monarch pcr and dna cleanup kit  (New England Biolabs)


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    Name:
    Monarch PCR DNA Cleanup Kit
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    Catalog Number:
    T1030
    Price:
    475
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    Other Kits
    Applications:
    DNA Manipulation
    Size:
    250 preps
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    New England Biolabs monarch pcr and dna cleanup kit
    Monarch PCR DNA Cleanup Kit

    https://www.bioz.com/result/monarch pcr and dna cleanup kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch pcr and dna cleanup kit - by Bioz Stars, 2021-09
    99/100 stars

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    1) Product Images from "Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics"

    Article Title: Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

    Journal: bioRxiv

    doi: 10.1101/2021.03.29.437587

    mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.
    Figure Legend Snippet: mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Techniques Used: Construct, Synthesized, Amplification, Polymerase Chain Reaction, In Vitro, Expressing, Staining, Fractionation, Transfection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing

    2) Product Images from "The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3"

    Article Title: The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.012518

    Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.
    Figure Legend Snippet: Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Techniques Used: Sequencing, Mutagenesis, Clone Assay, Generated, Polymerase Chain Reaction, Immunohistochemistry

    3) Product Images from "EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach"

    Article Title: EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

    Journal: Microorganisms

    doi: 10.3390/microorganisms9081649

    Efficiencies of two- and three-steps epicPCR protocols using different blocking primers (BPs) concentrations. The epicPCRs were run on SXT/R391 carrying bacteria from the Meurthe River water. A and C indicate wells with amplification products from two-steps epicPCR protocols (fusion-PCR on polyacrylamide beads + nested PCR). In the A lane, fusion-PCR products from the first step were used as template DNA in the second step without dilution whereas these products were diluted ten times in line C to circumvent the possible presence of PCR inhibitors. B indicates wells loaded with amplification products resulting from a three-steps epicPCR protocol (fusion PCR on polyacrylamide beads + blocking PCR with BPs as sole primers + nested PCR). The expected size of the final nested-PCR product is around 350 bp (depending on the 16S rRNA gene fragment polymorphism). The conditions of epicPCR as used in Hultman et al. (2018) and that we determined to be the best in our conditions (epicPCR 2.0) are indicated by black arrows. The minus symbols indicate negative controls with epicPCRs run without polyacrylamide beads-template.
    Figure Legend Snippet: Efficiencies of two- and three-steps epicPCR protocols using different blocking primers (BPs) concentrations. The epicPCRs were run on SXT/R391 carrying bacteria from the Meurthe River water. A and C indicate wells with amplification products from two-steps epicPCR protocols (fusion-PCR on polyacrylamide beads + nested PCR). In the A lane, fusion-PCR products from the first step were used as template DNA in the second step without dilution whereas these products were diluted ten times in line C to circumvent the possible presence of PCR inhibitors. B indicates wells loaded with amplification products resulting from a three-steps epicPCR protocol (fusion PCR on polyacrylamide beads + blocking PCR with BPs as sole primers + nested PCR). The expected size of the final nested-PCR product is around 350 bp (depending on the 16S rRNA gene fragment polymorphism). The conditions of epicPCR as used in Hultman et al. (2018) and that we determined to be the best in our conditions (epicPCR 2.0) are indicated by black arrows. The minus symbols indicate negative controls with epicPCRs run without polyacrylamide beads-template.

    Techniques Used: Blocking Assay, Amplification, Polymerase Chain Reaction, Nested PCR

    Control epicPCR amplifications targeting SXT/R391 ICEs performed on beads carrying E. coli MG1656::SXT MO10 as template. Each experiment was done in duplicates with a no-template DNA control, using either the Phusion DNA Polymerase GC or HF buffers. In the nested-PCR step, the use of blocking primers (BPs) has been done as depicted in Spencer et al. (2016), and usually performed so far. All these conditions are summarized in the table upper the gel. The expected size of the desired DNA fragment is 367 bp. Black arrows indicate epicPCR products obtained after performing fusion and nested PCRs using HF but not GC buffer.
    Figure Legend Snippet: Control epicPCR amplifications targeting SXT/R391 ICEs performed on beads carrying E. coli MG1656::SXT MO10 as template. Each experiment was done in duplicates with a no-template DNA control, using either the Phusion DNA Polymerase GC or HF buffers. In the nested-PCR step, the use of blocking primers (BPs) has been done as depicted in Spencer et al. (2016), and usually performed so far. All these conditions are summarized in the table upper the gel. The expected size of the desired DNA fragment is 367 bp. Black arrows indicate epicPCR products obtained after performing fusion and nested PCRs using HF but not GC buffer.

    Techniques Used: Nested PCR, Blocking Assay

    4) Product Images from "Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex"

    Article Title: Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex

    Journal: bioRxiv

    doi: 10.1101/2020.02.02.931477

    Determination of Bicc1 Binding Motifs in RNA a Schematic representation of RNA Bind-n-Seq (RBNS), which determines RNA motifs enriched by target proteins with the use of a random RNA sequence library. 293FT cells were transfected with a plasmid for overexpression (O/E) of FLAG-tagged Bicc1. Cell lysates containing the Bicc1-FLAG protein were then mixed with a random RNA sequence library, and resulting RNA-protein complexes were isolated by immunoprecipitation with magnetic bead–conjugated antibodies to FLAG. Finally, the isolated RNA sequences were converted to a DNA library by RT-PCR for deep sequencing. b Analysis of the RBNS data set. The number of each k-mer (where k = 4, 5, or 6) RNA sequence was compared between cells transfected with the Bicc1-FLAG expression plasmid and those subjected to mock transfection (control). c A motif logo generated from aligned hexamers significantly enriched by Bicc1-FLAG. d Enriched 4-mer and 5-mer sequences sorted by relative frequency determined by comparison of the Bicc1-FLAG and control RBNS data. e Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3’-UTR of mouse mRNAs. A total of 31,16 5 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. f Histogram of motif frequency revealed by metagene analysis. The vertical black and blue lines indicate the averaged frequency of each target motif and the frequency of each target motif in the 200-nucleotide proximal region of the 3’-UTR of Dand5 mRNA. g Maps of GAC-containing motifs in the 3’-UTR of Dand5 mRNAs for the indicated species.
    Figure Legend Snippet: Determination of Bicc1 Binding Motifs in RNA a Schematic representation of RNA Bind-n-Seq (RBNS), which determines RNA motifs enriched by target proteins with the use of a random RNA sequence library. 293FT cells were transfected with a plasmid for overexpression (O/E) of FLAG-tagged Bicc1. Cell lysates containing the Bicc1-FLAG protein were then mixed with a random RNA sequence library, and resulting RNA-protein complexes were isolated by immunoprecipitation with magnetic bead–conjugated antibodies to FLAG. Finally, the isolated RNA sequences were converted to a DNA library by RT-PCR for deep sequencing. b Analysis of the RBNS data set. The number of each k-mer (where k = 4, 5, or 6) RNA sequence was compared between cells transfected with the Bicc1-FLAG expression plasmid and those subjected to mock transfection (control). c A motif logo generated from aligned hexamers significantly enriched by Bicc1-FLAG. d Enriched 4-mer and 5-mer sequences sorted by relative frequency determined by comparison of the Bicc1-FLAG and control RBNS data. e Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3’-UTR of mouse mRNAs. A total of 31,16 5 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. f Histogram of motif frequency revealed by metagene analysis. The vertical black and blue lines indicate the averaged frequency of each target motif and the frequency of each target motif in the 200-nucleotide proximal region of the 3’-UTR of Dand5 mRNA. g Maps of GAC-containing motifs in the 3’-UTR of Dand5 mRNAs for the indicated species.

