) and 2i medium (d1 = 24 h 2i, d3 = 72 h 2i, d6 = 144 h 2i). Each column represents one CpG dyad, each row one sequenced chromosome. The very left column gives the mean methylation pattern over all analysed CpGs. Red = CpG dyad is modified on both DNA strands (BS = 5mC or 5hmC; oxBS = 5mC only); Dark green = CpG dyad is only modified on the plus strand (BS = 5mC or 5hmC; oxBS = 5mC only); Light green = CpG dyad is only modified on the lower strand (BS = 5mC or 5hmC; oxBS = 5mC only); Blue = CpG dyad is unmodified on both strands (BS = C only; oxBS = C or 5hmC); White = CpG dyad was not analysable.

    Techniques Used: Methylation, Modification

    Average modification level. Mean methylation level of BS (upper panel) and oxBS (middle panel) samples as well as the predicted 5hmC amount and distribution (lower panel). x-axis = days; y-axis = 5mC/5hmC level; red = CpG dyad is modified on both DNA strands (BS = 5mC or 5hmC; oxBS = 5mC only); dark green = CpG dyad is only modified on the plus strand (BS = 5mC or 5hmC; oxBS = 5mC only); light green = CpG dyad is only modified on the lower strand(BS = 5mC or 5hmC; oxBS = 5mC only); blue = CpG dyad is unmodified on both strands (BS = C only; oxBS = C or 5hmC).
    Figure Legend Snippet: Average modification level. Mean methylation level of BS (upper panel) and oxBS (middle panel) samples as well as the predicted 5hmC amount and distribution (lower panel). x-axis = days; y-axis = 5mC/5hmC level; red = CpG dyad is modified on both DNA strands (BS = 5mC or 5hmC; oxBS = 5mC only); dark green = CpG dyad is only modified on the plus strand (BS = 5mC or 5hmC; oxBS = 5mC only); light green = CpG dyad is only modified on the lower strand(BS = 5mC or 5hmC; oxBS = 5mC only); blue = CpG dyad is unmodified on both strands (BS = C only; oxBS = C or 5hmC).

    Techniques Used: Modification, Methylation

    14) Product Images from "Pharmacological or TRIB3-Mediated Suppression of ATF4 Transcriptional Activity Promotes Hepatoma Cell Resistance to Proteasome Inhibitor Bortezomib"

    Article Title: Pharmacological or TRIB3-Mediated Suppression of ATF4 Transcriptional Activity Promotes Hepatoma Cell Resistance to Proteasome Inhibitor Bortezomib

    Journal: Cancers

    doi: 10.3390/cancers13102341

    TRIB3 and ATF4 binding near genes that are regulated in response to bortezomib (Btz), nelfinavir (Nelf) and ISRIB in HepG2 cells. ( A ) Gene ontology enrichment analysis for genes with a TRIB3 ChIP-Seq peak within 2 kb of the transcription start site (TSS). The GO Biological Process database was used. ( B ) RNA-Seq differential expression results overlap with genes harboring TRIB3 ChIP-Seq peaks (TSS ± 2 kb). ISRIB-downregulated genes were obtained comparing ISRIB and Btz co-treatment to Btz alone. ( C ) Btz-upregulated genes with TSS-proximal (TSS ± 2 kb) TRIB3 ChIP-Seq peaks. Genes are ranked by the TRIB3 Btz ChIP-Seq signal strength. ( D ) ChIP-Seq coverage plots for selected loci. The αFlag neg. cells track represents ChIP-Seq of unedited HepG2 cells with anti-Flag antibody. ( E ) ChIP-qPCR validation for the binding of ATF4, TRIB3 (anti-Flag M2) and non-targeting IgG at selected loci detected in ChIP-Seq. A negative control locus is also shown (-Ctrl). The means ± SD of 2–5 ChIP experiments from two biological replicates are shown. The DNA quantity is presented as a percentage of input chromatin.
    Figure Legend Snippet: TRIB3 and ATF4 binding near genes that are regulated in response to bortezomib (Btz), nelfinavir (Nelf) and ISRIB in HepG2 cells. ( A ) Gene ontology enrichment analysis for genes with a TRIB3 ChIP-Seq peak within 2 kb of the transcription start site (TSS). The GO Biological Process database was used. ( B ) RNA-Seq differential expression results overlap with genes harboring TRIB3 ChIP-Seq peaks (TSS ± 2 kb). ISRIB-downregulated genes were obtained comparing ISRIB and Btz co-treatment to Btz alone. ( C ) Btz-upregulated genes with TSS-proximal (TSS ± 2 kb) TRIB3 ChIP-Seq peaks. Genes are ranked by the TRIB3 Btz ChIP-Seq signal strength. ( D ) ChIP-Seq coverage plots for selected loci. The αFlag neg. cells track represents ChIP-Seq of unedited HepG2 cells with anti-Flag antibody. ( E ) ChIP-qPCR validation for the binding of ATF4, TRIB3 (anti-Flag M2) and non-targeting IgG at selected loci detected in ChIP-Seq. A negative control locus is also shown (-Ctrl). The means ± SD of 2–5 ChIP experiments from two biological replicates are shown. The DNA quantity is presented as a percentage of input chromatin.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, Negative Control

