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    Monarch PCR and DNA Cleanup Kit
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    Monarch PCR and DNA Cleanup Kit 250 preps
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    New England Biolabs qpcr reactions
    Monarch PCR and DNA Cleanup Kit
    Monarch PCR and DNA Cleanup Kit 250 preps
    https://www.bioz.com/result/qpcr reactions/product/New England Biolabs
    Average 92 stars, based on 1869 article reviews
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    qpcr reactions - by Bioz Stars, 2021-01
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    Images

    1) Product Images from "DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia"

    Article Title: DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12692

    DAPK3‐mediated histone H3 threonine phosphorylation is required for faithful co‐transcriptional splicing. (A) EGR1/DUSP2 gene schematics showing introns and exons. Primer locations for qPCR and RT‐PCR indicated by vertical arrows and blue boxes. Numbers after gene names correspond to the position of the primers in and around the gene relative to the TSS, where + denotes 3′ of the TSS and − denotes 5′ of the TSS. Reverse transcription PCR (RT‐PCR) analysis comparing levels of processed EGR1/DUSP2 mRNA (top left) with EGR1/DUSP2 primary transcript levels (bottom left, top right, bottom right). CLL cells were pretreated with either 1 µ m ibrutinib or 25 µ m DAPKi for 1 h as indicated and then stimulated with anti‐IgM and sCD40L to activate IEG expression. Extracted RNA was reverse transcribed with either random primer (RP) or oligo‐dT, subjected to RT‐PCR and analysed by agarose gel electrophoresis. (B) qPCR analysis of EGR1/DUSP2 primary transcript (shades of blue) vs processed mRNA (red) from HBL1 cells pretreated with either 1 µ m ibrutinib or 25 µ m DAPKi for 1 h and then stimulated with or without anti‐IgM and CD40L for 1 h as indicated. Error bars represent the SD of n = 3 independent experiments. (C) Western blots of HBL1 cells transfected with siRNAs against DAPK3 (#1 and #2) and with a nonspecific control siRNA (siCtrl). On day 5 post‐transfection, cells were harvested and lysates probed with antibodies against DAPK3, DAPK3‐T265‐P, histone H3T6‐P and histone H3T11‐P. β‐actin was used as a loading control and for normalisation to siCtrl using image lab software (right). Blots are representative of three independent transfections. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test. P values for DAPK3 expression normalised to siCtrl = 0.0036 and 0.0004 for siCtrl vs. siDAPK3 #1 and siDAPK3 #2, respectively. (D) qPCR analysis of HBL1 cells transfected with siRNAs against DAPK3 (#1 and #2) and with a nonspecific control siRNA (siCtrl). On day 5 post‐transfection, HBL1 cells were stimulated with anti‐IgM for 1 h and then harvested for analysis. Expression changes were quantified using the ∆ C t method with TBP, GAPDH and PPP6C as control genes. Error bars represent the SD of three independent transfections. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM stimulated siCtrl as control. P values for EGR1, EGR1 +0.55 kb and EGR1 +1.0 kb = 0.0082 and 0.0009, 0.1163 and 0.2759, 0.0047 and 0.0270 for siCtrl vs. siDAPK3 #1 and siDAPK3 #2, respectively. Ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
    Figure Legend Snippet: DAPK3‐mediated histone H3 threonine phosphorylation is required for faithful co‐transcriptional splicing. (A) EGR1/DUSP2 gene schematics showing introns and exons. Primer locations for qPCR and RT‐PCR indicated by vertical arrows and blue boxes. Numbers after gene names correspond to the position of the primers in and around the gene relative to the TSS, where + denotes 3′ of the TSS and − denotes 5′ of the TSS. Reverse transcription PCR (RT‐PCR) analysis comparing levels of processed EGR1/DUSP2 mRNA (top left) with EGR1/DUSP2 primary transcript levels (bottom left, top right, bottom right). CLL cells were pretreated with either 1 µ m ibrutinib or 25 µ m DAPKi for 1 h as indicated and then stimulated with anti‐IgM and sCD40L to activate IEG expression. Extracted RNA was reverse transcribed with either random primer (RP) or oligo‐dT, subjected to RT‐PCR and analysed by agarose gel electrophoresis. (B) qPCR analysis of EGR1/DUSP2 primary transcript (shades of blue) vs processed mRNA (red) from HBL1 cells pretreated with either 1 µ m ibrutinib or 25 µ m DAPKi for 1 h and then stimulated with or without anti‐IgM and CD40L for 1 h as indicated. Error bars represent the SD of n = 3 independent experiments. (C) Western blots of HBL1 cells transfected with siRNAs against DAPK3 (#1 and #2) and with a nonspecific control siRNA (siCtrl). On day 5 post‐transfection, cells were harvested and lysates probed with antibodies against DAPK3, DAPK3‐T265‐P, histone H3T6‐P and histone H3T11‐P. β‐actin was used as a loading control and for normalisation to siCtrl using image lab software (right). Blots are representative of three independent transfections. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test. P values for DAPK3 expression normalised to siCtrl = 0.0036 and 0.0004 for siCtrl vs. siDAPK3 #1 and siDAPK3 #2, respectively. (D) qPCR analysis of HBL1 cells transfected with siRNAs against DAPK3 (#1 and #2) and with a nonspecific control siRNA (siCtrl). On day 5 post‐transfection, HBL1 cells were stimulated with anti‐IgM for 1 h and then harvested for analysis. Expression changes were quantified using the ∆ C t method with TBP, GAPDH and PPP6C as control genes. Error bars represent the SD of three independent transfections. Significant differences calculated using two‐way ANOVA followed by Dunnett's multiple comparison test with anti‐IgM stimulated siCtrl as control. P values for EGR1, EGR1 +0.55 kb and EGR1 +1.0 kb = 0.0082 and 0.0009, 0.1163 and 0.2759, 0.0047 and 0.0270 for siCtrl vs. siDAPK3 #1 and siDAPK3 #2, respectively. Ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

    Techniques Used: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Western Blot, Transfection, Software

    2) Product Images from "Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential"

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2020.00400

    Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .
    Figure Legend Snippet: Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Binding Assay, Positive Control

    3) Product Images from "Systematic Analysis of Splice-Site-Creating Mutations in Cancer"

    Article Title: Systematic Analysis of Splice-Site-Creating Mutations in Cancer

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.03.052

    Minigene Functional Assay of Splice-Site-Creating Mutations (A) Integrative genomics viewer (IGV) screenshot of the conventionally annotated synonymous mutation in PARP1 in exon 21. RNA-seq reads of the candidate splice-site-creating mutation reveal the creation of an alternative splice site (red reads) created by the conventionally annotated synonymous mutation. (B) Candidate recurrent splice-site-creating mutations in BAP1 . Conventionally annotated as synonymous variants, the BAP1 -mutated region shows alternatively spliced reads (red reads) in the IGV screenshot for each sample with the splice-site-creating mutation. (C) IGV screenshot of a conventionally annotated synonymous mutation in RAD51C in exon 2. (D) Maximum entropy score of the splice-site-creating variant before (purple) and after (red) the introduced mutation for each variant functionally validated in the mini-gene splicing assay. In silico predictions suggest all mutations strengthen the alternative splice site. (E) Candidate splice-site-creating mutations validated by the mini-gene splicing assay. Exons of interest were cloned into the pCAS2.1 vector and mutant (red); wild-type (purple) plasmids were transfected into 293T cells; and total RNA was extracted to identify mutation-induced alternatively spliced products.
    Figure Legend Snippet: Minigene Functional Assay of Splice-Site-Creating Mutations (A) Integrative genomics viewer (IGV) screenshot of the conventionally annotated synonymous mutation in PARP1 in exon 21. RNA-seq reads of the candidate splice-site-creating mutation reveal the creation of an alternative splice site (red reads) created by the conventionally annotated synonymous mutation. (B) Candidate recurrent splice-site-creating mutations in BAP1 . Conventionally annotated as synonymous variants, the BAP1 -mutated region shows alternatively spliced reads (red reads) in the IGV screenshot for each sample with the splice-site-creating mutation. (C) IGV screenshot of a conventionally annotated synonymous mutation in RAD51C in exon 2. (D) Maximum entropy score of the splice-site-creating variant before (purple) and after (red) the introduced mutation for each variant functionally validated in the mini-gene splicing assay. In silico predictions suggest all mutations strengthen the alternative splice site. (E) Candidate splice-site-creating mutations validated by the mini-gene splicing assay. Exons of interest were cloned into the pCAS2.1 vector and mutant (red); wild-type (purple) plasmids were transfected into 293T cells; and total RNA was extracted to identify mutation-induced alternatively spliced products.

    Techniques Used: Functional Assay, Mutagenesis, RNA Sequencing Assay, Variant Assay, Splicing Assay, In Silico, Clone Assay, Plasmid Preparation, Transfection

    4) Product Images from "Coordinated Changes in DNA Methylation in Antigen-Specific Memory CD4 T Cells"

    Article Title: Coordinated Changes in DNA Methylation in Antigen-Specific Memory CD4 T Cells

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1202267

    Transcriptional activity of a luciferase reporter gene in unmethylated and methylated DMR sequences from the introns of 15 genes. Transient transfections were performed with a control plasmid (pCpGL-EF1 promoter) or pCpGL-EF-DMR in P/I-treated EL-4 T
    Figure Legend Snippet: Transcriptional activity of a luciferase reporter gene in unmethylated and methylated DMR sequences from the introns of 15 genes. Transient transfections were performed with a control plasmid (pCpGL-EF1 promoter) or pCpGL-EF-DMR in P/I-treated EL-4 T

    Techniques Used: Activity Assay, Luciferase, Methylation, Transfection, Plasmid Preparation

    5) Product Images from "Scale-invariant patterning by size-dependent inhibition of Nodal signalling"

    Article Title: Scale-invariant patterning by size-dependent inhibition of Nodal signalling

    Journal: Nature cell biology

    doi: 10.1038/s41556-018-0155-7

    Lefty concentration increases in smaller embryos to allow scaling. (a) Increase in Lefty concentration over time in smaller embryos predicted by the size-dependent inhibition model. (b) Decrease in Lefty amount over time in smaller embryos predicted by the size-dependent inhibition model. (c) Animal pole views of maximum intensity confocal stack projections of WT untreated and extirpated embryos injected with lefty1-GFP mRNA in the YSL, and quantification of GFP intensity; **p
    Figure Legend Snippet: Lefty concentration increases in smaller embryos to allow scaling. (a) Increase in Lefty concentration over time in smaller embryos predicted by the size-dependent inhibition model. (b) Decrease in Lefty amount over time in smaller embryos predicted by the size-dependent inhibition model. (c) Animal pole views of maximum intensity confocal stack projections of WT untreated and extirpated embryos injected with lefty1-GFP mRNA in the YSL, and quantification of GFP intensity; **p

    Techniques Used: Concentration Assay, Inhibition, Injection

    High Lefty diffusivity is required for scaling. (a,b) Simulations of the model without feedback inhibition and hindered Lefty diffusion predict that a reduction in Lefty diffusivity – preventing Lefty from reaching the animal pole – should preclude scaling. (c) Schematic of morphotrap-mediated Lefty1-GFP diffusion hindrance in extirpated embryos. (d) Maximum intensity projections of confocal stacks of lft1 -/- ; lft2 -/- embryos injected with or without morphotrap (injected at the one-cell-stage) and lefty1-GFP mRNA in the YSL (injected at sphere stage). Lateral views. (e) Spatial distribution of Lefty1-GFP secreted from the YSL. The morphotrap prevents spreading of Lefty1-GFP towards the animal pole of the embryo. n[ lefty1-GFP mRNA injection]=6, n[ morphotrap + lefty1-GFP mRNA injection]=3, n[background values]=1, n[background values morphotrap]=2. The experimentally determined distributions of Lefty1-GFP with morphotrap-mediated diffusion hindrance resemble the simulation of the “Reduced Lefty diffusivity” scenario (b). Shaded regions: SEM. (f) Lateral views of representative 26 hpf lft1 -/- ; lft2 -/- embryos with different treatments. Numbers in the figure panel indicate the fraction of these representative embryos. (g) Phenotype distributions in lft1 -/- ; lft2 -/- embryos after different treatments (n[ lft1 -/- ; lft2 -/- ]=39; lft1 -/- ; lft2 -/- + lft1GFP : n[untreated]=137, n[extirpated]=44; lft1 -/- ; lft2 -/- + morphotrap + lft1GFP : n[untreated]=91, n[extirpated]=44). Embryos with partial rescue display imperfect tails and reduced cephalic structures (i.e. very mild Lefty-mutant phenotypes). (h) Fraction of treated lft1 -/- ; lft2 -/- embryos with low (
    Figure Legend Snippet: High Lefty diffusivity is required for scaling. (a,b) Simulations of the model without feedback inhibition and hindered Lefty diffusion predict that a reduction in Lefty diffusivity – preventing Lefty from reaching the animal pole – should preclude scaling. (c) Schematic of morphotrap-mediated Lefty1-GFP diffusion hindrance in extirpated embryos. (d) Maximum intensity projections of confocal stacks of lft1 -/- ; lft2 -/- embryos injected with or without morphotrap (injected at the one-cell-stage) and lefty1-GFP mRNA in the YSL (injected at sphere stage). Lateral views. (e) Spatial distribution of Lefty1-GFP secreted from the YSL. The morphotrap prevents spreading of Lefty1-GFP towards the animal pole of the embryo. n[ lefty1-GFP mRNA injection]=6, n[ morphotrap + lefty1-GFP mRNA injection]=3, n[background values]=1, n[background values morphotrap]=2. The experimentally determined distributions of Lefty1-GFP with morphotrap-mediated diffusion hindrance resemble the simulation of the “Reduced Lefty diffusivity” scenario (b). Shaded regions: SEM. (f) Lateral views of representative 26 hpf lft1 -/- ; lft2 -/- embryos with different treatments. Numbers in the figure panel indicate the fraction of these representative embryos. (g) Phenotype distributions in lft1 -/- ; lft2 -/- embryos after different treatments (n[ lft1 -/- ; lft2 -/- ]=39; lft1 -/- ; lft2 -/- + lft1GFP : n[untreated]=137, n[extirpated]=44; lft1 -/- ; lft2 -/- + morphotrap + lft1GFP : n[untreated]=91, n[extirpated]=44). Embryos with partial rescue display imperfect tails and reduced cephalic structures (i.e. very mild Lefty-mutant phenotypes). (h) Fraction of treated lft1 -/- ; lft2 -/- embryos with low (

    Techniques Used: Inhibition, Diffusion-based Assay, Injection, Mutagenesis

    6) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Figure Legend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    7) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    8) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    9) Product Images from "Analysis of somatic mutations identifies signs of selection during in vitro aging of primary dermal fibroblasts, et al. Analysis of somatic mutations identifies signs of selection during in vitro aging of primary dermal fibroblasts"

    Article Title: Analysis of somatic mutations identifies signs of selection during in vitro aging of primary dermal fibroblasts, et al. Analysis of somatic mutations identifies signs of selection during in vitro aging of primary dermal fibroblasts

    Journal: Aging Cell

    doi: 10.1111/acel.13010

    CDKN2A gene and its expression in the presence or absence of the c.53C > T mutation. (a) Schematic representation of the genomic structure and transcripts of the CDKN2A gene locus with c.53C > T mutation and primer locations. The exons are shown as rectangles and primers as arrows. Primers 1 and 5 were used for RFLP analysis (see figure c), primers 3 and 4 were used for ddPCR analysis (see figure b), and primers 2 and 5 were used for additional ddPCR analysis (see Figure S1 ). An EagI restriction site is present in a wild‐type carrier, but absent in the individual with the c.53C > T mutation. The size of the locus, transcripts, exons, and introns is not shown to scale. Of note, the size of the intron between exon 1a and exon 2 is approximately 3.5 kb. (b) Comparison of CDKN2A transcript copies after normalization to GAPDH in early and late passage primary fibroblasts from young and advanced age healthy subjects without the c.53C > T mutation and in early and late passage primary fibroblasts from the XPA patient where the c.53C > T mutation is present at an allele frequency of 55.3% in the late passage. Primers 3 and 4 were used for this ddPCR analysis. (c) RFLP analysis of CDKN2A transcript in early and late passage primary fibroblasts from young age healthy and advanced age healthy subjects without the c.53C > T mutation and in early and late passage primary fibroblasts from XPA patient where the c.53C > T mutation is present at an allele frequency of 55.3% in the late passage only. Primers 1 and 5 were first used to amplify the cDNA followed by digestion with restriction enzyme EagI (indicated by +). Part of the amplified sample was used as an undigested control (indicated by −). Undigested PCR product is 658 bp in size, while digested fragments are 274 bp and 384 bp in size
    Figure Legend Snippet: CDKN2A gene and its expression in the presence or absence of the c.53C > T mutation. (a) Schematic representation of the genomic structure and transcripts of the CDKN2A gene locus with c.53C > T mutation and primer locations. The exons are shown as rectangles and primers as arrows. Primers 1 and 5 were used for RFLP analysis (see figure c), primers 3 and 4 were used for ddPCR analysis (see figure b), and primers 2 and 5 were used for additional ddPCR analysis (see Figure S1 ). An EagI restriction site is present in a wild‐type carrier, but absent in the individual with the c.53C > T mutation. The size of the locus, transcripts, exons, and introns is not shown to scale. Of note, the size of the intron between exon 1a and exon 2 is approximately 3.5 kb. (b) Comparison of CDKN2A transcript copies after normalization to GAPDH in early and late passage primary fibroblasts from young and advanced age healthy subjects without the c.53C > T mutation and in early and late passage primary fibroblasts from the XPA patient where the c.53C > T mutation is present at an allele frequency of 55.3% in the late passage. Primers 3 and 4 were used for this ddPCR analysis. (c) RFLP analysis of CDKN2A transcript in early and late passage primary fibroblasts from young age healthy and advanced age healthy subjects without the c.53C > T mutation and in early and late passage primary fibroblasts from XPA patient where the c.53C > T mutation is present at an allele frequency of 55.3% in the late passage only. Primers 1 and 5 were first used to amplify the cDNA followed by digestion with restriction enzyme EagI (indicated by +). Part of the amplified sample was used as an undigested control (indicated by −). Undigested PCR product is 658 bp in size, while digested fragments are 274 bp and 384 bp in size