    Techniques Used: Binding Assay, Sequencing, Transfection, Plasmid Preparation, Over Expression, Isolation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Generated

    5) Product Images from "Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing"

    Article Title: Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing

    Journal: RNA Biology

    doi: 10.1080/15476286.2020.1813411

    Stably silenced and reverting cultures in aif5A, cdvA and slaB silencing experiments. A) Upper panel: Schematic representation of mRNAs of each gene and corresponding silencing miniCR constructs where arrows point to protospacers regions targeted by each construct (cf. Table 1 ). The positions of protospacers (PS) including PAS (coloured boxes with black tail) on respective mRNAs are given with respect to the gene length. Lower panel: Growth profiles (OD 600 ) of cells transformed with miniCR constructs leading to stably silenced cultures (coloured continuous lines)* and strong miniCR construct leading to reverted cultures (coloured discontinuous lines). Black lines (triangle) represent control cultures transformed with miniCR-Ctrl, devoid of a targeting spacer. Error bars, mean ± SD (n = 3). MiniCR-SB-23-T: culture transformed with miniCR-23 and transferred to fresh medium once. *Growth profiles of miniCR-SB-2/miniCR-aIF5A-II of another sample set have been published [ 31 , 32 ], but fresh transformants grown in parallel with the depicted revertant cultures are presented here. B) Left panel: Schematic representation of PCR products generated by culture PCR on stably silenced/reverted cultures using primers (prim-FW, prim-RV) binding up and downstream of the miniCR cassette, respectively. Amplicons of different lengths reflect the integrity of the miniCR array. Right panel: Agarose gels depicting culture PCR amplicons of miniCR cassettes in respective transformants (same cultures as in A sampled at OD 600 = 0.2 were used as templates). Band heights emerging from deletions in miniCR arrays are indicated by red arrows and often appear as multiple or fuzzy bands in reverted cultures. Position of DNA ladder is indicated (kb). Representative agarose gels of culture PCRs on miniCR-SB-123 and miniCR-aIF5A-I carrying revertant cultures can be found in refs. [ 31 ] and [ 32 ], respectively
    Figure Legend Snippet: Stably silenced and reverting cultures in aif5A, cdvA and slaB silencing experiments. A) Upper panel: Schematic representation of mRNAs of each gene and corresponding silencing miniCR constructs where arrows point to protospacers regions targeted by each construct (cf. Table 1 ). The positions of protospacers (PS) including PAS (coloured boxes with black tail) on respective mRNAs are given with respect to the gene length. Lower panel: Growth profiles (OD 600 ) of cells transformed with miniCR constructs leading to stably silenced cultures (coloured continuous lines)* and strong miniCR construct leading to reverted cultures (coloured discontinuous lines). Black lines (triangle) represent control cultures transformed with miniCR-Ctrl, devoid of a targeting spacer. Error bars, mean ± SD (n = 3). MiniCR-SB-23-T: culture transformed with miniCR-23 and transferred to fresh medium once. *Growth profiles of miniCR-SB-2/miniCR-aIF5A-II of another sample set have been published [ 31 , 32 ], but fresh transformants grown in parallel with the depicted revertant cultures are presented here. B) Left panel: Schematic representation of PCR products generated by culture PCR on stably silenced/reverted cultures using primers (prim-FW, prim-RV) binding up and downstream of the miniCR cassette, respectively. Amplicons of different lengths reflect the integrity of the miniCR array. Right panel: Agarose gels depicting culture PCR amplicons of miniCR cassettes in respective transformants (same cultures as in A sampled at OD 600 = 0.2 were used as templates). Band heights emerging from deletions in miniCR arrays are indicated by red arrows and often appear as multiple or fuzzy bands in reverted cultures. Position of DNA ladder is indicated (kb). Representative agarose gels of culture PCRs on miniCR-SB-123 and miniCR-aIF5A-I carrying revertant cultures can be found in refs. [ 31 ] and [ 32 ], respectively

    Techniques Used: Stable Transfection, Construct, Transformation Assay, Polymerase Chain Reaction, Generated, Binding Assay

    6) Product Images from "Duplex Proximity Sequencing (Pro-Seq): A method to improve DNA sequencing accuracy without the cost of molecular barcoding redundancy"

    Article Title: Duplex Proximity Sequencing (Pro-Seq): A method to improve DNA sequencing accuracy without the cost of molecular barcoding redundancy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0204265

    Overview of the targeted Pro-Seq workflow (described in detail in the Materials and methods ). In brief, double stranded DNA is loaded directly into droplets such that on average zero or one template molecule is incorporated in each droplet. Off-target DNA (not shown in figure) is also loaded into droplets, but does not amplify. Within each droplet are multiplexed gene-specific primers, and the Pro-Seq universal 5’ PEG-linked primers. The droplets are PCR cycled such that all copies of the starting template are linked to the universal linked primers (shown in detail in S2 Fig ). The emulsions are then broken, and the un-linked strands are digested and cleaned up. After quantification, the library is ready for sequencing.
    Figure Legend Snippet: Overview of the targeted Pro-Seq workflow (described in detail in the Materials and methods ). In brief, double stranded DNA is loaded directly into droplets such that on average zero or one template molecule is incorporated in each droplet. Off-target DNA (not shown in figure) is also loaded into droplets, but does not amplify. Within each droplet are multiplexed gene-specific primers, and the Pro-Seq universal 5’ PEG-linked primers. The droplets are PCR cycled such that all copies of the starting template are linked to the universal linked primers (shown in detail in S2 Fig ). The emulsions are then broken, and the un-linked strands are digested and cleaned up. After quantification, the library is ready for sequencing.