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    Solis BioDyne hot firepol dna polymerase
    TYK2 K923E is enzymatically inactive and generation of Tyk2 K923E mice. A. The in vitro kinase activity assay was performed in a TYK2-deficient cell line transiently transfected with plasmids encoding GFP, wild-type TYK2 or kinase-inactive TYK2 K923E . TYK2 and TYK2 K923E proteins were immunoprecipitated from cell extracts and subjected to an in vitro kinase assay using GST-IFNAR cyt as an exogenous substrate (left panel). TYK2 was immunoprecipitated from whole cell extracts and Western Blot analysis performed to detect phosphorylated TYK2 (pTyk2, upper right panel) or TYK2 protein (lower right panel). B. Scheme of the murine Tyk2 locus from exons 9-24 (black boxes). The point mutations introduced in exon 20 resulting in the amino acid exchange K > E and the introduction of the BspTI restriction endonuclease site are depicted. The neomycin resistance cassette ( neo r , white box) flanked by loxP sites (black triangles) was inserted into the intron sequence between exons 21 and 22. The lower scheme shows the targeted locus with the restriction sites important for Southern blot analysis. Note that after germline transmission the neo r cassette was excised to leave a single loxP site in the mutated allele. C. Southern blot analysis using a non-radioactively labelled 471 bp neo r probe verified correct targeting and lack of heterologous integration in the ES cell clone 1, whereas two other clones (2 and 3) were not correctly targeted. D. <t>DNA</t> from WT (+/+), heterozygous (+/m) or homozygous Tyk2 K923E (m/m) mouse tails was used to amplify a 710 bp fragment with primers surrounding exon 20. The amplicons were digested with BspTI resulting in a 498 bp and a 212 bp fragment only in the Tyk2 K923E alleles. E. Conventional genotyping of mouse tails results in a 678 bp fragment corresponding to the WT and a 778 bp fragment specific for Tyk2 K923E .
    Hot Firepol Dna Polymerase, supplied by Solis BioDyne, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TYK2 K923E is enzymatically inactive and generation of Tyk2 K923E mice. A. The in vitro kinase activity assay was performed in a TYK2-deficient cell line transiently transfected with plasmids encoding GFP, wild-type TYK2 or kinase-inactive TYK2 K923E . TYK2 and TYK2 K923E proteins were immunoprecipitated from cell extracts and subjected to an in vitro kinase assay using GST-IFNAR cyt as an exogenous substrate (left panel). TYK2 was immunoprecipitated from whole cell extracts and Western Blot analysis performed to detect phosphorylated TYK2 (pTyk2, upper right panel) or TYK2 protein (lower right panel). B. Scheme of the murine Tyk2 locus from exons 9-24 (black boxes). The point mutations introduced in exon 20 resulting in the amino acid exchange K > E and the introduction of the BspTI restriction endonuclease site are depicted. The neomycin resistance cassette ( neo r , white box) flanked by loxP sites (black triangles) was inserted into the intron sequence between exons 21 and 22. The lower scheme shows the targeted locus with the restriction sites important for Southern blot analysis. Note that after germline transmission the neo r cassette was excised to leave a single loxP site in the mutated allele. C. Southern blot analysis using a non-radioactively labelled 471 bp neo r probe verified correct targeting and lack of heterologous integration in the ES cell clone 1, whereas two other clones (2 and 3) were not correctly targeted. D. DNA from WT (+/+), heterozygous (+/m) or homozygous Tyk2 K923E (m/m) mouse tails was used to amplify a 710 bp fragment with primers surrounding exon 20. The amplicons were digested with BspTI resulting in a 498 bp and a 212 bp fragment only in the Tyk2 K923E alleles. E. Conventional genotyping of mouse tails results in a 678 bp fragment corresponding to the WT and a 778 bp fragment specific for Tyk2 K923E .