    Techniques Used: Expressing, Mutagenesis, Amplification, Polymerase Chain Reaction

    10) Product Images from "Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens"

    Article Title: Comparative Methods to Improve the Detection of BRAF V600 Mutations in Highly Pigmented Melanoma Specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158698

    Removal of the inhibitory effects of melanin on PCR amplification. (A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of BSA (ng/μl) were added to DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. (C) The effect of diluting DNA assessed in the presence of 40 ng/μl of melanin and either 1U or 2U of Taq polymerase. (D) NucleoSpin ® gDNA Clean-up XS Kit used on DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. PCR amplification of the DNA was monitored on 2% gel agarose electrophoresis with ethidium bromide staining. MW, molecular weight markers.
    Figure Legend Snippet: Removal of the inhibitory effects of melanin on PCR amplification. (A) Increasing concentrations of synthetic melanin were added to DNA extracted from 1676 melanoma cell lines. (B) Increasing concentrations of BSA (ng/μl) were added to DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. (C) The effect of diluting DNA assessed in the presence of 40 ng/μl of melanin and either 1U or 2U of Taq polymerase. (D) NucleoSpin ® gDNA Clean-up XS Kit used on DNA extracted from cultured cells containing 40 or 80 ng/μl of melanin. PCR amplification of the DNA was monitored on 2% gel agarose electrophoresis with ethidium bromide staining. MW, molecular weight markers.

    Techniques Used: Polymerase Chain Reaction, Amplification, Cell Culture, Electrophoresis, Staining, Molecular Weight

    11) Product Images from "Enzymatic Cleavage of 3’-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis"

    Article Title: Enzymatic Cleavage of 3’-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-64541-z

    Incorporation of 3’-Aep-dCMP by commercially available A-family DNA polymerases (AF-DNAPs). ( A ) Top, the schematic representation of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu. Bottom, DNA fragment analysis of the primer (N) and the primer plus an incorporated 3’-Aep-dCMP by BF (N + 1). ( B ) Activities of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu, respectively. The primer-extension assays were performed as described in the Methods using 0.1, 0.2, 0.4, 0.8, 2, 4, 10, 20, or 40 μM of 3’-Aep-dCTP in the reaction.
    Figure Legend Snippet: Incorporation of 3’-Aep-dCMP by commercially available A-family DNA polymerases (AF-DNAPs). ( A ) Top, the schematic representation of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu. Bottom, DNA fragment analysis of the primer (N) and the primer plus an incorporated 3’-Aep-dCMP by BF (N + 1). ( B ) Activities of single 3’-Aep-dCMP incorporation by Taq, Tth, Tfl, BF, KF, or Bsu, respectively. The primer-extension assays were performed as described in the Methods using 0.1, 0.2, 0.4, 0.8, 2, 4, 10, 20, or 40 μM of 3’-Aep-dCTP in the reaction.

    Techniques Used:

    12) Product Images from "RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops"

    Article Title: RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-018-0413-6

    Investigation of dosage effect using RNase I f -qPCR. The total RNAs isolated from distinct transgenic maize events from a segregating S1 population (including homozygous and hemizygous plants) of different constructs (listed on top) carrying v-ATPase C. The RNA were analyzed using the RNaseI f - qPCR assay. The transcripts level were analyzed by 95 °C (RNase I f ), and 70 °C (without RNase I f treatment) to quantify v-ATPase C dsRNA, and the endogenous TIP41 gene, respectively. The v-ATPase C dsRNA RTL for each biological replicate was normalized to endogenous gene (TIP41). The mean RTL ( N = 12) are denoted in the box plot
    Figure Legend Snippet: Investigation of dosage effect using RNase I f -qPCR. The total RNAs isolated from distinct transgenic maize events from a segregating S1 population (including homozygous and hemizygous plants) of different constructs (listed on top) carrying v-ATPase C. The RNA were analyzed using the RNaseI f - qPCR assay. The transcripts level were analyzed by 95 °C (RNase I f ), and 70 °C (without RNase I f treatment) to quantify v-ATPase C dsRNA, and the endogenous TIP41 gene, respectively. The v-ATPase C dsRNA RTL for each biological replicate was normalized to endogenous gene (TIP41). The mean RTL ( N = 12) are denoted in the box plot

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Transgenic Assay, Construct

    Cross comparison of RNase I f -qPCR and QuantiGene Plex (QGP) assay. The purified leaf RNA samples from distinct transgenic maize T0 events are analyzed by RNase I f -qPCR and QGP assay. The custom QGP assay detecting Dv v-ATPase C and TIP41 were performed and the data is shown on the x-axis. The RNase I f -qPCR data is analyzed from the R95 RTL (RNase I f treatment and cDNA from 95 °C incubation) and presented on the y-axis. The correlation coefficient R ( p
    Figure Legend Snippet: Cross comparison of RNase I f -qPCR and QuantiGene Plex (QGP) assay. The purified leaf RNA samples from distinct transgenic maize T0 events are analyzed by RNase I f -qPCR and QGP assay. The custom QGP assay detecting Dv v-ATPase C and TIP41 were performed and the data is shown on the x-axis. The RNase I f -qPCR data is analyzed from the R95 RTL (RNase I f treatment and cDNA from 95 °C incubation) and presented on the y-axis. The correlation coefficient R ( p

    Techniques Used: Real-time Polymerase Chain Reaction, Purification, Transgenic Assay, Incubation

    RNase I f -qPCR assay overview. The total RNA is isolated first ( a ) using the protocol described in materials and methods. Then the isolated total RNAs are treated with RNase I f and followed by RNA clean-up to obtain the purified dsRNA (loop sequence is digested) ( b ). The RNase I f -treated RNAs are proceeded for cDNA conversion. The RNAs are pre-incubated with random hexamers at 95 °C ( c ) and 70 °C ( d ), respectively, and followed by cDNA conversion. Blue strands represent the reversely transcribed cDNA for the RNA incubated in 95 °C. In contrast, no cDNA is produced from the RNA incubated in 70 °C if all ssRNA is digested by RNase I f . In addition, non-RNase I f -treated RNA was used for cDNA conversion at 95 °C ( e ) and 70 °C ( f ). For RNA pre-incubated in 95 °C. cDNAs (dark blue strand) are transcribed from both dsRNA and ssRNA. Oppositely, cDNAs are only converted from ssRNAs (including RNAi and endogenous gene). The qRT-PCR is performed after the cDNA conversion using the custom designed TaqMan™ assay
    Figure Legend Snippet: RNase I f -qPCR assay overview. The total RNA is isolated first ( a ) using the protocol described in materials and methods. Then the isolated total RNAs are treated with RNase I f and followed by RNA clean-up to obtain the purified dsRNA (loop sequence is digested) ( b ). The RNase I f -treated RNAs are proceeded for cDNA conversion. The RNAs are pre-incubated with random hexamers at 95 °C ( c ) and 70 °C ( d ), respectively, and followed by cDNA conversion. Blue strands represent the reversely transcribed cDNA for the RNA incubated in 95 °C. In contrast, no cDNA is produced from the RNA incubated in 70 °C if all ssRNA is digested by RNase I f . In addition, non-RNase I f -treated RNA was used for cDNA conversion at 95 °C ( e ) and 70 °C ( f ). For RNA pre-incubated in 95 °C. cDNAs (dark blue strand) are transcribed from both dsRNA and ssRNA. Oppositely, cDNAs are only converted from ssRNAs (including RNAi and endogenous gene). The qRT-PCR is performed after the cDNA conversion using the custom designed TaqMan™ assay

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Purification, Sequencing, Incubation, Produced, Quantitative RT-PCR, TaqMan Assay

    Northern blot to characterize the synthetic ssRNA and dsRNA. a RNAi targeted to Dv v-ATPase C transgene design cassette scheme. The transgene is comprised of a promoter and terminator between which an inversely-repeated sequence of the target gene is inserted with a spacer region between the repeats (promoter and terminator informaiton are described in Methods). Synthetic dsRNA and ssRNA are shown as diagram here. The thick grey line indicates the antisense RNA probe used for the RNA blot. The black arrows denote the primer location used in qRT-PCR. b Synthetic dsRNA and ssRNA (spiked into wild-type maize leaf B104 RNA) were treated with RNase I f . The non-treated RNA were used as control. The RNA blot was probed with the 149 bp DIG-labeled antisense RNA probe (shown in gray bar in A). The arrow on the right indicated the approximate molecular size
    Figure Legend Snippet: Northern blot to characterize the synthetic ssRNA and dsRNA. a RNAi targeted to Dv v-ATPase C transgene design cassette scheme. The transgene is comprised of a promoter and terminator between which an inversely-repeated sequence of the target gene is inserted with a spacer region between the repeats (promoter and terminator informaiton are described in Methods). Synthetic dsRNA and ssRNA are shown as diagram here. The thick grey line indicates the antisense RNA probe used for the RNA blot. The black arrows denote the primer location used in qRT-PCR. b Synthetic dsRNA and ssRNA (spiked into wild-type maize leaf B104 RNA) were treated with RNase I f . The non-treated RNA were used as control. The RNA blot was probed with the 149 bp DIG-labeled antisense RNA probe (shown in gray bar in A). The arrow on the right indicated the approximate molecular size

    Techniques Used: Northern Blot, Sequencing, Northern blot, Quantitative RT-PCR, Labeling

    RNase I f -qPCR assay linearity. 10-point standard of synthetic dsRNAs and sRNAs were analyzed using RNase I f -qPCR described in Fig. 1 . The synthetic RNAs were analyzed by 95 °C (RNase I f ), and 70 °C (without RNase I f ), to quantify dsRNAs ( a ), and ssRNAs ( b ), respectively. The relative transcript level (RTL) was obtained via normalization of target signal over the endogenous gene (TIP41). The correlation between log2 of RTL and RNA amounts are presented here. The equation of trend line and R 2 is shown
    Figure Legend Snippet: RNase I f -qPCR assay linearity. 10-point standard of synthetic dsRNAs and sRNAs were analyzed using RNase I f -qPCR described in Fig. 1 . The synthetic RNAs were analyzed by 95 °C (RNase I f ), and 70 °C (without RNase I f ), to quantify dsRNAs ( a ), and ssRNAs ( b ), respectively. The relative transcript level (RTL) was obtained via normalization of target signal over the endogenous gene (TIP41). The correlation between log2 of RTL and RNA amounts are presented here. The equation of trend line and R 2 is shown

    Techniques Used: Real-time Polymerase Chain Reaction

    13) Product Images from "CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes"

    Article Title: CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17180-w

    Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.
    Figure Legend Snippet: Plasmid toolbox for the construction of CRISPR/Cas9-HCAdV genomes. ( A ) Schematic presentation of intermediate CRISPR/Cas9 shuttle plasmids for simple gRNA manipulation and multiplexing and subsequent transfer of the customized CRISPR/Cas9 machinery into the HCAdV genome. Option 1: pShV-CBh-Cas9-gRNA for constitutive Cas9 expression. Option 2: pShV-TRE-Cas9-TeOn3G-gRNA for inducible Cas9 expression utilizing the TetOn3G system. Black arrowheads indicate unique restriction enzyme sites for insertion of further gRNA expression units. ( B ) Workflow for gRNA customization and multiplexing of the CRISPR/Cas9 machinery. Step1: Complementary annealed gRNA oligonucleotides are separately inserted between the Bsa I restriction enzyme sites resulting in pShV-CBh-Cas9-gRNA1, pShV-CBh-Cas9-gRNA2 and pShV-CBh-Cas9- gRNA3. Step 2: Customized gRNA expression units gRNA1 and gRNA2 are amplified by PCR using primers generating desired restriction enzyme sites. Step 3: gRNA1 and 2 are inserted into the respective restriction enzyme site within pShV-CBh-Cas9-gRNA1 resulting in pShV-CBh-Cas9-CBh-gRNA1-gRNA2-gRNA3. ( C ) Transfer of customized CRISPR/Cas9 transgenes into the HCAdV genomes. Option 1: Released CRISPR/Cas9 transgene cassettes flanked by homology arms are inserted into pHCAdV-HOM-CcdB-AMP-HOM replacing the CcdB-Amp R cassette. Option 2: Endonuclease guided cloning into pAd-FTC utilizing PI- Sce I and I- Ceu I. HOM, homology arms for homologous recombination into pHCAdV-HOM-CCBD-AMP-HOM; CBh-P, constitutive hybrid CMV enhancer/chicken β-actin promotor; TRE-P, inducible tetracycline responsible element promotor; TetOn3G, TetOn3G transactivator; Ef1-α-P, Ef1-α-Promotor; Cas9, Streptococcus pyogenes Cas9, gRNA, guide RNA expression unit; U6-P, U6 RNA polymerase III promotor, Kan R , Kanamycin resistance cassette; Amp R ; Ampicillin resistance cassette, Chl R , Chloramphenicol resistance cassette; CcdB, control of cell death B expression cassette; ITR, adenovirus serotype 5 inverted terminal repeat; Ψ, adenovirus serotype 5 packaging signal.

    Techniques Used: Plasmid Preparation, CRISPR, Multiplexing, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Homologous Recombination, RNA Expression

    14) Product Images from "Skin Characteristics in Patients with Pityriasis Versicolor Using Non-Invasive Method, MPA5"

    Article Title: Skin Characteristics in Patients with Pityriasis Versicolor Using Non-Invasive Method, MPA5

    Journal: Annals of Dermatology

    doi: 10.5021/ad.2012.24.4.444

    PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hyperpigmented lesions of 21 patients. Lanes: M: molecular Marker, 1: Malassezia globosa (CBS7966), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. globosa (CBS7966), 5: M. restricta (KCTC7848), 6: M. restricta (KCTC7848), 7: M. restricta (KCTC7848), 8: M. slooffiae (KCTC17431), 9: M. globosa (CBS7966), 10: M. globosa (CBS7966), 11: M. restricta (KCTC7848), 12: M. globosa (CBS7966), 13: M. globosa (CBS7966), 14: M. furfur (KCTC7743), 15: M. slooffiae (KCTC17431), 16: M. globosa (CBS7966), 17: M. globosa (CBS7966), 18: M. restricta (KCTC7848), 19: M. restricta (KCTC7848), 20: M. furfur (KCTC7743), 21: M. sympodialis (KCTC7985). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.
    Figure Legend Snippet: PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hyperpigmented lesions of 21 patients. Lanes: M: molecular Marker, 1: Malassezia globosa (CBS7966), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. globosa (CBS7966), 5: M. restricta (KCTC7848), 6: M. restricta (KCTC7848), 7: M. restricta (KCTC7848), 8: M. slooffiae (KCTC17431), 9: M. globosa (CBS7966), 10: M. globosa (CBS7966), 11: M. restricta (KCTC7848), 12: M. globosa (CBS7966), 13: M. globosa (CBS7966), 14: M. furfur (KCTC7743), 15: M. slooffiae (KCTC17431), 16: M. globosa (CBS7966), 17: M. globosa (CBS7966), 18: M. restricta (KCTC7848), 19: M. restricta (KCTC7848), 20: M. furfur (KCTC7743), 21: M. sympodialis (KCTC7985). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.