    Techniques Used: Polymerase Chain Reaction, Sequencing

    7) Product Images from "Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex"

    Article Title: Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

    Journal: Nature Communications

    doi: 10.1038/s41467-021-24295-2

    Screen for Bicc1-binding motifs in RNA. a Schematic representation of RNA Bind-n-Seq (RBNS), which determines RNA motifs enriched by target proteins with the use of a random RNA sequence library. 293FT cells were transfected with a plasmid for overexpression (O/E) of FLAG-tagged Bicc1. Cell lysates containing the Bicc1-FLAG protein were then mixed with a random RNA sequence library, and resulting RNA-protein complexes were isolated by immunoprecipitation with magnetic bead–conjugated antibodies to FLAG. Finally, the isolated RNA sequences were converted to a DNA library by RT-PCR for deep sequencing. b Analysis of the RBNS data set. The number of each k-mer (where k = 4, 5, or 6) RNA sequence was compared between cells transfected with the Bicc1-FLAG expression plasmid and those subjected to mock transfection (control). c Motif logos generated from aligned hexamers that were enriched by Bicc1-FLAG. d 4-mer, 5-mer, and 6-mer sequences ranked by their relative frequencies in Bicc1-FLAG versus control RBNS data. e Maps of GAC-containing motifs in the 3′-UTR of Dand5 mRNAs for the indicated species. f Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3′-UTR of mouse mRNAs. A total of 31,165 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. g Histogram of motif frequency revealed by metagene analysis. The vertical lines indicate the averaged frequency of each target motif (black) and the frequency of each target motif in the 200-nucleotide proximal region of the 3′-UTR of Dand5 mRNA (blue), respectively. h Multiple sequence alignment of a conserved segment within the proximal 200 nucleotides of mammalian, amphibian, and fish Dand5 3′ -UTRs . Colors highlight GAC motifs.
    Figure Legend Snippet: Screen for Bicc1-binding motifs in RNA. a Schematic representation of RNA Bind-n-Seq (RBNS), which determines RNA motifs enriched by target proteins with the use of a random RNA sequence library. 293FT cells were transfected with a plasmid for overexpression (O/E) of FLAG-tagged Bicc1. Cell lysates containing the Bicc1-FLAG protein were then mixed with a random RNA sequence library, and resulting RNA-protein complexes were isolated by immunoprecipitation with magnetic bead–conjugated antibodies to FLAG. Finally, the isolated RNA sequences were converted to a DNA library by RT-PCR for deep sequencing. b Analysis of the RBNS data set. The number of each k-mer (where k = 4, 5, or 6) RNA sequence was compared between cells transfected with the Bicc1-FLAG expression plasmid and those subjected to mock transfection (control). c Motif logos generated from aligned hexamers that were enriched by Bicc1-FLAG. d 4-mer, 5-mer, and 6-mer sequences ranked by their relative frequencies in Bicc1-FLAG versus control RBNS data. e Maps of GAC-containing motifs in the 3′-UTR of Dand5 mRNAs for the indicated species. f Schematic representation of metagene analysis for the 200-nucleotide proximal region of the 3′-UTR of mouse mRNAs. A total of 31,165 regions extracted from mouse genes (mm10) was searched with the indicated target motifs. g Histogram of motif frequency revealed by metagene analysis. The vertical lines indicate the averaged frequency of each target motif (black) and the frequency of each target motif in the 200-nucleotide proximal region of the 3′-UTR of Dand5 mRNA (blue), respectively. h Multiple sequence alignment of a conserved segment within the proximal 200 nucleotides of mammalian, amphibian, and fish Dand5 3′ -UTRs . Colors highlight GAC motifs.

    Techniques Used: Binding Assay, Sequencing, Transfection, Plasmid Preparation, Over Expression, Isolation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Generated, Fluorescence In Situ Hybridization

    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

    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

    9) Product Images from "Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing"

    Article Title: Single telomere length analysis in Ustilago maydis, a high-resolution tool for examining fungal telomere length distribution and C-strand 5’-end processing

    Journal: Microbial Cell

    doi: 10.15698/mic2018.09.645

    FIGURE 1: STELA protocol and investigation of UT4/5-containing telomeres. (A) Schematic illustration of the structure of UT4 and UT5-containing telomeres in U. maydis . The use of telorette oligos to modify the C-strand and the use of primers (UT4/5-F and teltail) to generate STELA products are also illustrated. (B) Four individual STELA PCR reactions for UT4/5 telomeres were performed using 2.5 pg of ligated wild type DNA as the template and shown on the left. A parallel Southern analysis is shown on the right. The same UT4/5 subtelomeric probe was used to detect telomere fragments in both analyses. (C) STELA assays were performed using 5 pg wild type DNA as the template, and the UT4/5-F and teltail oligos as primers. Following gel electrophoresis and transfer to a nylon membrane, the products were first detected using a UT4/5 subtelomeric probe (left panel). Subsequently, the UT4/5 probe was stripped from the membrane and the products re-analyzed using a TTAGGG repeat probe (middle panel). The sizes of the STELA fragments in the middle panel were determined using TESLA software. The lengths of the telomere tracts were then calculated by subtracting the subtelomere length (~630 bp), and then plotted (right). Error bars designate standard error of means.
    Figure Legend Snippet: FIGURE 1: STELA protocol and investigation of UT4/5-containing telomeres. (A) Schematic illustration of the structure of UT4 and UT5-containing telomeres in U. maydis . The use of telorette oligos to modify the C-strand and the use of primers (UT4/5-F and teltail) to generate STELA products are also illustrated. (B) Four individual STELA PCR reactions for UT4/5 telomeres were performed using 2.5 pg of ligated wild type DNA as the template and shown on the left. A parallel Southern analysis is shown on the right. The same UT4/5 subtelomeric probe was used to detect telomere fragments in both analyses. (C) STELA assays were performed using 5 pg wild type DNA as the template, and the UT4/5-F and teltail oligos as primers. Following gel electrophoresis and transfer to a nylon membrane, the products were first detected using a UT4/5 subtelomeric probe (left panel). Subsequently, the UT4/5 probe was stripped from the membrane and the products re-analyzed using a TTAGGG repeat probe (middle panel). The sizes of the STELA fragments in the middle panel were determined using TESLA software. The lengths of the telomere tracts were then calculated by subtracting the subtelomere length (~630 bp), and then plotted (right). Error bars designate standard error of means.