    Journal: PLoS ONE

    Article Title: TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

    doi: 10.1371/journal.pone.0039141

    Figure Lengend Snippet: TYK2 K923E is enzymatically inactive and generation of Tyk2 K923E mice. A. The in vitro kinase activity assay was performed in a TYK2-deficient cell line transiently transfected with plasmids encoding GFP, wild-type TYK2 or kinase-inactive TYK2 K923E . TYK2 and TYK2 K923E proteins were immunoprecipitated from cell extracts and subjected to an in vitro kinase assay using GST-IFNAR cyt as an exogenous substrate (left panel). TYK2 was immunoprecipitated from whole cell extracts and Western Blot analysis performed to detect phosphorylated TYK2 (pTyk2, upper right panel) or TYK2 protein (lower right panel). B. Scheme of the murine Tyk2 locus from exons 9-24 (black boxes). The point mutations introduced in exon 20 resulting in the amino acid exchange K > E and the introduction of the BspTI restriction endonuclease site are depicted. The neomycin resistance cassette ( neo r , white box) flanked by loxP sites (black triangles) was inserted into the intron sequence between exons 21 and 22. The lower scheme shows the targeted locus with the restriction sites important for Southern blot analysis. Note that after germline transmission the neo r cassette was excised to leave a single loxP site in the mutated allele. C. Southern blot analysis using a non-radioactively labelled 471 bp neo r probe verified correct targeting and lack of heterologous integration in the ES cell clone 1, whereas two other clones (2 and 3) were not correctly targeted. D. DNA from WT (+/+), heterozygous (+/m) or homozygous Tyk2 K923E (m/m) mouse tails was used to amplify a 710 bp fragment with primers surrounding exon 20. The amplicons were digested with BspTI resulting in a 498 bp and a 212 bp fragment only in the Tyk2 K923E alleles. E. Conventional genotyping of mouse tails results in a 678 bp fragment corresponding to the WT and a 778 bp fragment specific for Tyk2 K923E .

    Article Snippet: Hot FIREPol® DNA Polymerase (Solis Biodyne, Tartu Estonia), EvaGreen® (Biotium Inc, Hayward CA USA) and dNTP Set (Fermentas ThermoScientific Austria) were used according to manufactureŕs instructions.

    Techniques: Mouse Assay, In Vitro, Kinase Assay, Transfection, Immunoprecipitation, Western Blot, Sequencing, Southern Blot, Transmission Assay, Clone Assay, Genotyping Assay

    The secondary structure of the vrn - A1 promoter may prevent successful amplification and sequencing. ( a ) Schematic representation of vrn - A1 promoter variants with 137 bp, 181 bp and 194 bp deletions found in winter cultivars and the position of the G-quadruplex (G4). ( b ) Sequence motif of G4 found in Triple Dirk C (TDC, MH347747). ( c ) DNA fold prediction of G4 found in TDC.

    Journal: International Journal of Molecular Sciences

    Article Title: In-Depth Sequence Analysis of Bread Wheat VRN1 Genes

    doi: 10.3390/ijms222212284

    Figure Lengend Snippet: The secondary structure of the vrn - A1 promoter may prevent successful amplification and sequencing. ( a ) Schematic representation of vrn - A1 promoter variants with 137 bp, 181 bp and 194 bp deletions found in winter cultivars and the position of the G-quadruplex (G4). ( b ) Sequence motif of G4 found in Triple Dirk C (TDC, MH347747). ( c ) DNA fold prediction of G4 found in TDC.

    Article Snippet: Long amplicons (from 6 to 11 kb) were amplified by PrimeSTAR GXL DNA Polymerase (Takara Bio, Kusatsu, Japan) and Expand Long Range, dNTPack (Roche, Basel, Switzerland), and short amplicons (from 600 bp to 3 kb) were amplified by HOT FIREPol DNA Polymerase (Solis BioDyne, Tartu, Estonia), all according to the manufacturer’s instructions.

    Techniques: Amplification, Sequencing

    Examples of genes with known functional role in CD8+ T cells that display inverse correlation between gene expression and DNA methylation. ( A ) LGALS1 , ( B ) IFNG , ( C ) CCR7 and ( D ) SATB1 gene. Each panel is composed of three sections. The top sections display the different transcripts of the gene, marked according to the expression level fold change between younger and older individuals. The middle sections show the associated CpG sites that are differentially methylated. The scatter plots in the bottom sections illustrate the correspondence between expression and methylation levels for each site separately. The scatter plots are displayed in the same order as the sites in the middle section; the red and blue dots display levels detected in the younger and older individuals, respectively.