    Techniques Used: Polymerase Chain Reaction, Marker

    PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hypopigmented lesions of 9 patients. Lanes: M: molecular Marker , 1: Malassezia slooffiae (KCTC17431), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. restricta (KCTC7848), 5: M. globosa (CBS7966), 6: M. globosa (CBS7966), 7: M. globosa (CBS7966), 8: M. sympodialis (KCTC7985), 9: M. furfur (KCTC7743). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.
    Figure Legend Snippet: PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hypopigmented lesions of 9 patients. Lanes: M: molecular Marker , 1: Malassezia slooffiae (KCTC17431), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. restricta (KCTC7848), 5: M. globosa (CBS7966), 6: M. globosa (CBS7966), 7: M. globosa (CBS7966), 8: M. sympodialis (KCTC7985), 9: M. furfur (KCTC7743). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.

    Techniques Used: Polymerase Chain Reaction, Marker

    15) Product Images from "Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential"

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2020.00400

    Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .
    Figure Legend Snippet: Serum stability profiling: (A) PCR amplified DNA and (B) its single-stranded form before amplification, after treatment with 50%v/v normal human female serum. Effect of different salt concentrations on aptamer-CA125 binding: (C) negative controls and (D) treated aptamer (lane 1: ladder, lane 2: 0.2 M NaHCO 3 with 0.5 M NaCl, lane 3: 100 mM NaCl and 5 mM MgCl 2 , lane 4: milli-Q water as positive control; (E) Binding—saturation curve for determination of K D . The original gel images have been provided in Figures S4, S5 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Binding Assay, Positive Control

    16) Product Images from "A biosensor strategy for E. coli based on ligand-dependent stabilization"

    Article Title: A biosensor strategy for E. coli based on ligand-dependent stabilization

    Journal: ACS synthetic biology

    doi: 10.1021/acssynbio.8b00052

    Design and selection of E. coli biosensors based on ligand-dependent stability. A. A destabilized ligand-binding domain is fused between the Zif268 DNA-binding domain and RpoZ transcription-activating domain. In this biosensor scheme, the absence of ligand would lead to biosensor degradation by natural protein degradation processes, leading to weak transcriptional expression of HIS3 . However, in the presence of ligand, binding to ligand would stabilize the biosensor, leading to increased transcriptional expression of HIS3 . B. An error-prone PCR library of LacI is fused to Zif268 and RpoZ and subjected to positive selection in minimal media lacking histidine with IPTG ligand. E. coli with biosensor variants that activate expression of HIS3 grow in these selections. Individual biosensors are then screened for ligand-dependence by replica plating their E. coli host on minimal media lacking histidine with and without IPTG.
    Figure Legend Snippet: Design and selection of E. coli biosensors based on ligand-dependent stability. A. A destabilized ligand-binding domain is fused between the Zif268 DNA-binding domain and RpoZ transcription-activating domain. In this biosensor scheme, the absence of ligand would lead to biosensor degradation by natural protein degradation processes, leading to weak transcriptional expression of HIS3 . However, in the presence of ligand, binding to ligand would stabilize the biosensor, leading to increased transcriptional expression of HIS3 . B. An error-prone PCR library of LacI is fused to Zif268 and RpoZ and subjected to positive selection in minimal media lacking histidine with IPTG ligand. E. coli with biosensor variants that activate expression of HIS3 grow in these selections. Individual biosensors are then screened for ligand-dependence by replica plating their E. coli host on minimal media lacking histidine with and without IPTG.

    Techniques Used: Selection, Ligand Binding Assay, Binding Assay, Expressing, Polymerase Chain Reaction

    17) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Figure Legend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    18) Product Images from "Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling"

    Article Title: Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling

    Journal: bioRxiv

    doi: 10.1101/2020.06.25.171439

    IGV screenshots showing examples of repeat and retrotransposable element RNAs detected in plasma by TGIRT-seq. ( A-D ) Simple repeats, including centromeric and telomeric repeats. ( E-J ) Retrotransposable element RNAs. The repeat or transposable element RNA is named at the top with an arrow below indicating the 5’ to 3’ orientation of the RNA. The tracks below show coverage plots and read alignments for the peak region in combined datasets for NaOH-treated plasma DNA (n=4; combined top and bottom strands (turquois)) or DNase-treated plasma RNA (n=15; Top strand (T), pink and Bottom strand (B), purple) based on the number of deduplicated reads indicated to the right. Colors other than pink or purple in the read alignments indicate bases that do not match the reference sequence (red, green, blue, and brown indicate thymidine, adenosine, cytidine, and guanosine, respectively). The peak called by MACS2 is delineated by a bracketed line below the alignments with the peak ID and length indicated below. Close ups of simple repeat sequences (not to scale) are shown below the peak ID. AluSx, Alu subfamily Sx; FLAM_C, free left Alu monomer subfamily C; L1MCc, mammalian-wide LINE-1 (L1M) family Cc; LTR12C, LTR element 12C; MER52A, medium reiteration frequency interspersed repeat LTR from ERV1 endogenous retrovirus; L2c, LINE-2 family c; NTA/black boxes, non-templated nucleotides added by TGIRT-III to the 3’ end of cDNAs during TGIRT-seq library preparation, appearing at the 5’ end of the RNA sequence.
    Figure Legend Snippet: IGV screenshots showing examples of repeat and retrotransposable element RNAs detected in plasma by TGIRT-seq. ( A-D ) Simple repeats, including centromeric and telomeric repeats. ( E-J ) Retrotransposable element RNAs. The repeat or transposable element RNA is named at the top with an arrow below indicating the 5’ to 3’ orientation of the RNA. The tracks below show coverage plots and read alignments for the peak region in combined datasets for NaOH-treated plasma DNA (n=4; combined top and bottom strands (turquois)) or DNase-treated plasma RNA (n=15; Top strand (T), pink and Bottom strand (B), purple) based on the number of deduplicated reads indicated to the right. Colors other than pink or purple in the read alignments indicate bases that do not match the reference sequence (red, green, blue, and brown indicate thymidine, adenosine, cytidine, and guanosine, respectively). The peak called by MACS2 is delineated by a bracketed line below the alignments with the peak ID and length indicated below. Close ups of simple repeat sequences (not to scale) are shown below the peak ID. AluSx, Alu subfamily Sx; FLAM_C, free left Alu monomer subfamily C; L1MCc, mammalian-wide LINE-1 (L1M) family Cc; LTR12C, LTR element 12C; MER52A, medium reiteration frequency interspersed repeat LTR from ERV1 endogenous retrovirus; L2c, LINE-2 family c; NTA/black boxes, non-templated nucleotides added by TGIRT-III to the 3’ end of cDNAs during TGIRT-seq library preparation, appearing at the 5’ end of the RNA sequence.

    Techniques Used: Sequencing

    IGV screenshots showing examples of peaks corresponding to putatively structured, full-length excised intron RNAs detected in plasma by TGIRT-seq peak calling. ( A-C ) Full-length excised intron RNAs that correspond to annotated agotrons or mirtrons. ( D - F ) Full-length excised intron RNAs that do not correspond to annotated agotrons or mirtrons. Gene names are indicated at the top with the hg19 coordinates of the called peak in parentheses and an arrow below indicating the 5’ to 3’ orientation of the encoded RNA. The top track shows the gene map (exons, thick bars; intron, thin lines), with the relevant part of the gene map expanded below. The tracks below the gene map show coverage plots and read alignments for the peak region in combined datasets for NaOH-treated plasma DNA (n=4; combined top and bottom strands, turquoise) or DNase-treated plasma RNA (n=15; Top strand (T), pink and Bottom strand (B), purple) based on the number of deduplicated reads indicated at the right in the coverage tracks. Colors other than pink or purple in the read alignments indicate bases that do not match the reference sequence (red, green, blue, and brown indicate thymidine, adenosine, cytidine, and guanosine, respectively). The peak called by MACS2 is delineated by a bracketed line with the peak ID, length, and PhastCons score for 46 vertebrates including humans indicated below (see phylogenetic tree in Fig. 7 ). The most stable predicted secondary structure for the peak and its minimum free energy (MFE; ΔG) computed by RNAfold ( 88 ) are shown below the peak ID, with the annotated mature miRNA and passenger strand of mirtrons highlighted in red within the predicted secondary structure. Red boxes, short (1-2 nt) non-templated 3’ U or A tails; NTA/ black boxes, non-templated nucleotides added by TGIRT-III to the 3’ end of cDNAs during TGIRT-seq library preparation, appearing at 5’ end of the RNA sequence.
    Figure Legend Snippet: IGV screenshots showing examples of peaks corresponding to putatively structured, full-length excised intron RNAs detected in plasma by TGIRT-seq peak calling. ( A-C ) Full-length excised intron RNAs that correspond to annotated agotrons or mirtrons. ( D - F ) Full-length excised intron RNAs that do not correspond to annotated agotrons or mirtrons. Gene names are indicated at the top with the hg19 coordinates of the called peak in parentheses and an arrow below indicating the 5’ to 3’ orientation of the encoded RNA. The top track shows the gene map (exons, thick bars; intron, thin lines), with the relevant part of the gene map expanded below. The tracks below the gene map show coverage plots and read alignments for the peak region in combined datasets for NaOH-treated plasma DNA (n=4; combined top and bottom strands, turquoise) or DNase-treated plasma RNA (n=15; Top strand (T), pink and Bottom strand (B), purple) based on the number of deduplicated reads indicated at the right in the coverage tracks. Colors other than pink or purple in the read alignments indicate bases that do not match the reference sequence (red, green, blue, and brown indicate thymidine, adenosine, cytidine, and guanosine, respectively). The peak called by MACS2 is delineated by a bracketed line with the peak ID, length, and PhastCons score for 46 vertebrates including humans indicated below (see phylogenetic tree in Fig. 7 ). The most stable predicted secondary structure for the peak and its minimum free energy (MFE; ΔG) computed by RNAfold ( 88 ) are shown below the peak ID, with the annotated mature miRNA and passenger strand of mirtrons highlighted in red within the predicted secondary structure. Red boxes, short (1-2 nt) non-templated 3’ U or A tails; NTA/ black boxes, non-templated nucleotides added by TGIRT-III to the 3’ end of cDNAs during TGIRT-seq library preparation, appearing at 5’ end of the RNA sequence.

    Techniques Used: Sequencing

    Protein-coding gene transcripts detected by TGIRT-seq and ultra low-input SMART-Seq v4 in the same human plasma preparation. ( A ) Bioanalyzer traces of PCR-amplified cDNAs generated by SMART-Seq according to manufacturer’s protocol. Samples were analyzed using an Agilent 2100 Bioanalyzer with a High Sensitivity DNA kit. Traces are color-coded by sample type as shown in the Figure, with “Ladder” (dashed gray lines) indicating the DNA ladder supplied with the High Sensitivity DNA kit. The negative control was a SMART-Seq library prepared with water as input, and the cellular RNA control was the positive control RNA supplied with the SMART-Seq kit. SMART-Seq 1 and 2 indicate double-stranded DNAs generated by using the SMART-Seq kit from different samples of DNase I-treated plasma RNA. FU, fluorescence units. ( B ) Scatter plot comparing protein-coding gene transcripts detected in plasma by TGIRT-seq and SMART-Seq. Reads assigned to protein-coding genes transcripts (0.25 million deduplicated reads for TGIRT-seq and 2.18 million reads for SMART-Seq, respectively) were extracted from combined datasets obtained from DNase I-treated plasma RNA (n=12 for TGIRT-seq and n=4 for SMART-Seq) and quantified by Kallisto. The scatter plots compare average TPM values for protein-coding gene transcripts with each point representing one gene, color-coded by gene type as indicated in the Figure, with ribosomal protein mRNAs that are 5’ TOP mRNAs categorized as 5’ TOP. The marginal distributions of different color-coded mRNA species in the scatter plot are shown above for SMART-Seq and to the right for TGIRT-seq. The red line indicates identical TPM values for both methods. The Pearson’s correlation coefficient (r) is indicated at the bottom right. ( C ) Normalized 5’- to 3’- gene body coverage for protein-coding gene transcripts detected in plasma by TGIRT-seq and SMART-Seq. Gene body coverage was computed by Picard tools (Broad Institute) using the genomic alignment files generated by Kallisto. The plots show normalized gene coverage versus normalized gene length for all protein-coding transcripts in the indicated datasets color-coded as indicated in the Figure. The red horizontal line at y=1 indicates perfectly uniform 5’ to 3’ coverage. ( D ) IGV screen shots showing examples of protein-coding gene transcripts detected in DNase I-treated plasma RNA by SMART-Seq and TGIRT-seq. The gene name and length are indicated at the top with the arrow below indicating the 5’ to 3’ orientation of the mRNA. The gene model from RefSeq is shown below (exons, blue bars; introns, thin blue lines). Coverage tracks (gray) for the combined SMART-Seq and TGIRT-seq datasets are shown below the gene model followed by read alignments for the sense (pink) and antisense (purple) orientations down sampled to 100 reads for display when necessary. Colors other than pink or purple in the read alignments indicate bases that do not match the reference sequence (red, green, blue, and brown indicate thymidine, adenosine, cytidine, and guanosine, respectively). Spliced exons are connected by thin blue lines. The IGV alignment for ribosomal protein gene RPL10 shows an embedded snoRNA (SNORA70) that is detected by TGIRT-seq but not by SMART-Seq.
    Figure Legend Snippet: Protein-coding gene transcripts detected by TGIRT-seq and ultra low-input SMART-Seq v4 in the same human plasma preparation. ( A ) Bioanalyzer traces of PCR-amplified cDNAs generated by SMART-Seq according to manufacturer’s protocol. Samples were analyzed using an Agilent 2100 Bioanalyzer with a High Sensitivity DNA kit. Traces are color-coded by sample type as shown in the Figure, with “Ladder” (dashed gray lines) indicating the DNA ladder supplied with the High Sensitivity DNA kit. The negative control was a SMART-Seq library prepared with water as input, and the cellular RNA control was the positive control RNA supplied with the SMART-Seq kit. SMART-Seq 1 and 2 indicate double-stranded DNAs generated by using the SMART-Seq kit from different samples of DNase I-treated plasma RNA. FU, fluorescence units. ( B ) Scatter plot comparing protein-coding gene transcripts detected in plasma by TGIRT-seq and SMART-Seq. Reads assigned to protein-coding genes transcripts (0.25 million deduplicated reads for TGIRT-seq and 2.18 million reads for SMART-Seq, respectively) were extracted from combined datasets obtained from DNase I-treated plasma RNA (n=12 for TGIRT-seq and n=4 for SMART-Seq) and quantified by Kallisto. The scatter plots compare average TPM values for protein-coding gene transcripts with each point representing one gene, color-coded by gene type as indicated in the Figure, with ribosomal protein mRNAs that are 5’ TOP mRNAs categorized as 5’ TOP. The marginal distributions of different color-coded mRNA species in the scatter plot are shown above for SMART-Seq and to the right for TGIRT-seq. The red line indicates identical TPM values for both methods. The Pearson’s correlation coefficient (r) is indicated at the bottom right. ( C ) Normalized 5’- to 3’- gene body coverage for protein-coding gene transcripts detected in plasma by TGIRT-seq and SMART-Seq. Gene body coverage was computed by Picard tools (Broad Institute) using the genomic alignment files generated by Kallisto. The plots show normalized gene coverage versus normalized gene length for all protein-coding transcripts in the indicated datasets color-coded as indicated in the Figure. The red horizontal line at y=1 indicates perfectly uniform 5’ to 3’ coverage. ( D ) IGV screen shots showing examples of protein-coding gene transcripts detected in DNase I-treated plasma RNA by SMART-Seq and TGIRT-seq. The gene name and length are indicated at the top with the arrow below indicating the 5’ to 3’ orientation of the mRNA. The gene model from RefSeq is shown below (exons, blue bars; introns, thin blue lines). Coverage tracks (gray) for the combined SMART-Seq and TGIRT-seq datasets are shown below the gene model followed by read alignments for the sense (pink) and antisense (purple) orientations down sampled to 100 reads for display when necessary. Colors other than pink or purple in the read alignments indicate bases that do not match the reference sequence (red, green, blue, and brown indicate thymidine, adenosine, cytidine, and guanosine, respectively). Spliced exons are connected by thin blue lines. The IGV alignment for ribosomal protein gene RPL10 shows an embedded snoRNA (SNORA70) that is detected by TGIRT-seq but not by SMART-Seq.