    Techniques Used: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Software

    10) Product Images from "Using weapons instead of perfume – chemical association strategies of the myrmecophilous bug Scolopostethus pacificus (Rhyparochromidae)"

    Article Title: Using weapons instead of perfume – chemical association strategies of the myrmecophilous bug Scolopostethus pacificus (Rhyparochromidae)

    Journal: bioRxiv

    doi: 10.1101/2020.12.08.412577

    Molecular gut content analysis of Scolopostethus pacificus . Standard ITS2 (CAS5ps+CAS28s) and ant-specific Loc1 and Loc2 primers were used for PCR amplification using S. pacificus body-DNA, gut-DNA and Liometopum occidentale -DNA. While ITS2 amplified in all cases, no ITS2 fragment was amplified with ant-specific ITS (Loc 1/2) was amplified from dissected bugs’ guts.
    Figure Legend Snippet: Molecular gut content analysis of Scolopostethus pacificus . Standard ITS2 (CAS5ps+CAS28s) and ant-specific Loc1 and Loc2 primers were used for PCR amplification using S. pacificus body-DNA, gut-DNA and Liometopum occidentale -DNA. While ITS2 amplified in all cases, no ITS2 fragment was amplified with ant-specific ITS (Loc 1/2) was amplified from dissected bugs’ guts.

    Techniques Used: Polymerase Chain Reaction, Amplification

    11) Product Images from "Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction"

    Article Title: Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction

    Journal: Communications Biology

    doi: 10.1038/s42003-021-01661-w

    epi-gSCAR workflow schematics and methylation readout for the DLX4 locus. a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8 ). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3 ), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.
    Figure Legend Snippet: epi-gSCAR workflow schematics and methylation readout for the DLX4 locus. a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8 ). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3 ), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.

    Techniques Used: Methylation, Single-cell Isolation, Lysis, Variant Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing, DNA Methylation Assay, Next-Generation Sequencing, Methylation Sequencing

    12) Product Images from "Fitness landscape of a dynamic RNA structure"

    Article Title: Fitness landscape of a dynamic RNA structure

    Journal: bioRxiv

    doi: 10.1101/2020.06.06.130575

    Generation of mutant library using site-saturation mutagenesis via a two-step PCR. Solid arrows denote oligonucleotides. In the first step, two pairs of oligonucleotides containing mixed bases ( https://www.idtdna.com/pages/products/custom-dna-rna/mixed-bases ) are used to amplify two separate fragments (purple and pink) containing one P1ex sub-region each. The 3’ end of the purple fragment and the 5’ end of the pink fragment share a 12-bp overlapping region, which allow self-annealing and subsequent 3’ extension during the second PCR. As a result, the assembled amplicons contain two varying sub-regions. The purple fragment is amplified using primers Frag1-f and Frag1-r, whereas the pink fragment is amplified using primers Frag2-f and Frag2-r ( Table S3 ).
    Figure Legend Snippet: Generation of mutant library using site-saturation mutagenesis via a two-step PCR. Solid arrows denote oligonucleotides. In the first step, two pairs of oligonucleotides containing mixed bases ( https://www.idtdna.com/pages/products/custom-dna-rna/mixed-bases ) are used to amplify two separate fragments (purple and pink) containing one P1ex sub-region each. The 3’ end of the purple fragment and the 5’ end of the pink fragment share a 12-bp overlapping region, which allow self-annealing and subsequent 3’ extension during the second PCR. As a result, the assembled amplicons contain two varying sub-regions. The purple fragment is amplified using primers Frag1-f and Frag1-r, whereas the pink fragment is amplified using primers Frag2-f and Frag2-r ( Table S3 ).

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification

    13) Product Images from "eDNAir: proof of concept that animal DNA can be collected from air sampling"

    Article Title: eDNAir: proof of concept that animal DNA can be collected from air sampling

    Journal: PeerJ

    doi: 10.7717/peerj.11030

    PCR results for filtered air. DNA was extracted from filters and amplified with primers targeting the 16S mitochondrial region (A) using primers designed for mammals ( Taylor, 1996 ) and using vertebrate primers commonly applied in aquatic eDNA ( Riaz et al., 2011 ) for the 12S mitochondrial region (B). In each well 5 µl of PCR reaction was run on a 1% agarose gel. Fast DNA ladder from New England BioLabs was used as a size standard.
    Figure Legend Snippet: PCR results for filtered air. DNA was extracted from filters and amplified with primers targeting the 16S mitochondrial region (A) using primers designed for mammals ( Taylor, 1996 ) and using vertebrate primers commonly applied in aquatic eDNA ( Riaz et al., 2011 ) for the 12S mitochondrial region (B). In each well 5 µl of PCR reaction was run on a 1% agarose gel. Fast DNA ladder from New England BioLabs was used as a size standard.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    14) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats

    Journal: Genome Biology

    doi: 10.1186/s13059-020-02124-x

    Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. a Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structure annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template, or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. b Extended and stalled products were then analysed by denaturing poly acrylamide gel electrophoresis (PAGE), recovered from the gel matrix and prepared for high-throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence from the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, is reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear over time

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Polyacrylamide Gel Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

    15) 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

    16) 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

    17) Product Images from "Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction"

    Article Title: Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction

    Journal: Communications Biology

    doi: 10.1038/s42003-021-01661-w

    epi-gSCAR workflow schematics and methylation readout for the DLX4 locus. a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8 ). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3 ), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.
    Figure Legend Snippet: epi-gSCAR workflow schematics and methylation readout for the DLX4 locus. a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8 ). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3 ), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.