    Journal: Scientific Reports

    Article Title: Age-related profiling of DNA methylation in CD8+ T cells reveals changes in immune response and transcriptional regulator genes

    doi: 10.1038/srep13107

    Figure Lengend Snippet: Examples of genes with known functional role in CD8+ T cells that display inverse correlation between gene expression and DNA methylation. ( A ) LGALS1 , ( B ) IFNG , ( C ) CCR7 and ( D ) SATB1 gene. Each panel is composed of three sections. The top sections display the different transcripts of the gene, marked according to the expression level fold change between younger and older individuals. The middle sections show the associated CpG sites that are differentially methylated. The scatter plots in the bottom sections illustrate the correspondence between expression and methylation levels for each site separately. The scatter plots are displayed in the same order as the sites in the middle section; the red and blue dots display levels detected in the younger and older individuals, respectively.

    Article Snippet: DNA was amplified using bisulfite-converted DNA, Hot Start DNA Polymerase (Solis BioDyne) and specific primers, according to the EpiTYPER protocol.

    Techniques: Functional Assay, Expressing, DNA Methylation Assay, Methylation

    TRIB3 and ATF4 binding near genes that are regulated in response to bortezomib (Btz), nelfinavir (Nelf) and ISRIB in HepG2 cells. ( A ) Gene ontology enrichment analysis for genes with a TRIB3 ChIP-Seq peak within 2 kb of the transcription start site (TSS). The GO Biological Process database was used. ( B ) RNA-Seq differential expression results overlap with genes harboring TRIB3 ChIP-Seq peaks (TSS ± 2 kb). ISRIB-downregulated genes were obtained comparing ISRIB and Btz co-treatment to Btz alone. ( C ) Btz-upregulated genes with TSS-proximal (TSS ± 2 kb) TRIB3 ChIP-Seq peaks. Genes are ranked by the TRIB3 Btz ChIP-Seq signal strength. ( D ) ChIP-Seq coverage plots for selected loci. The αFlag neg. cells track represents ChIP-Seq of unedited HepG2 cells with anti-Flag antibody. ( E ) ChIP-qPCR validation for the binding of ATF4, TRIB3 (anti-Flag M2) and non-targeting IgG at selected loci detected in ChIP-Seq. A negative control locus is also shown (-Ctrl). The means ± SD of 2–5 ChIP experiments from two biological replicates are shown. The DNA quantity is presented as a percentage of input chromatin.

    Journal: Cancers

    Article Title: Pharmacological or TRIB3-Mediated Suppression of ATF4 Transcriptional Activity Promotes Hepatoma Cell Resistance to Proteasome Inhibitor Bortezomib

    doi: 10.3390/cancers13102341

    Figure Lengend Snippet: TRIB3 and ATF4 binding near genes that are regulated in response to bortezomib (Btz), nelfinavir (Nelf) and ISRIB in HepG2 cells. ( A ) Gene ontology enrichment analysis for genes with a TRIB3 ChIP-Seq peak within 2 kb of the transcription start site (TSS). The GO Biological Process database was used. ( B ) RNA-Seq differential expression results overlap with genes harboring TRIB3 ChIP-Seq peaks (TSS ± 2 kb). ISRIB-downregulated genes were obtained comparing ISRIB and Btz co-treatment to Btz alone. ( C ) Btz-upregulated genes with TSS-proximal (TSS ± 2 kb) TRIB3 ChIP-Seq peaks. Genes are ranked by the TRIB3 Btz ChIP-Seq signal strength. ( D ) ChIP-Seq coverage plots for selected loci. The αFlag neg. cells track represents ChIP-Seq of unedited HepG2 cells with anti-Flag antibody. ( E ) ChIP-qPCR validation for the binding of ATF4, TRIB3 (anti-Flag M2) and non-targeting IgG at selected loci detected in ChIP-Seq. A negative control locus is also shown (-Ctrl). The means ± SD of 2–5 ChIP experiments from two biological replicates are shown. The DNA quantity is presented as a percentage of input chromatin.

    Article Snippet: XBP1 splicing was detected by RT-PCR using HOT FIREPol DNA Polymerase (Solis BioDyne) and primers that span the IRE1-mediated splicing event on the XBP1 mRNA ( ).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, RNA Sequencing Assay, Expressing, Real-time Polymerase Chain Reaction, Negative Control