    Techniques Used: Polymerase Chain Reaction, Amplification, Generated, Negative Control, Positive Control, Fluorescence, Sequencing

    19) Product Images from "A high rate of polymerization during synthesis of mouse mammary tumor virus DNA alleviates hypermutation by APOBEC3 proteins"

    Article Title: A high rate of polymerization during synthesis of mouse mammary tumor virus DNA alleviates hypermutation by APOBEC3 proteins

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007533

    MMTV RT is more processive and faster than HIV-1 RT. RT enzymes were extracted from preparations containing equal virion levels (quantified by RTqPCR and verified by physical particle counting (EM, S3 Fig ). (A) To determine RTs processivity an MS2 cDNA was synthesized in the presence of an excess of the template and a trap to limit re-association of RTs with cDNAs. The cDNA products were A-tailed and amplified using anchor- and MS2-specific primers. A schematic diagram of the method is shown in the upper panel. The length distribution of the cDNA products was analyzed on 1.5% agarose gels (lower panel); lane 1: MassRuler DNA ladder (Fermentas), lane 2: extension terminated after 0 min, lane 3: extension terminated after 15 min. The assay was repeated three times with similar outcome. (B) Kinetics of MS2 cDNA synthesis was assayed in the absence of the trap and with a limited amount of the MS2 RNA/primer template. The cDNA polymerization was terminated after the various amount of time and the presence of 0.4 kb- or 1.4 kb-long cDNA determined by PCR with MS2-specific primers. PCR products were analyzed on agarose gels (marker: 2-Log DNA ladder (NEB)). The Fig shows a representative example of three assays.
    Figure Legend Snippet: MMTV RT is more processive and faster than HIV-1 RT. RT enzymes were extracted from preparations containing equal virion levels (quantified by RTqPCR and verified by physical particle counting (EM, S3 Fig ). (A) To determine RTs processivity an MS2 cDNA was synthesized in the presence of an excess of the template and a trap to limit re-association of RTs with cDNAs. The cDNA products were A-tailed and amplified using anchor- and MS2-specific primers. A schematic diagram of the method is shown in the upper panel. The length distribution of the cDNA products was analyzed on 1.5% agarose gels (lower panel); lane 1: MassRuler DNA ladder (Fermentas), lane 2: extension terminated after 0 min, lane 3: extension terminated after 15 min. The assay was repeated three times with similar outcome. (B) Kinetics of MS2 cDNA synthesis was assayed in the absence of the trap and with a limited amount of the MS2 RNA/primer template. The cDNA polymerization was terminated after the various amount of time and the presence of 0.4 kb- or 1.4 kb-long cDNA determined by PCR with MS2-specific primers. PCR products were analyzed on agarose gels (marker: 2-Log DNA ladder (NEB)). The Fig shows a representative example of three assays.

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

    20) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    21) Product Images from "High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments"

    Article Title: High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz851

    Experimental strategies to assemble long DNA and RNA hairpins. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled. ( A ) DNA hairpin construct using LNC: linear or plasmid DNA is used as template for PCR reactions; amplified fragments are purified and digested; fragments are then submitted to three rounds of purification and ligation (L1, L2, L3) to obtain the desired final product. ( B ) DNA hairpin construct, annealing method (ANC): template DNA is amplified by PCR and purified (pur.); one strand of the amplified fragments is nicked with enzymes Nb.BbvCI or Nt.BbvCI, gel purified and annealed (ann.) to obtain the final construct. ( C ) RNA hairpin construct: template DNA is amplified by PCR and purified, stem is amplified in three separate parts; RNA products are obtained by IVTR, purified and monophosphorylated (mP); products are then annealed and ligated to obtained the final construct.
    Figure Legend Snippet: Experimental strategies to assemble long DNA and RNA hairpins. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled. ( A ) DNA hairpin construct using LNC: linear or plasmid DNA is used as template for PCR reactions; amplified fragments are purified and digested; fragments are then submitted to three rounds of purification and ligation (L1, L2, L3) to obtain the desired final product. ( B ) DNA hairpin construct, annealing method (ANC): template DNA is amplified by PCR and purified (pur.); one strand of the amplified fragments is nicked with enzymes Nb.BbvCI or Nt.BbvCI, gel purified and annealed (ann.) to obtain the final construct. ( C ) RNA hairpin construct: template DNA is amplified by PCR and purified, stem is amplified in three separate parts; RNA products are obtained by IVTR, purified and monophosphorylated (mP); products are then annealed and ligated to obtained the final construct.

    Techniques Used: Labeling, Construct, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Purification, Ligation

    Experimental strategies to assemble linear DNA and RNA constructs. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled ( A ) DNA construct, ligation method (LNC): linear or plasmid DNA is used as a template for restriction digestions and PCR reactions; fragments are purified (pur.) and ligated (lig.) to obtain the desired final product. ( B ) DNA construct, annealing method (ANC): plasmid DNA is used as template for PCR reactions; one strand is nicked and removed; complementary single strands are annealed (ann.) to obtain the desired final product. ( C ) RNA construct, coilable (ANC): RNA strands are obtained by run-off in vitro transcription reaction (IVTR), then purified and annealed. Single strands are monophosphorylated (mP) prior to annealing and then ligated (lig.) to obtain a coilable product. ( D ) RNA construct, non-coilable (ANC): template DNA is amplified by PCR and purified; RNA single strands are obtained as in (C) and annealed.
    Figure Legend Snippet: Experimental strategies to assemble linear DNA and RNA constructs. The colored lines represent different nucleic acid strands. BIO and DIG are respectively biotin- and digoxygenin-labeled ( A ) DNA construct, ligation method (LNC): linear or plasmid DNA is used as a template for restriction digestions and PCR reactions; fragments are purified (pur.) and ligated (lig.) to obtain the desired final product. ( B ) DNA construct, annealing method (ANC): plasmid DNA is used as template for PCR reactions; one strand is nicked and removed; complementary single strands are annealed (ann.) to obtain the desired final product. ( C ) RNA construct, coilable (ANC): RNA strands are obtained by run-off in vitro transcription reaction (IVTR), then purified and annealed. Single strands are monophosphorylated (mP) prior to annealing and then ligated (lig.) to obtain a coilable product. ( D ) RNA construct, non-coilable (ANC): template DNA is amplified by PCR and purified; RNA single strands are obtained as in (C) and annealed.

    Techniques Used: Construct, Labeling, Ligation, Plasmid Preparation, Polymerase Chain Reaction, Purification, In Vitro, Amplification

    22) Product Images from "Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway"

    Article Title: Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    Journal: Scientific Reports

    doi: 10.1038/srep08572

    Targeted integration into Mgat1 locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in Mgat1 locus. (b) Indel frequency in Mgat1 locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNAs. The genomic regions covering sgRNA target sites were amplified, then subjected to Miseq analysis. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. (c) Agarose gel of 5′/3′ junction PCR on transiently transfected cells and stable cell pools. (++) Stable cells expressing both mCherry and Zsgreen1-DR; (+-) stable cells expressing only mCherry. (d) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pools were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Population analysis of Mgat1 disrupted cells by F-RCA-I staining. Based on red/green fluorescence intensity of stable cell pools, which was further selected with RCA-I or not, alteration of fluorescence intensity was analyzed upon F-RCA-I staining. Each scatter plot was divided by four quadrants, denoted by Q1 to Q4. Q3 and Q4 populations, marked by red squares, represent negative stained cells with F-RCA-I, indicating functional knockout of Mgat1 locus. Numerals below the red squares show the percentage of Q3 and Q4. (f) Agarose gel of out-out PCR results of stable pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (2.0 kb for sgRNA1 target site; 1.9 kb for sgRNA5 target site) or targeted integration (5.6 kb for sgRNA1 target site; 5.5 kb for sgRNA5 target site). (g) Relative copy number of Mgat1 and mCherry regions in clonal cells, as described in Fig. 1(e) . ( Top ) sgRNA1 target site ( Bottom ) sgRNA5 target site.
    Figure Legend Snippet: Targeted integration into Mgat1 locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in Mgat1 locus. (b) Indel frequency in Mgat1 locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNAs. The genomic regions covering sgRNA target sites were amplified, then subjected to Miseq analysis. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. (c) Agarose gel of 5′/3′ junction PCR on transiently transfected cells and stable cell pools. (++) Stable cells expressing both mCherry and Zsgreen1-DR; (+-) stable cells expressing only mCherry. (d) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pools were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Population analysis of Mgat1 disrupted cells by F-RCA-I staining. Based on red/green fluorescence intensity of stable cell pools, which was further selected with RCA-I or not, alteration of fluorescence intensity was analyzed upon F-RCA-I staining. Each scatter plot was divided by four quadrants, denoted by Q1 to Q4. Q3 and Q4 populations, marked by red squares, represent negative stained cells with F-RCA-I, indicating functional knockout of Mgat1 locus. Numerals below the red squares show the percentage of Q3 and Q4. (f) Agarose gel of out-out PCR results of stable pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (2.0 kb for sgRNA1 target site; 1.9 kb for sgRNA5 target site) or targeted integration (5.6 kb for sgRNA1 target site; 5.5 kb for sgRNA5 target site). (g) Relative copy number of Mgat1 and mCherry regions in clonal cells, as described in Fig. 1(e) . ( Top ) sgRNA1 target site ( Bottom ) sgRNA5 target site.

    Techniques Used: CRISPR, Sequencing, Transfection, Expressing, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Stable Transfection, Purification, Staining, Fluorescence, Functional Assay, Knock-Out

    Targeted integration into COSMC locus using CRISPR/Cas9. (a) Schematic illustration of the targeting strategy for the specific locus of interest. Donor plasmid consists of three parts: short homology arms flanking sgRNA target site cleaved by Cas9 (red triangle), mCherry and neomycin resistance gene expression cassettes inside homology arms, and ZsGreen1-DR expression cassette outside homology arms. Upon DSBs induced by CRISPR/Cas9, HDR-mediated repair can be used to insert a total size of 3.7 kb of expression cassettes through recombination of the target locus with donor plasmids. Primer position for 5′/3′ junction PCR is indicated. (b) Agarose gel of 5′/3′ junction PCR on transiently transfected cells and stable cell pools. An asterisk indicates the use of linearized donor plasmid. M, 1 kb DNA ladder (c) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. The chromatogram sequence of junction PCR amplicon was compared with the reference sequence at the genome-donor boundaries. (d) Agarose gel of out-out PCR results of stable cell pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (1.6 kb) or targeted integration (5.3 kb). (e) Relative copy number of COSMC and mCherry regions in clonal cells. Each plot shows the relative copy number of each region in comparison to the reference sample. Genomic DNA of wild type CHO-S and Clone #1 and was used as the reference for COSMC and mCherry region, respectively (shown in red). The error bars represent the standard deviations (n ≥ 3).
    Figure Legend Snippet: Targeted integration into COSMC locus using CRISPR/Cas9. (a) Schematic illustration of the targeting strategy for the specific locus of interest. Donor plasmid consists of three parts: short homology arms flanking sgRNA target site cleaved by Cas9 (red triangle), mCherry and neomycin resistance gene expression cassettes inside homology arms, and ZsGreen1-DR expression cassette outside homology arms. Upon DSBs induced by CRISPR/Cas9, HDR-mediated repair can be used to insert a total size of 3.7 kb of expression cassettes through recombination of the target locus with donor plasmids. Primer position for 5′/3′ junction PCR is indicated. (b) Agarose gel of 5′/3′ junction PCR on transiently transfected cells and stable cell pools. An asterisk indicates the use of linearized donor plasmid. M, 1 kb DNA ladder (c) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. The chromatogram sequence of junction PCR amplicon was compared with the reference sequence at the genome-donor boundaries. (d) Agarose gel of out-out PCR results of stable cell pools or clonal cells. Primer pairs annealing to genomic DNA region were used resulting in PCR products of either wild type (1.6 kb) or targeted integration (5.3 kb). (e) Relative copy number of COSMC and mCherry regions in clonal cells. Each plot shows the relative copy number of each region in comparison to the reference sample. Genomic DNA of wild type CHO-S and Clone #1 and was used as the reference for COSMC and mCherry region, respectively (shown in red). The error bars represent the standard deviations (n ≥ 3).

    Techniques Used: CRISPR, Plasmid Preparation, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Stable Transfection, Sequencing, Purification, Amplification

    Targeted integration into LdhA locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in LdhA locus. (b) Indel frequency in LdhA locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNA. The genomic regions covering sgRNA target sites were amplified, then subjected to deep sequencing analysis using Miseq. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. Investigation of target specific knock-in in transiently transfected and stable cell pools analyzed by (c) Agarose gel of 5′/3′ junction PCR (d) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Relative copy number of LdhA and mCherry regions in clonal cells, as described in Fig. 1(e) .
    Figure Legend Snippet: Targeted integration into LdhA locus using CRISPR/Cas9. (a) Illustration of the five sgRNA target genomic sites in LdhA locus. (b) Indel frequency in LdhA locus analyzed by deep sequencing. Genomic DNA was extracted 3 days after transfection with plasmids expressing Cas9 gene and sgRNA. The genomic regions covering sgRNA target sites were amplified, then subjected to deep sequencing analysis using Miseq. The percentage of wt and indel sequences are described in the bar plot. The values from control samples transfected only with plasmid expressing Cas9 were subtracted from test samples. Investigation of target specific knock-in in transiently transfected and stable cell pools analyzed by (c) Agarose gel of 5′/3′ junction PCR (d) Sanger sequencing of the 5′/3′ junction PCR amplicons. Amplicons from the stable cell pool were purified and directly sequenced after PCR amplification. M, 1 kb DNA ladder. (e) Relative copy number of LdhA and mCherry regions in clonal cells, as described in Fig. 1(e) .

    Techniques Used: CRISPR, Sequencing, Transfection, Expressing, Amplification, Plasmid Preparation, Knock-In, Stable Transfection, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Purification

    23) Product Images from "NLRP3 Activation Was Regulated by DNA Methylation Modification during Mycobacterium tuberculosis Infection"

    Article Title: NLRP3 Activation Was Regulated by DNA Methylation Modification during Mycobacterium tuberculosis Infection

    Journal: BioMed Research International

    doi: 10.1155/2016/4323281

    Construction and identification of NLRP3 promoter. (a) Two potential promoter regions of NLRP3 were amplified from genome and identified by double restriction enzyme digestion. 1 and 2 were PCR products from human genomic DNA. P1 and P2 were pNLPR3-P1 and pNLPR3-P2, respectively. P1E and P2E exhibited the enzyme digested products of pNLPR3-P1 and pNLPR3-P2 digested by Xho I and Mlu I. M1 and M2 were 250 bp and DL2000 DNA marker, respectively. (b) The activities of two potential NLRP3 promoters were identified by Dual-Luciferase Reporter Assay. HEK293T cells were plated in 24-well cell culture plate and cotransfected with firefly luciferase report plasmids pNLRP3-P1/P2 and Renilla luciferase report plasmid pRL. The relative promoter activity was measured 48 h after transfection as in Materials and Methods. Firefly luciferase activity was normalized to Renilla luciferase activity, and the promoter activity was expressed as the mean ± SD of at least triplicate wells ( ∗∗ p
    Figure Legend Snippet: Construction and identification of NLRP3 promoter. (a) Two potential promoter regions of NLRP3 were amplified from genome and identified by double restriction enzyme digestion. 1 and 2 were PCR products from human genomic DNA. P1 and P2 were pNLPR3-P1 and pNLPR3-P2, respectively. P1E and P2E exhibited the enzyme digested products of pNLPR3-P1 and pNLPR3-P2 digested by Xho I and Mlu I. M1 and M2 were 250 bp and DL2000 DNA marker, respectively. (b) The activities of two potential NLRP3 promoters were identified by Dual-Luciferase Reporter Assay. HEK293T cells were plated in 24-well cell culture plate and cotransfected with firefly luciferase report plasmids pNLRP3-P1/P2 and Renilla luciferase report plasmid pRL. The relative promoter activity was measured 48 h after transfection as in Materials and Methods. Firefly luciferase activity was normalized to Renilla luciferase activity, and the promoter activity was expressed as the mean ± SD of at least triplicate wells ( ∗∗ p

    Techniques Used: Amplification, Polymerase Chain Reaction, Marker, Luciferase, Reporter Assay, Cell Culture, Plasmid Preparation, Activity Assay, Transfection

    DNA methylation modification regulated NLRP3 activation in vitro . (a) The methylated ( Sss I treated) or unmethylated pNLRP3-P1/P2 was transfected into HEK293T cells together with pRL, respectively, and each relative promoter activity was measured 48 h after transfection. The mRNA relative expression levels of NLRP3 (b) and IL-1 β (c) in PMA stimulated THP-1 cells were detected 48 h after treatment with 100 nM DAC. (d) Supernatants of PMA stimulated THP-1 cells were collected 48 h after treatment by different concentrations of DAC, and the cell viability was analyzed using CCK-8 kit ( ∗∗ p
    Figure Legend Snippet: DNA methylation modification regulated NLRP3 activation in vitro . (a) The methylated ( Sss I treated) or unmethylated pNLRP3-P1/P2 was transfected into HEK293T cells together with pRL, respectively, and each relative promoter activity was measured 48 h after transfection. The mRNA relative expression levels of NLRP3 (b) and IL-1 β (c) in PMA stimulated THP-1 cells were detected 48 h after treatment with 100 nM DAC. (d) Supernatants of PMA stimulated THP-1 cells were collected 48 h after treatment by different concentrations of DAC, and the cell viability was analyzed using CCK-8 kit ( ∗∗ p

    Techniques Used: DNA Methylation Assay, Modification, Activation Assay, In Vitro, Methylation, Transfection, Activity Assay, Expressing, CCK-8 Assay

    24) Product Images from "Skin Characteristics in Patients with Pityriasis Versicolor Using Non-Invasive Method, MPA5"

    Article Title: Skin Characteristics in Patients with Pityriasis Versicolor Using Non-Invasive Method, MPA5

    Journal: Annals of Dermatology

    doi: 10.5021/ad.2012.24.4.444

    PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hyperpigmented lesions of 21 patients. Lanes: M: molecular Marker, 1: Malassezia globosa (CBS7966), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. globosa (CBS7966), 5: M. restricta (KCTC7848), 6: M. restricta (KCTC7848), 7: M. restricta (KCTC7848), 8: M. slooffiae (KCTC17431), 9: M. globosa (CBS7966), 10: M. globosa (CBS7966), 11: M. restricta (KCTC7848), 12: M. globosa (CBS7966), 13: M. globosa (CBS7966), 14: M. furfur (KCTC7743), 15: M. slooffiae (KCTC17431), 16: M. globosa (CBS7966), 17: M. globosa (CBS7966), 18: M. restricta (KCTC7848), 19: M. restricta (KCTC7848), 20: M. furfur (KCTC7743), 21: M. sympodialis (KCTC7985). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.
    Figure Legend Snippet: PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hyperpigmented lesions of 21 patients. Lanes: M: molecular Marker, 1: Malassezia globosa (CBS7966), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. globosa (CBS7966), 5: M. restricta (KCTC7848), 6: M. restricta (KCTC7848), 7: M. restricta (KCTC7848), 8: M. slooffiae (KCTC17431), 9: M. globosa (CBS7966), 10: M. globosa (CBS7966), 11: M. restricta (KCTC7848), 12: M. globosa (CBS7966), 13: M. globosa (CBS7966), 14: M. furfur (KCTC7743), 15: M. slooffiae (KCTC17431), 16: M. globosa (CBS7966), 17: M. globosa (CBS7966), 18: M. restricta (KCTC7848), 19: M. restricta (KCTC7848), 20: M. furfur (KCTC7743), 21: M. sympodialis (KCTC7985). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.