    Techniques Used: Methylation, Single-cell Isolation, Lysis, Variant Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing, DNA Methylation Assay, Next-Generation Sequencing, Methylation Sequencing

    18) Product Images from "Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction"

    Article Title: Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction

    Journal: Communications Biology

    doi: 10.1038/s42003-021-01661-w

    epi-gSCAR workflow schematics and methylation readout for the DLX4 locus. a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8 ). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3 ), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.
    Figure Legend Snippet: epi-gSCAR workflow schematics and methylation readout for the DLX4 locus. a epi-gSCAR workflow: single-cell isolation is followed by lysis and chromatin digestion to render DNA accessible for methylation-sensitive restriction enzyme (MSRE) digestion with HhaI (i). Cleavage of methylated HhaI sites (light blue) is blocked, while unmethylated sites (dark blue) are cleaved; the resulting DNA ends are tagged with poly(d)A tails (red) (ii). Poly(d)A tails are primed by anchored (GAT-oligo(dT)12-CG, blue–green–gray, assay variant A) or non-anchored adapters (GAT-oligo(dT)12, green–gray, assay variant B). Anchored adapters were used to limit the length of poly(d)A tails in the library (Supplementary Fig. 8 ). This is followed by gap filling and ligation, which results in tagged restriction enzyme scars (iii). Random priming by 7N-GAT adapters (orange–gray) facilitates quasilinear amplification of the genome (iv). PCR generates amplicons carrying genetic and epigenetic information (v). b HhaI sites in CGIs and around TSSs across 100 bp windows and 3 kb upstream and downstream. c Methylation analysis of the DLX4 locus by step-out PCR followed by Sanger sequencing. DLX4 locus with CGI1 (green) and CGI2 (red), CpGs (red) and HhaI sites (blue), primer map for analysis of HhaI sites 1–10 (CGI1) and 3–21 (CGI2), and corresponding sequencing reads. Magnification of reads obtained from single cell K_05 (selected for analysis as it demonstrated satisfactory results in initial suppression PCR experiments) corresponding to HhaI sites 4–6 in CGI2 showing intact and tagged-scar HhaI sites: intact HhaI sites are called as having been methylated and poly(d)A-tailed HhaI scar sites unmethylated; presence of both suggests heterozygous methylation. d DNA methylation in single cells K_01–K_07 at individual HhaI sites for CGI1 (green) and CGI2 (red) of DLX4 assessed by PCR and/or NGS (Supplementary Fig. 3 ), and comparison with Kasumi-1 cell-bulk whole-genome bisulfite sequencing (WGBS) data. Using step-out PCR on single cell K_05, CGI1 was unmethylated at all analyzed HhaI sites (6/6). CGI2 featured high level of heterozygous methylation (14/19 methylated; 5/19 heterozygous methylation). e Mean methylation levels of CGI1 (green) and CGI2 (red) for single cells K_01–K_07 (NGS and PCR), Kasumi-1 WGBS and Illumina 450 K array.

    Techniques Used: Methylation, Single-cell Isolation, Lysis, Variant Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing, DNA Methylation Assay, Next-Generation Sequencing, Methylation Sequencing

    19) Product Images from "Zika virus noncoding RNA suppresses apoptosis and is required for virus transmission by mosquitoes"

    Article Title: Zika virus noncoding RNA suppresses apoptosis and is required for virus transmission by mosquitoes

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16086-y

    sfRNA facilitates replication and transmission of ZIKV in vivo. Ae. aegypti mosquitoes were exposed to an infectious blood meal containing 10 8 FFU/ml of each virus (≈1:5 mixture of virus stock and defibrinated sheep blood). At 7 days post infection (dpi), the percentage of infected mosquitoes ( a ) and viral titres in the bodies ( b ) were determined to serve as indicators of the infection rate and of initial viral replication. At 14 dpi ( c ), the infection, dissemination and transmission rates were determined as percentage of ZIKV-positive bodies, legs and wings, and saliva samples, respectively. ZIKV titres at 14 dpi ( d ) indicate viral replication efficiency and viral loads in saliva. e Production of ZIKV sfRNAs in infected mosquitoes. RNA was isolated from the pools of 10 mosquitoes at 10 days after i.t. injection and used for Northern blot hybridization with radioactively labelled DNA oligo complementary to the viral 3ʹUTR. The bottom panel shows a polyacrylamide gel with Et-Br-stained ribosomal RNA as loading control. Figure shows representative images of two independent experiments that produced similar results. f ZIKV genomic RNA levels in RNA samples used for Northern blot in ( e ) values are the means of three technical replicates +/− standard errors of the means. Viral RNA abundance was determined using qRT-PCR and the standard curve quantification method. Values are the means ± SEM of three technical replicates. g Infection and transmission rates in mosquitoes infected by i.t. injection. h ZIKV titres in mosquitoes inoculated by i.t. injection. Mosquitoes in ( e – h ) were injected with 200 nl of the inoculum containing 10 4 FFU/ml of each virus. Viral titres were determined by IPA on C6/36 cells ( a – d ) or Vero 76 cells ( g , h ). Sample sizes ( n ) for all statistical tests indicated in the panels refer to biologically independent mosquitoes. Panels ( b , d , h ) show individual and median (horizontal line) values. Statistical differences were determined using chi-squared ( a , c , g ) or Mann–Whitney U tests ( b , d , h ), all P -values are two-sided, no multiple comparisons were performed in each test.
    Figure Legend Snippet: sfRNA facilitates replication and transmission of ZIKV in vivo. Ae. aegypti mosquitoes were exposed to an infectious blood meal containing 10 8 FFU/ml of each virus (≈1:5 mixture of virus stock and defibrinated sheep blood). At 7 days post infection (dpi), the percentage of infected mosquitoes ( a ) and viral titres in the bodies ( b ) were determined to serve as indicators of the infection rate and of initial viral replication. At 14 dpi ( c ), the infection, dissemination and transmission rates were determined as percentage of ZIKV-positive bodies, legs and wings, and saliva samples, respectively. ZIKV titres at 14 dpi ( d ) indicate viral replication efficiency and viral loads in saliva. e Production of ZIKV sfRNAs in infected mosquitoes. RNA was isolated from the pools of 10 mosquitoes at 10 days after i.t. injection and used for Northern blot hybridization with radioactively labelled DNA oligo complementary to the viral 3ʹUTR. The bottom panel shows a polyacrylamide gel with Et-Br-stained ribosomal RNA as loading control. Figure shows representative images of two independent experiments that produced similar results. f ZIKV genomic RNA levels in RNA samples used for Northern blot in ( e ) values are the means of three technical replicates +/− standard errors of the means. Viral RNA abundance was determined using qRT-PCR and the standard curve quantification method. Values are the means ± SEM of three technical replicates. g Infection and transmission rates in mosquitoes infected by i.t. injection. h ZIKV titres in mosquitoes inoculated by i.t. injection. Mosquitoes in ( e – h ) were injected with 200 nl of the inoculum containing 10 4 FFU/ml of each virus. Viral titres were determined by IPA on C6/36 cells ( a – d ) or Vero 76 cells ( g , h ). Sample sizes ( n ) for all statistical tests indicated in the panels refer to biologically independent mosquitoes. Panels ( b , d , h ) show individual and median (horizontal line) values. Statistical differences were determined using chi-squared ( a , c , g ) or Mann–Whitney U tests ( b , d , h ), all P -values are two-sided, no multiple comparisons were performed in each test.