    Techniques Used: Polymerase Chain Reaction, Marker

    PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hypopigmented lesions of 9 patients. Lanes: M: molecular Marker , 1: Malassezia slooffiae (KCTC17431), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. restricta (KCTC7848), 5: M. globosa (CBS7966), 6: M. globosa (CBS7966), 7: M. globosa (CBS7966), 8: M. sympodialis (KCTC7985), 9: M. furfur (KCTC7743). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.
    Figure Legend Snippet: PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hypopigmented lesions of 9 patients. Lanes: M: molecular Marker , 1: Malassezia slooffiae (KCTC17431), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. restricta (KCTC7848), 5: M. globosa (CBS7966), 6: M. globosa (CBS7966), 7: M. globosa (CBS7966), 8: M. sympodialis (KCTC7985), 9: M. furfur (KCTC7743). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.

    Techniques Used: Polymerase Chain Reaction, Marker

    25) Product Images from "Evolutionary conserved NSL complex/BRD4 axis controls transcription activation via histone acetylation"

    Article Title: Evolutionary conserved NSL complex/BRD4 axis controls transcription activation via histone acetylation

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16103-0

    RNAi screen identifies functional NSL complex interactome. a Scheme of primary genome-wide screen. Plasmids containing a fusion construct, nsl3 gene fused to Gal4 DNA-binding domain ( Gal4-DBD ), a firefly luciferase reporter containing Gal4 DNA-binding upstream activating sequence elements (5xUAS) and a constitutively active hsp70 Renilla luciferase reporter were co-transfected into S2 cells. Effect of RNAi of a candidate (X) on reporter signal is assayed. Renilla signal serves a control for transfection efficiency. b Data distribution of primary RNAi screen. Scatterplot of Z-scores and luciferase signal (average of two replicates) are plotted for each knockdown (within the Z-score range of −70 to +6). Data points from grey shaded areas were used for secondary screen. Grey datapoints: candidates excluded due to strong effect on Renilla signal (see Methods for more details on filtering and analysis of RNAi screen), orange datapoints: positive control knockdowns, blue datapoints: negative control knockdowns (GST, GFP and Diap1), green triangles: other candidates (dBRD4, Nurf complex and PAF complex). c Scheme of secondary screen reporter assay. Upper part as in a , lower part: Gal4-DBD fused to Gal4 activation domain ( Gal4-AD ) represents the canonical full length Gal4 . Full length Gal4 it is used as control, to discriminate NSL unspecific transcription factors. d Venn diagram depicting overlap of candidates that scored in the primary and secondary Nsl3-Gal4-DBD screens. The same thresholds for firefly luciferase signal relative to Renilla luciferase signal were applied for both primary and secondary screens. e Heatmap of log-scaled fold changes of normalized luciferase signal in the primary and secondary RNAi screens. Results for the 367 knockdowns performed in the secondary assays are plotted. The order of genes was generated by unsupervised hierarchical clustering. f Z-scores of genome-wide RNAi screen for several complexes and protein categories are listed. If a gene was targeted by multiple dsRNAs, an average of the respective Z-scores is given.
    Figure Legend Snippet: RNAi screen identifies functional NSL complex interactome. a Scheme of primary genome-wide screen. Plasmids containing a fusion construct, nsl3 gene fused to Gal4 DNA-binding domain ( Gal4-DBD ), a firefly luciferase reporter containing Gal4 DNA-binding upstream activating sequence elements (5xUAS) and a constitutively active hsp70 Renilla luciferase reporter were co-transfected into S2 cells. Effect of RNAi of a candidate (X) on reporter signal is assayed. Renilla signal serves a control for transfection efficiency. b Data distribution of primary RNAi screen. Scatterplot of Z-scores and luciferase signal (average of two replicates) are plotted for each knockdown (within the Z-score range of −70 to +6). Data points from grey shaded areas were used for secondary screen. Grey datapoints: candidates excluded due to strong effect on Renilla signal (see Methods for more details on filtering and analysis of RNAi screen), orange datapoints: positive control knockdowns, blue datapoints: negative control knockdowns (GST, GFP and Diap1), green triangles: other candidates (dBRD4, Nurf complex and PAF complex). c Scheme of secondary screen reporter assay. Upper part as in a , lower part: Gal4-DBD fused to Gal4 activation domain ( Gal4-AD ) represents the canonical full length Gal4 . Full length Gal4 it is used as control, to discriminate NSL unspecific transcription factors. d Venn diagram depicting overlap of candidates that scored in the primary and secondary Nsl3-Gal4-DBD screens. The same thresholds for firefly luciferase signal relative to Renilla luciferase signal were applied for both primary and secondary screens. e Heatmap of log-scaled fold changes of normalized luciferase signal in the primary and secondary RNAi screens. Results for the 367 knockdowns performed in the secondary assays are plotted. The order of genes was generated by unsupervised hierarchical clustering. f Z-scores of genome-wide RNAi screen for several complexes and protein categories are listed. If a gene was targeted by multiple dsRNAs, an average of the respective Z-scores is given.

    Techniques Used: Functional Assay, Genome Wide, Construct, Binding Assay, Luciferase, Sequencing, Transfection, Positive Control, Negative Control, Reporter Assay, Activation Assay, Generated

    26) Product Images from "Single-stranded DNA and RNA origami"

    Article Title: Single-stranded DNA and RNA origami

    Journal: Science (New York, N.Y.)

    doi: 10.1126/science.aao2648

    Schematic of ssOrigami synthesis and replication by in vitro PCR and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind III. ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).
    Figure Legend Snippet: Schematic of ssOrigami synthesis and replication by in vitro PCR and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind III. ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).

    Techniques Used: In Vitro, Polymerase Chain Reaction, In Vivo, Clone Assay, Sequencing, Modification, Ligation, Plasmid Preparation, Transformation Assay, Amplification, Produced

    27) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

    Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube
    Figure Legend Snippet: Storage condition of purified ChIP DNA is important. Purified ChIP DNA was adjusted to a concentration of 1 ng/μL ( a ) or 0.1 ng/μL ( b ), aliquoted into 4 different types of microcentrifuge tubes in 15 μL volume, and stored at −20 °C. DNA was quantified using Qubit dsDNA High Sensitivity assay at the indicated time points and expressed as a percentage of the amount measured at day 0. Three independent DNA samples were used in the experiment and DNA concentration from five tubes were measured at each time point. MaxyClear, Axygen® 1.7 mL MaxyClear Snaplock Microcentrifuge Tube; LoBind, Eppendorf DNA LoBind Snap Cap PCR Tube; Siliconized, Fisherbrand™ Siliconized Low-Retention Microcentrifuge Tube; Premium, Fisherbrand™ Premium Microcentrifuge Tube

    Techniques Used: Purification, Chromatin Immunoprecipitation, Concentration Assay, Sensitive Assay, Polymerase Chain Reaction

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Figure Legend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Techniques Used: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    28) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    29) Product Images from "Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis"

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

    Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).
    Figure Legend Snippet: Darwin Assembly using a θ oligonucleotide. Here, a single θ oligonucleotide is used in place of the two boundary oligonucleotides allowing enzymatic cleanup after the assembly reaction. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the nicked strand degraded by exonuclease III (1). Inner oligonucleotides and a single θ oligonucleotide are annealed to the ssDNA plasmid (2). The θ oligonucleotide encodes both assembly priming and termination sequences linked by a flexible linker such that successful assembly of the mutated strand results in a closed circle (3). The template plasmid can now be linearized (e.g. at the yellow dot, by adding a targeting oligonucleotide and appropriate restriction endonuclease) and both exonuclease I and exonuclease III added to degrade any non-circular DNA (4). The mutated gene can now be amplified from the closed circle by PCR (5) and cloned into a fresh vector (6) using the type IIS restriction sites (white dots).

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay

    Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.
    Figure Legend Snippet: Darwin assembled TgoT DNA polymerase library. ( A ) Five separate sequencing reactions (range and reads shown in blue) were required to sample the diversity introduced across the eight target residues (shown in red along the TgoT gene). Mutations included focused degeneracies (e.g. YWC used against Y384) or ‘small intelligent’ (S-int) diversity (NDT, VMA, ATG and TGG oligonucleotides mixed in a 12:6:1:1 ratio). Resulting incorporation is shown in box plots with outliers explicitly labelled. Wild-type contamination was determined from positions where diversity excluded those sequences (N.A.: not applicable). As with the T7 RNA polymerase library, incorporation trends and biases were analysed to identify any biases in assembly. Ranked incorporation frequencies are shown for the residues targeted with ‘small intelligent’ diversity, and the top three highest (greens) and lowest (oranges) ranked codons (based on a straight sum of ranks) are highlighted ( B ). Outnest PCR of the TgoT DNA polymerase library (expected product of 2501 bp) showing that the final PCR can be carried out with either A- (MyTaq) or B-family (Q5) polymerases ( C ). MW: 1 kb ladder (NEB). NT: no template PCR control.

    Techniques Used: Sequencing, Polymerase Chain Reaction

    Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).
    Figure Legend Snippet: Principles of Darwin Assembly. Plasmid DNA (black, with the gene of interest in orange) is nicked by a nicking endonuclease (at the purple dot) and the cut strand degraded by exonuclease III (1). Boundary and inner (mutagenic) oligonucleotides are annealed to the ssDNA plasmid (2). Key features of the oligonucleotides are highlighted: 5′-boundary oligonucleotide is 5′-biotinylated; non-complementary overhangs are shown in blue with Type IIs endonuclease recognition sites shown in white; mutations are shown as red X in the inner oligonucleotides; 3′-boundary oligonucleotide is protected at its 3′-end. After annealing, primers are extended and ligated in an isothermal assembly reaction (3). The assembled strand can be isolated by paramagnetic streptavidin-coated beads (4) and purified by alkali washing prior to PCR using outnested priming sites (5) and cloning (6) using the type IIS restriction sites (white dots). The purification step (4) is not always necessary but we found it improved PCR performance, especially for long assembly reactions ( > 1 kb).

    Techniques Used: Plasmid Preparation, Isolation, Purification, Polymerase Chain Reaction, Clone Assay

    30) Product Images from "Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens"

    Article Title: Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0235222

    Distribution of the total extracted DNA from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® PCR DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.
    Figure Legend Snippet: Distribution of the total extracted DNA from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® PCR DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.

    Techniques Used: Polymerase Chain Reaction

    Comparison of fragment sizes between two protocols from three specimens. Electropherograms from the DNeasy extraction Kit (blue) and the Monarch PCR DNA Clean-up Kit (red). The specimens individual MTD-TW numbers are as follows: A 9248, B 9252, C 9251. See Supplementary Table 1 for more details on the DNA yield from each extraction.
    Figure Legend Snippet: Comparison of fragment sizes between two protocols from three specimens. Electropherograms from the DNeasy extraction Kit (blue) and the Monarch PCR DNA Clean-up Kit (red). The specimens individual MTD-TW numbers are as follows: A 9248, B 9252, C 9251. See Supplementary Table 1 for more details on the DNA yield from each extraction.

    Techniques Used: Polymerase Chain Reaction

    31) Product Images from "Detection of Astrovirus in Historical Cases of European Sporadic Bovine Encephalitis, Switzerland 1958–1976"

    Article Title: Detection of Astrovirus in Historical Cases of European Sporadic Bovine Encephalitis, Switzerland 1958–1976

    Journal: Frontiers in Veterinary Science

    doi: 10.3389/fvets.2016.00091

    Results of RT-PCR and sequencing of astrovirus RNA extracted from case 3466 . A scheme of the BoAstV CH13 genome is presented. The viral genome is organized in three open-reading frames (ORF). The red bar indicates the target sequence for RT-PCR using primers MA2/MA4 within ORF 1b, which encodes for the RNA-dependent RNA polymerase. The blue and black bars show target sequences of in situ hybridization probes A and B, respectively, within ORF2 that encodes for structural capsid proteins. The nested RT-PCR protocol with primers bAV3/bAV4 (first round) and bAV1/bAV2 (second round) was designed to yield amplicons in the target region of ISH probe B (black bar). Sequence comparisons of the MA2/MA4 and of the bAV1/bAV2 amplicons of case 3466 with the BoAstV CH13 reference sequence (GenBank accession number: NC_024498) are shown. Alignments were generated with the Geneious R9 software, version 9.0.4 (Biomatters).
    Figure Legend Snippet: Results of RT-PCR and sequencing of astrovirus RNA extracted from case 3466 . A scheme of the BoAstV CH13 genome is presented. The viral genome is organized in three open-reading frames (ORF). The red bar indicates the target sequence for RT-PCR using primers MA2/MA4 within ORF 1b, which encodes for the RNA-dependent RNA polymerase. The blue and black bars show target sequences of in situ hybridization probes A and B, respectively, within ORF2 that encodes for structural capsid proteins. The nested RT-PCR protocol with primers bAV3/bAV4 (first round) and bAV1/bAV2 (second round) was designed to yield amplicons in the target region of ISH probe B (black bar). Sequence comparisons of the MA2/MA4 and of the bAV1/bAV2 amplicons of case 3466 with the BoAstV CH13 reference sequence (GenBank accession number: NC_024498) are shown. Alignments were generated with the Geneious R9 software, version 9.0.4 (Biomatters).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing, In Situ Hybridization, Generated, Software

    32) Product Images from "Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery"

    Article Title: Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery

    Journal: Nature Communications

    doi: 10.1038/ncomms15790

    Activity of a high-fidelity base editor (HF-BE3) in human cells. ( a – c ) On- and off-target editing associated with plasmid transfection of BE3 and HF-BE3 was assayed using high-throughput sequencing of genomic DNA from HEK293T cells treated with sgRNAs targeting non-repetitive genomic loci EMX1 ( a ), FANCF ( b ) and HEK293 site 3 ( c ). On- and off-target loci associated with each sgRNA are separated by a vertical line. ( d ) On- and off-target editing associated with the highly repetitive sgRNA targeting VEGFA site 2. Values and error bars reflect mean±s.d. of three independent biological replicates performed on different days. For a – c , stars indicate significant editing based on a comparison between the treated sample and an untreated control. * P ≤0.05, ** P ≤0.01 and *** P ≤0.001 (Student's two-tailed t -test). For d , asterisks are not shown since all treated samples displayed significant editing relative to the control. Individual P values are listed in Supplementary Table 1 .
    Figure Legend Snippet: Activity of a high-fidelity base editor (HF-BE3) in human cells. ( a – c ) On- and off-target editing associated with plasmid transfection of BE3 and HF-BE3 was assayed using high-throughput sequencing of genomic DNA from HEK293T cells treated with sgRNAs targeting non-repetitive genomic loci EMX1 ( a ), FANCF ( b ) and HEK293 site 3 ( c ). On- and off-target loci associated with each sgRNA are separated by a vertical line. ( d ) On- and off-target editing associated with the highly repetitive sgRNA targeting VEGFA site 2. Values and error bars reflect mean±s.d. of three independent biological replicates performed on different days. For a – c , stars indicate significant editing based on a comparison between the treated sample and an untreated control. * P ≤0.05, ** P ≤0.01 and *** P ≤0.001 (Student's two-tailed t -test). For d , asterisks are not shown since all treated samples displayed significant editing relative to the control. Individual P values are listed in Supplementary Table 1 .

    Techniques Used: Activity Assay, Plasmid Preparation, Transfection, Next-Generation Sequencing, Two Tailed Test

    DNA-free in vivo base editing in zebrafish embryos and in the inner ear of live mice using RNP delivery of BE3. ( a ) On-target genome editing in zebrafish harvested 4 days after injection of BE3 complexed with indicated sgRNA. Values and error bars reflect mean±s.d. of three injected and three control zebrafish. Controls were injected with BE3 complexed with an unrelated sgRNA. ( b ) Schematic showing in vivo injection of BE3:sgRNA complexes encapsulated into cationic lipid nanoparticles. ( c ) Base editing of cytosine residues in the base editor window at the VEGFA site 2 genomic locus. ( d ) On-target editing at each cytosine in the base-editing window of the VEGFA site 2 target locus. ( c , d ) Values and error bars reflect mean±s.e.m. of three mice injected with sgRNA targeting VEGFA site 2, three uninjected mice and one mouse injected with unrelated sgRNA.
    Figure Legend Snippet: DNA-free in vivo base editing in zebrafish embryos and in the inner ear of live mice using RNP delivery of BE3. ( a ) On-target genome editing in zebrafish harvested 4 days after injection of BE3 complexed with indicated sgRNA. Values and error bars reflect mean±s.d. of three injected and three control zebrafish. Controls were injected with BE3 complexed with an unrelated sgRNA. ( b ) Schematic showing in vivo injection of BE3:sgRNA complexes encapsulated into cationic lipid nanoparticles. ( c ) Base editing of cytosine residues in the base editor window at the VEGFA site 2 genomic locus. ( d ) On-target editing at each cytosine in the base-editing window of the VEGFA site 2 target locus. ( c , d ) Values and error bars reflect mean±s.e.m. of three mice injected with sgRNA targeting VEGFA site 2, three uninjected mice and one mouse injected with unrelated sgRNA.

    Techniques Used: In Vivo, Mouse Assay, Injection

    33) Product Images from "Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor"

    Article Title: Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20199071

    Illustration of the biosensor concept to measure glycolytic fluxes in single S. cerevisiae cells Expression of the bacterial transcriptional repressor CggR at constant levels, i.e., independent of growth rate and substrates. Binding of CggR as a dimer of dimers to the operator (CggRO) of the synthetic cis‐regulatory region, forming the CggR–DNA complex repressing transcription. At high glycolytic fluxes, fructose‐1,6‐bisphosphate (FBP) levels are high and FBP binds to CggR disrupting the dimer–dimer contacts, which induces a conformational change in the repressor, such that transcription of the reporter gene (YFP) can occur. The binding of FBP to CggR and consequent transcription is dependent on the FBP concentration, which correlates with glycolytic flux. The activity of the glycolytic flux biosensor is measured by quantifying YFP expression. YFP expression levels are normalized through a second reporter, mCherry, under the control of TEF1 mutant 8 promoter (P TEFmut8 ), to control for global variation in protein expression activity.
    Figure Legend Snippet: Illustration of the biosensor concept to measure glycolytic fluxes in single S. cerevisiae cells Expression of the bacterial transcriptional repressor CggR at constant levels, i.e., independent of growth rate and substrates. Binding of CggR as a dimer of dimers to the operator (CggRO) of the synthetic cis‐regulatory region, forming the CggR–DNA complex repressing transcription. At high glycolytic fluxes, fructose‐1,6‐bisphosphate (FBP) levels are high and FBP binds to CggR disrupting the dimer–dimer contacts, which induces a conformational change in the repressor, such that transcription of the reporter gene (YFP) can occur. The binding of FBP to CggR and consequent transcription is dependent on the FBP concentration, which correlates with glycolytic flux. The activity of the glycolytic flux biosensor is measured by quantifying YFP expression. YFP expression levels are normalized through a second reporter, mCherry, under the control of TEF1 mutant 8 promoter (P TEFmut8 ), to control for global variation in protein expression activity.

    Techniques Used: Expressing, Binding Assay, Concentration Assay, Activity Assay, Mutagenesis

    The glycolytic flux sensor can measure glycolytic flux in individual cells Subpopulations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. Histogram of single‐cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a subpopulation of minimally 5% can be distinguished. Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6‐phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al , 2019 ). The boxplot horizontal line indicates the median and the box extends from the 25 th to 75 th percentiles. Plotted points are outliers that are higher or lower than the upper and lower whiskers, respectively. YFP/mCherry ratio measured by microscopy in co‐existing dividing (high flux) versus non‐dividing (low flux) isogenic TM6 cells on 10 g/l glucose. Each data point corresponds to data from a single cell. Brightfield (BF), YFP, and mCherry microscopy images for a co‐existing dividing (high flux) and a non‐dividing (low flux) TM6 cell expressing the flux sensor in 10 g/l glucose minimal medium. The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor‐expressing strain (F), which reflects the cell‐cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell‐cycle trajectories as the difference between the YFP and mCherry production rates normalized to have the same scale (see more details in Materials and methods; Appendix Fig S10 ). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5,000 iterations). We smoothed the single‐cell‐cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent‐protein maturation in a first‐order kinetics model. To align the cell‐cycle trajectories and to calculate the phase, we used the array of three cell‐cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. Specifically, we computed the average cell‐cycle‐relative timing for each of these events φ ¯ e in the following way: ∀ e ∈ E φ ¯ e = 1 N ∑ c c = 1 N t cc e − t cc cyt t cc n e x t c y t − t cc cyt , where N is the number of cell cycles in the replicate of interest, and t cc e is the time in minutes when the event e happens in the cell cycle cc . The orange vertical lines denote φ ¯ budding for both replicates. In the aligned cell cycles, we converted the time in minutes t to the phase φ cc in the following way: φ cc = φ ¯ E [ i + 1 ] − φ ¯ E [ i ] t − t cc E [ i ] t cc E [ i + 1 ] − t cc E [ i ] + φ ¯ E [ i ] for t ∈ t cc E [ i ] , t cc E [ i + 1 ] if E [ i ] = cyt or t ∈ t cc E [ i ] , t cc E [ i + 1 ] if E [ i ] ≠ cyt, where i is the index number of an event in the array E . The cell cycles used for the analysis had the duration in the interval between 150 and 300 min, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 g/l glucose in the microfluidic device.
    Figure Legend Snippet: The glycolytic flux sensor can measure glycolytic flux in individual cells Subpopulations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. Histogram of single‐cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a subpopulation of minimally 5% can be distinguished. Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6‐phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al , 2019 ). The boxplot horizontal line indicates the median and the box extends from the 25 th to 75 th percentiles. Plotted points are outliers that are higher or lower than the upper and lower whiskers, respectively. YFP/mCherry ratio measured by microscopy in co‐existing dividing (high flux) versus non‐dividing (low flux) isogenic TM6 cells on 10 g/l glucose. Each data point corresponds to data from a single cell. Brightfield (BF), YFP, and mCherry microscopy images for a co‐existing dividing (high flux) and a non‐dividing (low flux) TM6 cell expressing the flux sensor in 10 g/l glucose minimal medium. The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor‐expressing strain (F), which reflects the cell‐cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell‐cycle trajectories as the difference between the YFP and mCherry production rates normalized to have the same scale (see more details in Materials and methods; Appendix Fig S10 ). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5,000 iterations). We smoothed the single‐cell‐cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent‐protein maturation in a first‐order kinetics model. To align the cell‐cycle trajectories and to calculate the phase, we used the array of three cell‐cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. Specifically, we computed the average cell‐cycle‐relative timing for each of these events φ ¯ e in the following way: ∀ e ∈ E φ ¯ e = 1 N ∑ c c = 1 N t cc e − t cc cyt t cc n e x t c y t − t cc cyt , where N is the number of cell cycles in the replicate of interest, and t cc e is the time in minutes when the event e happens in the cell cycle cc . The orange vertical lines denote φ ¯ budding for both replicates. In the aligned cell cycles, we converted the time in minutes t to the phase φ cc in the following way: φ cc = φ ¯ E [ i + 1 ] − φ ¯ E [ i ] t − t cc E [ i ] t cc E [ i + 1 ] − t cc E [ i ] + φ ¯ E [ i ] for t ∈ t cc E [ i ] , t cc E [ i + 1 ] if E [ i ] = cyt or t ∈ t cc E [ i ] , t cc E [ i + 1 ] if E [ i ] ≠ cyt, where i is the index number of an event in the array E . The cell cycles used for the analysis had the duration in the interval between 150 and 300 min, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 g/l glucose in the microfluidic device.

    Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Microscopy, Expressing, Concentration Assay

    The engineered flux‐sensor reports glycolytic flux with a high dynamic flux range Overview about the different design steps in our promotor engineering strategy (cf. also Appendix Fig S4 ). The four reporter plasmids were transferred to the wild‐type strain containing the CggR (R250A) under the control of the P TEFmut7 . The strength of the four promoters was assessed by quantifying YFP (FL1) and mCherry (FL3) fluorescence in exponentially growing wild‐type cells in minimal medium with 10 g/l glucose. The FL1 and FL3 fluorescence shown is the non‐background‐corrected median of 100,000 cell events. The non‐FL control is the signal from a wild‐type strain grown under the same conditions. Error bars represent the standard deviation of three independent experiments. The background fluorescence, assessed by the wild‐type harboring the YCplac33 plasmid, was subtracted from FL1 and FL3. The final reporter activity is the ratio of the background‐corrected YFP and mCherry values. Error bars represent the standard deviation of three independently determined ratios from three replicate experiments. Reporter activity of the sensor across (D) multiple FBP levels and (E) glycolytic fluxes. The glycolytic flux is reported as the flux between the metabolites fructose 6‐phosphate (F6P) and fructose‐1,6‐bisphosphate (FBP). Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al , 2019 ). Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid ( Appendix Table S8 ). The control is the wild type and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments. Fraction of CggR bound to FBP across FBP concentrations. The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant. The percentage of CggR molecules bound to FBP was calculated after normalizing the T m values for unbound/bound state using the T m at 0 mM FBP as unbound and at 36 mM (corresponding to maximum FBP concentration used) as total bound states. The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one‐site specific binding model in GraphPad. The solid line corresponds to the wild‐type CggR and the dashed line to the R250A variant. Vertical lines delimit the physiological FBP range.
    Figure Legend Snippet: The engineered flux‐sensor reports glycolytic flux with a high dynamic flux range Overview about the different design steps in our promotor engineering strategy (cf. also Appendix Fig S4 ). The four reporter plasmids were transferred to the wild‐type strain containing the CggR (R250A) under the control of the P TEFmut7 . The strength of the four promoters was assessed by quantifying YFP (FL1) and mCherry (FL3) fluorescence in exponentially growing wild‐type cells in minimal medium with 10 g/l glucose. The FL1 and FL3 fluorescence shown is the non‐background‐corrected median of 100,000 cell events. The non‐FL control is the signal from a wild‐type strain grown under the same conditions. Error bars represent the standard deviation of three independent experiments. The background fluorescence, assessed by the wild‐type harboring the YCplac33 plasmid, was subtracted from FL1 and FL3. The final reporter activity is the ratio of the background‐corrected YFP and mCherry values. Error bars represent the standard deviation of three independently determined ratios from three replicate experiments. Reporter activity of the sensor across (D) multiple FBP levels and (E) glycolytic fluxes. The glycolytic flux is reported as the flux between the metabolites fructose 6‐phosphate (F6P) and fructose‐1,6‐bisphosphate (FBP). Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al , 2019 ). Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid ( Appendix Table S8 ). The control is the wild type and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments. Fraction of CggR bound to FBP across FBP concentrations. The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant. The percentage of CggR molecules bound to FBP was calculated after normalizing the T m values for unbound/bound state using the T m at 0 mM FBP as unbound and at 36 mM (corresponding to maximum FBP concentration used) as total bound states. The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one‐site specific binding model in GraphPad. The solid line corresponds to the wild‐type CggR and the dashed line to the R250A variant. Vertical lines delimit the physiological FBP range.

    Techniques Used: Fluorescence, Standard Deviation, Plasmid Preparation, Activity Assay, Flow Cytometry, Cytometry, Expressing, Variant Assay, Concentration Assay, Binding Assay

    34) Product Images from "Engineered cartilage from human chondrocytes with homozygous knockout of cell cycle inhibitor p21"

    Article Title: Engineered cartilage from human chondrocytes with homozygous knockout of cell cycle inhibitor p21

    Journal: bioRxiv

    doi: 10.1101/731216

    Screening and efficiency of homozygous p21 knockout. A) Sequence of p21 targeted by editing, with start of coding region in green, guide RNA target sites underlined, PAM sites in blue, expected cut sites denoted by red arrows. B) Representative gel showing the emergence of the ∼222 base pair shorter product with the inclusion of guide RNAs targeting p21. DNA from bulk population of targeted cells (left) or individual colonies (right). C) 80 colonies from donors 2 and 3 were screened for editing outcomes by PCR. D) Sanger sequencing of representative colonies with predicted and actual reads noted.
    Figure Legend Snippet: Screening and efficiency of homozygous p21 knockout. A) Sequence of p21 targeted by editing, with start of coding region in green, guide RNA target sites underlined, PAM sites in blue, expected cut sites denoted by red arrows. B) Representative gel showing the emergence of the ∼222 base pair shorter product with the inclusion of guide RNAs targeting p21. DNA from bulk population of targeted cells (left) or individual colonies (right). C) 80 colonies from donors 2 and 3 were screened for editing outcomes by PCR. D) Sanger sequencing of representative colonies with predicted and actual reads noted.

    Techniques Used: Knock-Out, Sequencing, Polymerase Chain Reaction

    35) Product Images from "Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens"

    Article Title: Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0235222

    Distribution of the total extracted DNA from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® PCR DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.
    Figure Legend Snippet: Distribution of the total extracted DNA from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® PCR DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.

    Techniques Used: Polymerase Chain Reaction

    Comparison of fragment sizes between two protocols from three specimens. Electropherograms from the DNeasy extraction Kit (blue) and the Monarch PCR DNA Clean-up Kit (red). The specimens individual MTD-TW numbers are as follows: A 9248, B 9252, C 9251. See Supplementary Table 1 for more details on the DNA yield from each extraction.
    Figure Legend Snippet: Comparison of fragment sizes between two protocols from three specimens. Electropherograms from the DNeasy extraction Kit (blue) and the Monarch PCR DNA Clean-up Kit (red). The specimens individual MTD-TW numbers are as follows: A 9248, B 9252, C 9251. See Supplementary Table 1 for more details on the DNA yield from each extraction.