    Techniques Used: Transmission Assay, In Vivo, Infection, Isolation, Injection, Northern Blot, Hybridization, Staining, Produced, Quantitative RT-PCR, Indirect Immunoperoxidase Assay, MANN-WHITNEY

    20) Product Images from "Bacterial defenses against a natural antibiotic promote collateral resilience to clinical antibiotics"

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

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3001093

    PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. (A) Structures of PYO, 2 representative fluoroquinolones (CIP and LVX) and 2 representative aminoglycosides (GEN and TOB). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not [ 21 , 22 ]. 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; n = 3) and PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI-opmD systems (right; n = 3). For full qRT-PCR dataset, see S1 – S3 Figs. (C) Effect of PYO on tolerance to CIP (1 μg/mL), LVX (1 μg/mL), and CST (16 μg/mL) in GMM ( n = 4). (D) Effect of PYO on tolerance to CIP (1 μg/mL) and TOB (40 μg/mL) in SCFM ( n = 4). PYO itself was not toxic under the experimental conditions [ 16 ] ( S4C Fig ). WT made 50–80 μM PYO as measured by absorbance of the culture supernatant at 691 nm. See S5A Fig for experimental design. (E) Effect on tolerance to CIP (1 μg/mL) in GMM caused by the presence of the 4 main phenazines produced by P . aeruginosa (PYO, PCA, PCN, and 1-OH-PHZ) ( n = 4). For this experiment, a Δ phz * strain that cannot produce or modify any phenazine was used (see Methods ). (F, G) Effect of PYO on lag during outgrowth after exposure to CIP in GMM. A representative field of view over different time points (F; magenta = WT::mApple, green = Δ phz ::GFP; see S1 Movie ) is shown together with the quantification of growth area on the agarose pads at time 0 hour and 15 hours (G). For these experiments, a culture of each strain tested was grown and exposed to CIP (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 CIP (see Methods and S5D Fig 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 S4E Fig . See S4C Fig for complementary data about effects of PYO on lag. Scale bar: 20 μm. (H) Tolerance of Δ phz to CIP (1 μg/mL) in GMM in the presence of different concentrations of PYO ( n = 4). (G) Tolerance of Δ phz to CIP (1 μg/mL) in GMM 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, D, E, H—1-way ANOVA with Tukey HSD multiple comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); G, I—Welch unpaired t test (* p
    Figure Legend Snippet: PYO induces expression of specific efflux systems, conferring cross-tolerance to fluoroquinolones. (A) Structures of PYO, 2 representative fluoroquinolones (CIP and LVX) and 2 representative aminoglycosides (GEN and TOB). PYO and fluoroquinolones are pumped by MexEF-OprN and MexGHI-OpmD, while aminoglycosides are not [ 21 , 22 ]. 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; n = 3) and PYO-dose-dependent changes in expression of mexEF-oprN and mexGHI-opmD systems (right; n = 3). For full qRT-PCR dataset, see S1 – S3 Figs. (C) Effect of PYO on tolerance to CIP (1 μg/mL), LVX (1 μg/mL), and CST (16 μg/mL) in GMM ( n = 4). (D) Effect of PYO on tolerance to CIP (1 μg/mL) and TOB (40 μg/mL) in SCFM ( n = 4). PYO itself was not toxic under the experimental conditions [ 16 ] ( S4C Fig ). WT made 50–80 μM PYO as measured by absorbance of the culture supernatant at 691 nm. See S5A Fig for experimental design. (E) Effect on tolerance to CIP (1 μg/mL) in GMM caused by the presence of the 4 main phenazines produced by P . aeruginosa (PYO, PCA, PCN, and 1-OH-PHZ) ( n = 4). For this experiment, a Δ phz * strain that cannot produce or modify any phenazine was used (see Methods ). (F, G) Effect of PYO on lag during outgrowth after exposure to CIP in GMM. A representative field of view over different time points (F; magenta = WT::mApple, green = Δ phz ::GFP; see S1 Movie ) is shown together with the quantification of growth area on the agarose pads at time 0 hour and 15 hours (G). For these experiments, a culture of each strain tested was grown and exposed to CIP (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 CIP (see Methods and S5D Fig 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 S4E Fig . See S4C Fig for complementary data about effects of PYO on lag. Scale bar: 20 μm. (H) Tolerance of Δ phz to CIP (1 μg/mL) in GMM in the presence of different concentrations of PYO ( n = 4). (G) Tolerance of Δ phz to CIP (1 μg/mL) in GMM 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, D, E, H—1-way ANOVA with Tukey HSD multiple comparison test, with asterisks showing significant differences relative to untreated Δ phz (no PYO); G, I—Welch unpaired t test (* p

    Techniques Used: Expressing, Quantitative RT-PCR, Produced

    21) Product Images from "Efficient coralline algal psbA mini barcoding and High Resolution Melt (HRM) analysis using a simple custom DNA preparation"