    Techniques Used: Polymerase Chain Reaction

    36) Product Images from "Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence"

    Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-12-47

    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.
    Figure Legend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Incubation, Labeling

    Assessment of various DNA polymerases for their ability to incorporate BrdU . Complete and incomplete specific incorporation reactions (Figure 1) were carried out with 5 DNA Polymerases: Bst exo - (thermophilic), T4 (mesophilic), Taq (thermophilic), OptiTaq (thermophilic blend) and Pfu (hyperthermophilic) in the presence of BrdUTP. Lanes M, Perfect 100 bp Ladder; lane 1, PCR 1 fragment (379 bp); lane 2, BsaI-cleaved PCR 1 fragment; lane 3, PCR 2 fragment (625 bp); lane 4, BsaI-cleaved PCR 2 fragment; lane 5, BsaI restriction fragments: I (363 bp) and III (609 bp). Lanes 6-18 reactions with specified DNA Polymerases: lane 6, restriction fragments: I and III, T4; lane 7, restriction fragments: I and III, Bst exo - ; lane 8, restriction fragments: I and III, Bst exo - , T4 DNA Ligase; lane 9, restriction fragments: I and III, T4; lane 10, restriction fragments: I and III, T4, T4 DNA Ligase; lane 11, restriction fragments: I and III, Taq; lane 12, restriction fragments: I and III, Taq, T4 DNA Ligase; lane 13, restriction fragments: I and III, OptiTaq; lane 14, restriction fragments: I and III, OptiTaq, T4 DNA Ligase; lane 15, restriction fragments: I and III, Tfl; lane 16, restriction fragments: I and III, Tfl, T4 DNA Ligase; lane 17, restriction fragments: I and III, Pfu; lane 18, restriction fragments: I and III, Pfu, T4 DNA Ligase. I, III BsaI restriction fragments numbered as in Figure 1.
    Figure Legend Snippet: Assessment of various DNA polymerases for their ability to incorporate BrdU . Complete and incomplete specific incorporation reactions (Figure 1) were carried out with 5 DNA Polymerases: Bst exo - (thermophilic), T4 (mesophilic), Taq (thermophilic), OptiTaq (thermophilic blend) and Pfu (hyperthermophilic) in the presence of BrdUTP. Lanes M, Perfect 100 bp Ladder; lane 1, PCR 1 fragment (379 bp); lane 2, BsaI-cleaved PCR 1 fragment; lane 3, PCR 2 fragment (625 bp); lane 4, BsaI-cleaved PCR 2 fragment; lane 5, BsaI restriction fragments: I (363 bp) and III (609 bp). Lanes 6-18 reactions with specified DNA Polymerases: lane 6, restriction fragments: I and III, T4; lane 7, restriction fragments: I and III, Bst exo - ; lane 8, restriction fragments: I and III, Bst exo - , T4 DNA Ligase; lane 9, restriction fragments: I and III, T4; lane 10, restriction fragments: I and III, T4, T4 DNA Ligase; lane 11, restriction fragments: I and III, Taq; lane 12, restriction fragments: I and III, Taq, T4 DNA Ligase; lane 13, restriction fragments: I and III, OptiTaq; lane 14, restriction fragments: I and III, OptiTaq, T4 DNA Ligase; lane 15, restriction fragments: I and III, Tfl; lane 16, restriction fragments: I and III, Tfl, T4 DNA Ligase; lane 17, restriction fragments: I and III, Pfu; lane 18, restriction fragments: I and III, Pfu, T4 DNA Ligase. I, III BsaI restriction fragments numbered as in Figure 1.

    Techniques Used: Polymerase Chain Reaction

    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 441 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked); lane 1, 260 bp BsaI-cleaved PCR (restriction fragment I); lane 2, 208 bp BsaI-cleaved PCR (restriction fragment III); lane 3, BrdUTP-filled restriction fragments I and III, T4 DNA ligase; lane 4, BrdUTP-filled restriction fragments I and III; lane 5, dTTP-filled restriction fragment I and BrdUTP-filled restriction fragment III, T4 DNA ligase; lane 6, dTTP-filled restriction fragment I and BrdU-filled restriction fragment III. Lanes 7-9, controls of enzymes functional purity: lane 7, control PCR fragment with internal BsaI site; lane 8, BsaI-cleaved control PCR fragment; lane 9, BsaI-cleaved control PCR fragment after addition of T4 DNA Ligase; lane M, Perfect 100 bp Ladder. I, III BsaI restriction fragments numbered as in Figure 1.
    Figure Legend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 441 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked); lane 1, 260 bp BsaI-cleaved PCR (restriction fragment I); lane 2, 208 bp BsaI-cleaved PCR (restriction fragment III); lane 3, BrdUTP-filled restriction fragments I and III, T4 DNA ligase; lane 4, BrdUTP-filled restriction fragments I and III; lane 5, dTTP-filled restriction fragment I and BrdUTP-filled restriction fragment III, T4 DNA ligase; lane 6, dTTP-filled restriction fragment I and BrdU-filled restriction fragment III. Lanes 7-9, controls of enzymes functional purity: lane 7, control PCR fragment with internal BsaI site; lane 8, BsaI-cleaved control PCR fragment; lane 9, BsaI-cleaved control PCR fragment after addition of T4 DNA Ligase; lane M, Perfect 100 bp Ladder. I, III BsaI restriction fragments numbered as in Figure 1.

    Techniques Used: Polymerase Chain Reaction, Functional Assay

    37) Product Images from "Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum"

    Article Title: Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2014.00186

    O. tsutsugamushi transcriptionally expresses Ank, T1SS, and T4SS genes during infection of mammalian host cells . RT-PCR using primers targeting genes encoding O. tsutsugamushi Anks (A) , T1SS components (B) , T4SS components (C) , and the 16S rRNA gene (infection control) (A) was performed on DNA-free total RNA isolated from infected L929 cells (RT lanes). O. tsutsugamushi genomic DNA and water served as positive (+) and negative (−) controls, respectively. Results are representative of two experiments with similar results.
    Figure Legend Snippet: O. tsutsugamushi transcriptionally expresses Ank, T1SS, and T4SS genes during infection of mammalian host cells . RT-PCR using primers targeting genes encoding O. tsutsugamushi Anks (A) , T1SS components (B) , T4SS components (C) , and the 16S rRNA gene (infection control) (A) was performed on DNA-free total RNA isolated from infected L929 cells (RT lanes). O. tsutsugamushi genomic DNA and water served as positive (+) and negative (−) controls, respectively. Results are representative of two experiments with similar results.

    Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Isolation

    38) Product Images from "FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli"

    Article Title: FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0129547

    Construction of FabV and FabI-containing plasmid vectors. The bla gene (AmpR) was removed and restriction enzyme ( Cla I and Sma I) sites were introduced downstream P3 promoter of β-lactamase by whole-plasmid PCR of pUC19, pSA-HP24, and pBR322 vectors. Inserts were prepared by PCR amplifying fab V and fab I using Vibrio cholerae O1 El Tor or E . coli BL21(DE3) genomic DNA respectively as DNA template. Vectors and inserts were restricted using Cla I and Sma I, gel purified, and ligated at 1:3 vector/insert ratio. These manipulations resulted in the construction of (A) FabV-containing pUC19, (B) FabI-containing pUC19, (C) FabV-containing pSA-HP24, and (D) FabI-containing pBR322 plasmid vectors.
    Figure Legend Snippet: Construction of FabV and FabI-containing plasmid vectors. The bla gene (AmpR) was removed and restriction enzyme ( Cla I and Sma I) sites were introduced downstream P3 promoter of β-lactamase by whole-plasmid PCR of pUC19, pSA-HP24, and pBR322 vectors. Inserts were prepared by PCR amplifying fab V and fab I using Vibrio cholerae O1 El Tor or E . coli BL21(DE3) genomic DNA respectively as DNA template. Vectors and inserts were restricted using Cla I and Sma I, gel purified, and ligated at 1:3 vector/insert ratio. These manipulations resulted in the construction of (A) FabV-containing pUC19, (B) FabI-containing pUC19, (C) FabV-containing pSA-HP24, and (D) FabI-containing pBR322 plasmid vectors.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Purification

    39) Product Images from "Assembled Plastid and Mitochondrial Genomes, as well as Nuclear Genes, Place the Parasite Family Cynomoriaceae in the Saxifragales"

    Article Title: Assembled Plastid and Mitochondrial Genomes, as well as Nuclear Genes, Place the Parasite Family Cynomoriaceae in the Saxifragales

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evw147

    Gene losses and rearrangements in the plastome of Cynomorium compared to that of Liquidambar formosana (GenBank accession KC588388). Colored lines link homologous genes between the two plastomes. The LSC of Cynomorium is divided in two parts (linked by the dotted line) to help visualization. Maps were drawn using OGDraw ( Lohse et al. 2013 ). GC content and coverage of the Cynomorium plastome are depicted in blue and red, respectively. (The pattern is the same when using less stringent mapping parameters; Results.).
    Figure Legend Snippet: Gene losses and rearrangements in the plastome of Cynomorium compared to that of Liquidambar formosana (GenBank accession KC588388). Colored lines link homologous genes between the two plastomes. The LSC of Cynomorium is divided in two parts (linked by the dotted line) to help visualization. Maps were drawn using OGDraw ( Lohse et al. 2013 ). GC content and coverage of the Cynomorium plastome are depicted in blue and red, respectively. (The pattern is the same when using less stringent mapping parameters; Results.).

    Techniques Used:

    40) Product Images from "One‐step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock‐in mice"

    Article Title: One‐step CRISPR/Cas9 method for the rapid generation of human antibody heavy chain knock‐in mice

    Journal: The EMBO Journal

    doi: 10.15252/embj.201899243

    Characterization of PGT121 KI mice Schematic of the TaqMan probes and their targeting sites within the WT IgH and PGT121 IgH. T: TaqMan probe. Schematic showing the annealing sites of primers used to validate PGT121 KI animals. Fo.1F and Fo.2F primers were targeted at promoter region and PGT121 region, respectively, and combined with Re.1R primer targeted to the genomic region after homologous 3′ Arm. KI alleles are predicted to result in the amplification of a Fo.1 fragment (3.3 kb) and Fo.2 fragment (2.8 kb). Genomic DNA was extracted from the F0 founders born after CRISPR injection or from a C57BL/6 (WT) mouse. Long‐range PCR was performed to detect the insertion of the PGT121 VDJ sequences at the correct genomic locus. Table showing the frequency of the different genotypes of mice generated after CRISPR injection with plasmid donors containing long or short homology arms. # of HDR occurrence indicates the integration of the PGT121 heavy chain in the mouse IgH locus. # of Cas9‐mediated D 4 ‐J 4 deletions indicates the efficiency of our sgRNA‐directed Cas9 double‐stranded breaks. HC: heavy chain.
    Figure Legend Snippet: Characterization of PGT121 KI mice Schematic of the TaqMan probes and their targeting sites within the WT IgH and PGT121 IgH. T: TaqMan probe. Schematic showing the annealing sites of primers used to validate PGT121 KI animals. Fo.1F and Fo.2F primers were targeted at promoter region and PGT121 region, respectively, and combined with Re.1R primer targeted to the genomic region after homologous 3′ Arm. KI alleles are predicted to result in the amplification of a Fo.1 fragment (3.3 kb) and Fo.2 fragment (2.8 kb). Genomic DNA was extracted from the F0 founders born after CRISPR injection or from a C57BL/6 (WT) mouse. Long‐range PCR was performed to detect the insertion of the PGT121 VDJ sequences at the correct genomic locus. Table showing the frequency of the different genotypes of mice generated after CRISPR injection with plasmid donors containing long or short homology arms. # of HDR occurrence indicates the integration of the PGT121 heavy chain in the mouse IgH locus. # of Cas9‐mediated D 4 ‐J 4 deletions indicates the efficiency of our sgRNA‐directed Cas9 double‐stranded breaks. HC: heavy chain.

    Techniques Used: Mouse Assay, Amplification, CRISPR, Injection, Polymerase Chain Reaction, Generated, Plasmid Preparation

    One‐step CRISPR zygote injection to generate mice carrying PGT121 heavy chain in the mouse IgH locus Schematic depicting CRISPR/Cas9 injection. A circular plasmid bearing germline PGT121‐gH VDJ sequences, two guide RNAs, and Cas9 protein were injected into zygotes and implanted into pseudopregnant mice. Cas9‐induced double‐stranded breaks in the genome of zygotes are used to insert germline PGT121 VDJ sequences flanked by homologous arms on each side of the cut site via HDR. After 3 weeks, F0 founder mice are born, some of which bear the human bnAbs germline precursor. Strategy for insertion of PGT121 rearranged VDJ into mouse IgH locus. Targeting DNA donor with 5′ (3.9 kb) and 3′ (2.6 kb) homology arms to the C57BL/6 WT mouse IgH locus, murine VHJ558 promoter, leader, and the human PGT121 heavy chain VDJ sequences are located between two homology arms. CRISPR/Cas9‐mediated HDR leads to the insertion of the promoter and PGT121 sequences into the C57BL/6 mouse genome. P: murine VHJ558 promoter; HDR: homology‐directed repair; bnAbs: broadly neutralizing antibodies. sgRNA targeting sites are indicated in red. Three distinct fragments of genomic DNA were amplified by PCR, and in vitro digestion assay was performed with each of the sgRNAs to validate the efficiency of Cas9‐mediated cleavage. Analysis sgRNA off‐target effects in unrelated genes. Amplicons corresponding to Aakt, Map3K10, and Nop9 were generated by PCR by using gene‐specific primers. In vitro digestion assay was performed to measure the Cas9‐directed cleavage efficiency.
    Figure Legend Snippet: One‐step CRISPR zygote injection to generate mice carrying PGT121 heavy chain in the mouse IgH locus Schematic depicting CRISPR/Cas9 injection. A circular plasmid bearing germline PGT121‐gH VDJ sequences, two guide RNAs, and Cas9 protein were injected into zygotes and implanted into pseudopregnant mice. Cas9‐induced double‐stranded breaks in the genome of zygotes are used to insert germline PGT121 VDJ sequences flanked by homologous arms on each side of the cut site via HDR. After 3 weeks, F0 founder mice are born, some of which bear the human bnAbs germline precursor. Strategy for insertion of PGT121 rearranged VDJ into mouse IgH locus. Targeting DNA donor with 5′ (3.9 kb) and 3′ (2.6 kb) homology arms to the C57BL/6 WT mouse IgH locus, murine VHJ558 promoter, leader, and the human PGT121 heavy chain VDJ sequences are located between two homology arms. CRISPR/Cas9‐mediated HDR leads to the insertion of the promoter and PGT121 sequences into the C57BL/6 mouse genome. P: murine VHJ558 promoter; HDR: homology‐directed repair; bnAbs: broadly neutralizing antibodies. sgRNA targeting sites are indicated in red. Three distinct fragments of genomic DNA were amplified by PCR, and in vitro digestion assay was performed with each of the sgRNAs to validate the efficiency of Cas9‐mediated cleavage. Analysis sgRNA off‐target effects in unrelated genes. Amplicons corresponding to Aakt, Map3K10, and Nop9 were generated by PCR by using gene‐specific primers. In vitro digestion assay was performed to measure the Cas9‐directed cleavage efficiency.

    Techniques Used: CRISPR, Injection, Mouse Assay, Plasmid Preparation, Amplification, Polymerase Chain Reaction, In Vitro, Generated

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    Polymerase Chain Reaction:

    Article Title: High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments
    Article Snippet: .. Other DNA fragments were obtained by PCR with the Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA USA), except for the fragment containing the stem-loop (S-SL), which was obtained with the LA Taq DNA polymerase (Takara Bio Europe) using the GC buffer I. PCR products were purified using either the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), the Wizard® SV Gel and PCR Clean-Up System (Promega GmbH, Mannheim, Germany) or the Monarch™ PCR & DNA Cleanup Kit (NEB). .. Unless otherwise stated, plasmids were obtained from the GeneArt Gene Synthesis Service (Thermo Fisher Scientific).

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential
    Article Snippet: .. Monarch PCR & DNA clean up kit (5 μg) and Monarch DNA gel extraction kit was purchased from New England Biolabs, India. .. Hot start Taq Polymerase was procured from Thermo Fisher Scientific, and all membranes were purchased from MDI, India.

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters
    Article Snippet: .. The amplicons were purified with NEB Monarch PCR & DNA Cleanup Kit and quantified with Nanodrop. .. 200 ng were checked on a gel and 500 ng were sent to GENEWIZ to be sequenced with NGS-based amplicon sequencing.

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. After 25 μl pilot PCRs, PCRs were scaled up (usually to 2 × 50 μl reactions) and gel purified using Monarch PCR and DNA cleanup kits (NEB). ..

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats
    Article Snippet: .. To prepare the vector for cloning and amplification, 1.5 μg of the pBluescript II SK (–) phagemid (Agilent) was cut with Hind III and Bam HI (NEB) for 1h at 37 °C, treated with alkaline phosphatase (Roche) and purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. .. The DNA library was then ligated to the linearised pBluescript II SK (–) plasmid using T4 DNA ligase (NEB) at a 3:1 insert to vector ratio using 200 ng of the linearised vector.

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats
    Article Snippet: .. Digested DNA was purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. .. To prepare the vector for cloning and amplification, 1.5 μg of the pBluescript II SK (–) phagemid (Agilent) was cut with Hind III and Bam HI (NEB) for 1h at 37 °C, treated with alkaline phosphatase (Roche) and purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions.

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats
    Article Snippet: .. The PCR products from all reactions were pooled and purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. .. Ligation and transformation Purified DNA library (250 ng) was cut with the Hind III and Bam HI restriction enzymes (NEB) for 1h at 37 °C in a reaction mixture containing 1X of the NEB cut smart buffer.

    Article Title: Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis
    Article Snippet: .. PCR products were purified using GeneJET PCR Purification Kits (Thermo Fisher Scientific, Waltham MA, USA), Nucleospin Gel and PCR Clean-up (Machery-Nagel GmbH, Düren, Germany) or Monarch PCR and DNA Cleanup kits (NEB). .. Gel purification was carried out using Monarch DNA Gel Extraction Kit (NEB).

    Gel Extraction:

    Article Title: Novel ssDNA Ligand Against Ovarian Cancer Biomarker CA125 With Promising Diagnostic Potential
    Article Snippet: .. Monarch PCR & DNA clean up kit (5 μg) and Monarch DNA gel extraction kit was purchased from New England Biolabs, India. .. Hot start Taq Polymerase was procured from Thermo Fisher Scientific, and all membranes were purchased from MDI, India.

    Plasmid Preparation:

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats
    Article Snippet: .. To prepare the vector for cloning and amplification, 1.5 μg of the pBluescript II SK (–) phagemid (Agilent) was cut with Hind III and Bam HI (NEB) for 1h at 37 °C, treated with alkaline phosphatase (Roche) and purified with the Monarch PCR & DNA Cleanup Kit (NEB) according to the manufacturer’s instructions. .. The DNA library was then ligated to the linearised pBluescript II SK (–) plasmid using T4 DNA ligase (NEB) at a 3:1 insert to vector ratio using 200 ng of the linearised vector.

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  • 96
    New England Biolabs monarch pcr purification kit
    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . Rings with an aromatic character are highlighted in red. B . Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P. <t>aeruginosa</t> (left). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full <t>qRT-PCR</t> dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (10 µg/mL) separately, then cells of both cultures were washed, mixed and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CP (see Methods and Fig. S5D for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) upon artificial induction of the mexGHI-opmD operon with arabinose (n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p
    Monarch Pcr Purification Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    New England Biolabs monarch pcr dna clean up kit
    Distribution of the total extracted <t>DNA</t> from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® <t>PCR</t> DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.
    Monarch Pcr Dna Clean Up Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . Rings with an aromatic character are highlighted in red. B . Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P. aeruginosa (left). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (10 µg/mL) separately, then cells of both cultures were washed, mixed and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CP (see Methods and Fig. S5D for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) upon artificial induction of the mexGHI-opmD operon with arabinose (n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p

    Journal: bioRxiv

    Article Title: Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics

    doi: 10.1101/2020.04.20.049437

    Figure Lengend Snippet: PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. A . Structures of PYO, two representative fluoroquinolones (CP = ciprofloxacin, LV = levofloxacin) and two representative aminoglycosides (GM = gentamicin, TM = tobramycin). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not 11 . Rings with an aromatic character are highlighted in red. B . Normalized cDNA levels for genes within operons coding for the 11 main RND efflux systems in P. aeruginosa (left). PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI- opmD systems (right; n = 3). For full qRT-PCR dataset, see Figs. S2, S3 and S4. C . Effect of PYO on tolerance to CP and LV in glucose minimal medium (left), and to CP in SCFM (right) (all 1 µg/mL) (n = 4). PYO itself was not toxic under the experimental conditions 8 . WT made 50-80 µM PYO as measured by absorbance of the culture supernatant at 691 nm. See Fig. S5A for experimental design. D-E . Effect of PYO on lag during outgrowth after exposure to CP. A representative field of view over different time points (D; magenta = WT::mApple, green = Δ phz ::GFP; see Movie S1) is shown together with the quantification of growth area on the agarose pads at time 0 hrs and 15 hrs (E). For these experiments, a culture of each strain tested was grown and exposed to CP (10 µg/mL) separately, then cells of both cultures were washed, mixed and placed together on a pad and imaged during outgrowth. The pads did not contain any PYO or CP (see Methods and Fig. S5D for details). White arrows in the displayed images point to regions with faster recovery of WT growth. The field of view displayed is marked with a black arrow in the quantification plot. The results for the experiment with swapped fluorescent proteins are shown in Fig. S5E. Scale bar: 20 µm. F . Tolerance of Δ phz to CP (1 µg/mL) in stationary phase in the presence of different concentrations of PYO (n = 4). G . Tolerance of Δ phz to CP (1 µg/mL) upon artificial induction of the mexGHI-opmD operon with arabinose (n = 4). The dashed green line marks the average survival of PYO-producing WT under similar conditions (without arabinose). Statistics: C, F – One-way ANOVA with Tukey’s HSD multiple-comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); E, G – Welch’s unpaired t- test (* p

    Article Snippet: Fragments amplified from P. aeruginosa PA14 genomic DNA (gDNA) and cleaned up using the Monarch PCR Purification kit (New England Biolabs) were used for Gibson assembly together with pMQ30 cut with SacI and HindIII.

    Techniques: Expressing, Quantitative RT-PCR

    Distribution of the total extracted DNA from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® PCR DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.

    Journal: PLoS ONE

    Article Title: Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens

    doi: 10.1371/journal.pone.0235222

    Figure Lengend Snippet: Distribution of the total extracted DNA from the three kits. (A) samples extracted with the DNeasy extraction Kit (Qiagen); dots in green are the samples extracted using the innuPREP DNA Mini Kit (Analytik Jena), and the dots in magenta are samples extracted with the Monarch® PCR DNA Clean-up Kit (New England Biolabs). A trend line was included for each protocol for visualisation of overall distribution.

    Article Snippet: Minimal-destructive DNA extraction from type specimensAs shown in the section above, the Oligonucleotide Clean-up protocol of the Monarch® PCR & DNA Clean-up Kit enabled us to extract higher amounts of DNA in old museum specimens ( > 20 years).

    Techniques: Polymerase Chain Reaction

    Comparison of fragment sizes between two protocols from three specimens. Electropherograms from the DNeasy extraction Kit (blue) and the Monarch PCR DNA Clean-up Kit (red). The specimens individual MTD-TW numbers are as follows: A 9248, B 9252, C 9251. See Supplementary Table 1 for more details on the DNA yield from each extraction.

    Journal: PLoS ONE

    Article Title: Advantages of an easy-to-use DNA extraction method for minimal-destructive analysis of collection specimens

    doi: 10.1371/journal.pone.0235222

    Figure Lengend Snippet: Comparison of fragment sizes between two protocols from three specimens. Electropherograms from the DNeasy extraction Kit (blue) and the Monarch PCR DNA Clean-up Kit (red). The specimens individual MTD-TW numbers are as follows: A 9248, B 9252, C 9251. See Supplementary Table 1 for more details on the DNA yield from each extraction.

    Article Snippet: Minimal-destructive DNA extraction from type specimensAs shown in the section above, the Oligonucleotide Clean-up protocol of the Monarch® PCR & DNA Clean-up Kit enabled us to extract higher amounts of DNA in old museum specimens ( > 20 years).

    Techniques: Polymerase Chain Reaction

    Single‐cell quantification of RNA expression by sm FISH highlights strong heterogeneity of p53 target gene expression p53 has been shown to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown. We selected p53 target genes that are involved in different cell fate programs ranging from apoptosis (BAX), DNA repair (DDB2) cell cycle arrest (CDKN1A), proliferation control (SESN1), and the regulation of the p53 network itself (PPM1D and MDM2). Induction of selected p53 target genes after DNA damage induction in A549 wild‐type and p53 knockdown cells. RNA levels were measured by qRT–PCR before and 3 h after treatment with 10 Gy IR. Fold changes relative to basal levels are shown for each cell line as mean and standard deviation from technical triplicates. Fluorescence microscopy images of smFISH probes labeled with CAL Fluor 610 (gray) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 μm distance; images were contrast‐ and brightness‐enhanced for better visualization. Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets show broad variability of RNA counts per cell for all genes in basal conditions. Dashed line: median; solid line: probability density estimate (see Data visualization section), CV: coefficient of variation, Fano: Fano factor, m: median, n : number of cells analyzed. Source data are available online for this figure.

    Journal: Molecular Systems Biology

    Article Title: Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency

    doi: 10.15252/msb.20199068

    Figure Lengend Snippet: Single‐cell quantification of RNA expression by sm FISH highlights strong heterogeneity of p53 target gene expression p53 has been shown to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown. We selected p53 target genes that are involved in different cell fate programs ranging from apoptosis (BAX), DNA repair (DDB2) cell cycle arrest (CDKN1A), proliferation control (SESN1), and the regulation of the p53 network itself (PPM1D and MDM2). Induction of selected p53 target genes after DNA damage induction in A549 wild‐type and p53 knockdown cells. RNA levels were measured by qRT–PCR before and 3 h after treatment with 10 Gy IR. Fold changes relative to basal levels are shown for each cell line as mean and standard deviation from technical triplicates. Fluorescence microscopy images of smFISH probes labeled with CAL Fluor 610 (gray) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 μm distance; images were contrast‐ and brightness‐enhanced for better visualization. Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets show broad variability of RNA counts per cell for all genes in basal conditions. Dashed line: median; solid line: probability density estimate (see Data visualization section), CV: coefficient of variation, Fano: Fano factor, m: median, n : number of cells analyzed. Source data are available online for this figure.

    Article Snippet: The DNA was cleaned up using the Monarch® PCR & DNA Cleanup Kit (NEB).

    Techniques: RNA Expression, Fluorescence In Situ Hybridization, Expressing, Irradiation, Quantitative RT-PCR, Standard Deviation, Fluorescence, Microscopy, Labeling, Staining

    Smyd2 and Set8 activities affect p53 nuclear dynamics and promoter binding Western blot of acetylated p53 (K370/K382) in A549 Smyd2 and Set8 knockdown cells compared to wild‐type cell lines shows an increase in acetylation specifically at later time points in the DNA damage response. Dynamics of total p53 remained pulse like. GAPDH is shown as loading control. Amount of p53 bound to CDKN1A and MDM2 promoters in A549 Smyd2 (B) and Set8 (C) knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from two biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We observed an increase in promoter binding at later time points similar to the results after Nutlin‐3 treatment.

    Journal: Molecular Systems Biology

    Article Title: Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency

    doi: 10.15252/msb.20199068

    Figure Lengend Snippet: Smyd2 and Set8 activities affect p53 nuclear dynamics and promoter binding Western blot of acetylated p53 (K370/K382) in A549 Smyd2 and Set8 knockdown cells compared to wild‐type cell lines shows an increase in acetylation specifically at later time points in the DNA damage response. Dynamics of total p53 remained pulse like. GAPDH is shown as loading control. Amount of p53 bound to CDKN1A and MDM2 promoters in A549 Smyd2 (B) and Set8 (C) knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from two biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We observed an increase in promoter binding at later time points similar to the results after Nutlin‐3 treatment.

    Article Snippet: The DNA was cleaned up using the Monarch® PCR & DNA Cleanup Kit (NEB).

    Techniques: Binding Assay, Western Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR

    Sm FISH ‐based analysis at the first and second p53 pulse after IR reveals gene‐specific stochastic expression patterns Schematic illustration of the life cycle of an mRNA and the rate constants that influence RNA abundance due to stochastic bursting according to previously published models of promoter activity. While burst frequency (bf) describes the switching of a promoter between a transcriptionally active and inactive state with the rate constants k on and k off, the burst size (bs) describes the number of RNAs transcribed in an active period. Additionally, degradation (δ) further influences RNA levels by reducing the cytoplasmic RNA pool. Illustration of promoter activity according to the random telegraph model. An increase in RNA levels per cell can be due to a higher burst frequency (more active promoter periods, a higher rate of transcription initiation), or an increase in burst size (a higher rate of RNA transcription in an active period). Additionally, also mixtures of both scenarios are possible. We used smFISH data to calculated promoter activity based on previously published models. An overview of the calculations characterizing stochastic gene expression is shown. X RNA : number of quantified RNAs/cell, n : number of genomic loci, f : fraction of active promoters (proxy for burst frequency bf), μ: transcription rate per cell [RNA/h] (proxy for burst size bs), δ RNA : RNA degradation rate per cell [1/h], M : polymerase occupancy [RNAs/h], v : RNAP2 speed (estimated as 3 kb/min), l : gene length, TSS: active TSS at the moment of measurement. Further details can be found in Materials and Methods section. Quantification of stochastic gene expression for the indicated p53 target genes before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (μ; proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity ( > 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates μ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2 C). Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene. Based on promoter activity, we allocated target gene promoters along three archetypical expression patterns illustrated by a schematic triangle. Amount of p53 bound to indicated target gene promoters before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from 3 to 4 biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.) Source data are available online for this figure.

    Journal: Molecular Systems Biology

    Article Title: Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency

    doi: 10.15252/msb.20199068

    Figure Lengend Snippet: Sm FISH ‐based analysis at the first and second p53 pulse after IR reveals gene‐specific stochastic expression patterns Schematic illustration of the life cycle of an mRNA and the rate constants that influence RNA abundance due to stochastic bursting according to previously published models of promoter activity. While burst frequency (bf) describes the switching of a promoter between a transcriptionally active and inactive state with the rate constants k on and k off, the burst size (bs) describes the number of RNAs transcribed in an active period. Additionally, degradation (δ) further influences RNA levels by reducing the cytoplasmic RNA pool. Illustration of promoter activity according to the random telegraph model. An increase in RNA levels per cell can be due to a higher burst frequency (more active promoter periods, a higher rate of transcription initiation), or an increase in burst size (a higher rate of RNA transcription in an active period). Additionally, also mixtures of both scenarios are possible. We used smFISH data to calculated promoter activity based on previously published models. An overview of the calculations characterizing stochastic gene expression is shown. X RNA : number of quantified RNAs/cell, n : number of genomic loci, f : fraction of active promoters (proxy for burst frequency bf), μ: transcription rate per cell [RNA/h] (proxy for burst size bs), δ RNA : RNA degradation rate per cell [1/h], M : polymerase occupancy [RNAs/h], v : RNAP2 speed (estimated as 3 kb/min), l : gene length, TSS: active TSS at the moment of measurement. Further details can be found in Materials and Methods section. Quantification of stochastic gene expression for the indicated p53 target genes before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (μ; proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity ( > 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates μ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2 C). Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene. Based on promoter activity, we allocated target gene promoters along three archetypical expression patterns illustrated by a schematic triangle. Amount of p53 bound to indicated target gene promoters before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT–PCR measurements) from 3 to 4 biological repeats are shown as dots; mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.) Source data are available online for this figure.

    Article Snippet: The DNA was cleaned up using the Monarch® PCR & DNA Cleanup Kit (NEB).

    Techniques: Fluorescence In Situ Hybridization, Expressing, Activity Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Binding Assay

    The interplay of p53's C‐terminal lysine acetylation and methylation regulates transiently expressed target genes in response to IR A schematic illustration of p53's C‐terminal modifications and described functional implications, including key regulatory enzymes. Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML‐277, central lanes), and sustained p53 (10 Gy IR + Nutlin‐3, right lanes). See Fig 3 and Materials and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western blot and normalized to the abundance 3 h post‐IR. Means and propagated standard errors from three independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12 . The p53‐K370 methylase Smyd2 was down‐regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild‐type and knockdown cells by qRT–PCR. Mean levels and standard deviation from technical triplicates are indicated. Promoter activity of CDKN1A (E) and MDM2 (F) was quantified in Smyd2 knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong ( > 75% of TSS, solid colors) and weak (

    Journal: Molecular Systems Biology

    Article Title: Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency

    doi: 10.15252/msb.20199068

    Figure Lengend Snippet: The interplay of p53's C‐terminal lysine acetylation and methylation regulates transiently expressed target genes in response to IR A schematic illustration of p53's C‐terminal modifications and described functional implications, including key regulatory enzymes. Total p53, p53 acetylated at K382 and K370 as well as GAPDH were measured by Western blot at indicated time points in the context of different p53 dynamics: pulsing p53 (10 Gy IR), transient p53 (10 Gy IR + BML‐277, central lanes), and sustained p53 (10 Gy IR + Nutlin‐3, right lanes). See Fig 3 and Materials and Methods section for details. The relative change in p53 acetylation at K370 (light green) and K382 (dark green) was quantified from Western blot and normalized to the abundance 3 h post‐IR. Means and propagated standard errors from three independent experiments are indicated. Acetylation increased over time in the context of sustained p53. See also Appendix Fig S12 . The p53‐K370 methylase Smyd2 was down‐regulated in a clonal stable A549 cell line expressing a corresponding shRNA. Transcript levels were measured in wild‐type and knockdown cells by qRT–PCR. Mean levels and standard deviation from technical triplicates are indicated. Promoter activity of CDKN1A (E) and MDM2 (F) was quantified in Smyd2 knockdown cells before (basal, gray) and 3 h (red), 6 h (blue), and 9 h (orange) after DNA damage (10 Gy IR). Left panel: The percentage of cells with active TSS, subdivided into populations with strong ( > 75% of TSS, solid colors) and weak (

    Article Snippet: The DNA was cleaned up using the Monarch® PCR & DNA Cleanup Kit (NEB).

    Techniques: Methylation, Functional Assay, Western Blot, Expressing, shRNA, Quantitative RT-PCR, Standard Deviation, Activity Assay