    Article Title: Efficient coralline algal psbA mini barcoding and High Resolution Melt (HRM) analysis using a simple custom DNA preparation

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36998-6

    DNA preparation (3 methods) and PCR (4 protocols) comparisons. 1 Kb DNA ladder (L). Grinded sample with QuickExtract followed with Monarch purification, eluate diluted 10 −1 in Di H 2 O (A1, A2, A3 A4). Whole sample with QuickExtract, eluate diluted 10 −2 in Di H 2 O (B1, B2, B3 B4). Whole sample with QuickExtract followed with Monarch purification, eluate diluted 10 −1 in Di H 2 O (C1, C2, C3 C4). Primers GazF1 GazR1 producing a 610 bp amplicon (A1, B1 C1). Primers psbA21-350F psbA22-350R producing a 350 bp amplicon (A2, B2 C2). Primers psbA-F1 psbA-R1 producing a 957 bp amplicon (A3, B3 C3). Primers psbA-F1 psbA-R600 producing a 600 bp amplicon (A4, B4 C4). The full-length gel is presented in Supplementary Figure S3 .
    Figure Legend Snippet: DNA preparation (3 methods) and PCR (4 protocols) comparisons. 1 Kb DNA ladder (L). Grinded sample with QuickExtract followed with Monarch purification, eluate diluted 10 −1 in Di H 2 O (A1, A2, A3 A4). Whole sample with QuickExtract, eluate diluted 10 −2 in Di H 2 O (B1, B2, B3 B4). Whole sample with QuickExtract followed with Monarch purification, eluate diluted 10 −1 in Di H 2 O (C1, C2, C3 C4). Primers GazF1 GazR1 producing a 610 bp amplicon (A1, B1 C1). Primers psbA21-350F psbA22-350R producing a 350 bp amplicon (A2, B2 C2). Primers psbA-F1 psbA-R1 producing a 957 bp amplicon (A3, B3 C3). Primers psbA-F1 psbA-R600 producing a 600 bp amplicon (A4, B4 C4). The full-length gel is presented in Supplementary Figure S3 .

    Techniques Used: Polymerase Chain Reaction, Purification, Amplification

    22) 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

    23) Product Images from "A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters"

    Article Title: A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters

    Journal: bioRxiv

    doi: 10.1101/834630

    (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p
    Figure Legend Snippet: (A) Assay to detect CRISPR/Cas9-mediated cleavage in vitro . A typical region of the Muc14a gene containing at least 2 binding sites for each of the gRNAs: Muc14a _3, Muc14a_4 , Muc14a_5 and Muc14a_6 (top). The PCR amplified DNA fragment was used as a digestion target for Cas9/gRNA cleavage reactions in vitro (bottom). Reactions were run on a gel to detect cleavage. A control without gRNA was included. (B) Analysis of combinations of gRNAs and Cas9 sources for X-shredding. Average male frequencies in the F1 progeny are shown for each parental genotype with a single copy of βtub85Dtub85D-cas9 transgene (1X), two copies of βtub85Dtub85D-cas9 transgene (2X) or one copy of nos-cas9 (grey bars). All lines were crossed to wild type w individuals. The reciprocal cross (female ctrl) or heterozygote βtub85Dtub85D-cas9/ + or nos-cas9/ + without gRNA (no gRNA) were used as control. The black arrow indicates gRNAs in the multiplex array and the red dotted line indicates an unbiased sex-ratio. Crosses were set as pools of males and females or as multiple male single crosses in which case error bars indicate the mean ± SD for a minimum of ten independent single crosses. For all crosses n indicates the total number of individuals (males + females) in the F1 progeny counted. (C) Developmental survival analysis of the F1 progeny of Muc14a_6/βtub85Dtub85D-cas9 males crossed to w females compared to w and βtub85Dtub85D-cas9/ + control males crossed to w females. n indicates the number of individuals recorded at every developmental stage (males + females) in the F1 progeny. Bars indicate means ± SD for at least ten independent single crosses. Statistical significance was calculated with a t test assuming unequal variance. ** p

    Techniques Used: CRISPR, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Multiplex Assay

    24) Product Images from "DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats"

    Article Title: DNA polymerase stalling at structured DNA constrains the expansion of Short Tandem Repeats

    Journal: bioRxiv

    doi: 10.1101/2020.06.20.162743

    Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.
    Figure Legend Snippet: Pooled measurement of DNA polymerase stalling at STRs. (a) Overview of the high-throughput primer extension assay used to monitor DNA synthesis at designed sequences. A library of 20,000 sequences comprising all STR permutations at three different lengths together with control structured DNA sequences was synthesised on a programmable microarray, eluted and inserted into a phagemid vector. After PCR amplification, insertion into a phagemid vector and bacterial amplification, circular single-stranded DNA templates were produced using a M13KO7 helper phage. Fluorescently labelled primer (P3) and structures annealing were performed before initiating DNA synthesis through the addition of T7 DNA polymerase. Primers are then either fully extended to the length of the circular template or the extension is stopped within STRs if the DNA polymerases stall at structured DNAs. (b) Extended and stalled products were then analysed by denaturing Poly Acrylamide Gel (PAGE) electrophoresis, recovered from the gel matrix and prepared for high throughput sequencing. DNA polymerase stalling was then quantified by analysing the enrichment of each sequence form the library in the stalled and extended fractions. Representative fluorescence gel imaging of primer extension reactions on templates containing a G-quadruplex (G4) structure, a mutated G4 or the entire DNA library, stopped after the indicated times, are reported for comparison. Blue and red arrows indicate the position of the extended and stalled products respectively. The green line highlights the presence of transient stall sites that disappear overtime.

    Techniques Used: High Throughput Screening Assay, Primer Extension Assay, DNA Synthesis, Microarray, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Produced, Acrylamide Gel Assay, Polyacrylamide Gel Electrophoresis, Electrophoresis, Next-Generation Sequencing, Sequencing, Fluorescence, Imaging

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    New England Biolabs monarch gdna blood lysis buffer
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    Monarch Gdna Blood Lysis Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs monarch pcr dna cleanup kit
    Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic <t>DNA.</t> Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing <t>PCR</t> products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.
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    SHK-1 cells were electroporated with 1.4 μM Cas9:gRNA RNP and transferred to 2 separate 96-well plates. (A) After 48h, cell survival (plate1) was calculated using CellTiter Glo 2.0. (B) genomic DNA (plate 2) was extracted at 7 dpt, and the target sequence amplified by PCR and editing efficiency estimated using Sanger sequencing. (C) Using 1.4 μM RNP, the editing efficiency after 7 and 14 ddpt was estimated for different electroporation settings. (D) All the sequencing data, obtained from ICE analysis of Sanger sequencing of the intergenic target region from optimisation experiments (n=55) were pooled and plotted according to edit pattern.

    Journal: bioRxiv

    Article Title: Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes

    doi: 10.1101/2020.04.03.022038

    Figure Lengend Snippet: SHK-1 cells were electroporated with 1.4 μM Cas9:gRNA RNP and transferred to 2 separate 96-well plates. (A) After 48h, cell survival (plate1) was calculated using CellTiter Glo 2.0. (B) genomic DNA (plate 2) was extracted at 7 dpt, and the target sequence amplified by PCR and editing efficiency estimated using Sanger sequencing. (C) Using 1.4 μM RNP, the editing efficiency after 7 and 14 ddpt was estimated for different electroporation settings. (D) All the sequencing data, obtained from ICE analysis of Sanger sequencing of the intergenic target region from optimisation experiments (n=55) were pooled and plotted according to edit pattern.

    Article Snippet: Genomic DNA (gDNA) was extracted with QuickExtract buffer (Lucigen, Middleton, USA) by adding 30 μL to a well of a 96-well plate and incubating for 5 min.

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Electroporation

    Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Journal: The Journal of Biological Chemistry

    Article Title: The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3

    doi: 10.1074/jbc.RA120.012518

    Figure Lengend Snippet: Mutations were detected in FKH domains of FOXP3 transcripts in HCC. A , representative sequencing chromatograms of point mutations. B , representative sequencing chromatograms of the complicated mutation status. Upper left panel , FKH sequences in mRNA. Upper right panel , sequences of the corresponding regions in genomic DNA. Lower panel , sequences of individual TA clones which were generated to examine individual sequences in multiple-mutation bearing PCR products. C , representative immunohistochemical results of FOXP3-positive lymphocyte distribution in tumors and corresponding nontumorous tissues. Black arrow , FOXP3-positive lymphocytes.

    Article Snippet: The 1.2-kb PCR products were purified with Monarch PCR & DNA Cleanup Kit (New England Biolabs) and sent to BGI (Shenzhen, China) for sequencing with primer 5′-GTAGCCATGGAAACAGCACA-3′.

    Techniques: Sequencing, Mutagenesis, Clone Assay, Generated, Polymerase Chain Reaction, Immunohistochemistry

    mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Journal: bioRxiv

    Article Title: Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

    doi: 10.1101/2021.03.29.437587

    Figure Lengend Snippet: mRNA reporter design and in-cell and in-solution workflows with in-cell polysome validation. (A) Schematic for the 3’ UTR-barcoded mRNA reporter used to screen mRNA performance in a pooled format. The constant regions and barcode, which flank a variable 3’ UTR, were instrumental for amplifying and identifying hundreds of constructs simultaneously in each of the pooled experiments that comprise PERSIST-seq. The DNA templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled PCR using primers complementary to the constant region (T7 promoter) preceding the variable 5’ UTR, and to the ‘constant3’ region following the variable 3’ UTR. (B) Summary of the workflow to progress from the individually synthesized DNA templates to the in vitro synthesized mRNA pool of 233 different constructs. We then use the same mRNA pool to screen mRNA performance in a three-pronged set of in-cell and in-solution expression and stability analyses. (C) Quality control of the 233-mRNA pool on a 1.2% formaldehyde (FA) gel stained with ethidium bromide (EtBr) after 3 hrs of in vitro transcription (IVT). The mRNA pool was analyzed before and after capping and polyadenylation. Pooled IVT is equally efficient with the starting template DNA pool with or without PCR-amplification of the DNA template pool. The three major bands corresponding to the three CDS types are indicated. The RiboRuler High Range RNA ladder (Thermo Fisher) is loaded for reference. (D) Polysome fractionation analysis of a transfected mRNA reporter. As an example, the distribution of an mRNA with short scrambled 5’ and 3’ UTRs 6 hrs after transfection into HEK293T cells was compared to the distribution of endogenous human ActB mRNA. RNA was extracted from fractions and quantified by qPCR with a RNA spike-in for normalization. Values are plotted as mRNA normalized per fraction. (E) In-solution RNA degradation strategy of barcoded mRNAs containing CDS variants with hHBB 5’ and 3’ UTRs. The differential degradation of CDS variants depends on their individual CDS structures. mRNA pools are degraded in solution by nucleophilic attack (red circle). After degradation, RT-PCR is performed to selectively amplify mRNAs that remain intact along their full length. Then, the barcode regions of these full-length mRNAs are PCR-amplified, adaptor-ligated, and prepared for Illumina sequencing.

    Article Snippet: PCR reactions were purified with Monarch PCR & DNA Cleanup Kit (NEB, T1030L).

    Techniques: Construct, Synthesized, Amplification, Polymerase Chain Reaction, In Vitro, Expressing, Staining, Fractionation, Transfection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Efficiencies of two- and three-steps epicPCR protocols using different blocking primers (BPs) concentrations. The epicPCRs were run on SXT/R391 carrying bacteria from the Meurthe River water. A and C indicate wells with amplification products from two-steps epicPCR protocols (fusion-PCR on polyacrylamide beads + nested PCR). In the A lane, fusion-PCR products from the first step were used as template DNA in the second step without dilution whereas these products were diluted ten times in line C to circumvent the possible presence of PCR inhibitors. B indicates wells loaded with amplification products resulting from a three-steps epicPCR protocol (fusion PCR on polyacrylamide beads + blocking PCR with BPs as sole primers + nested PCR). The expected size of the final nested-PCR product is around 350 bp (depending on the 16S rRNA gene fragment polymorphism). The conditions of epicPCR as used in Hultman et al. (2018) and that we determined to be the best in our conditions (epicPCR 2.0) are indicated by black arrows. The minus symbols indicate negative controls with epicPCRs run without polyacrylamide beads-template.

    Journal: Microorganisms

    Article Title: EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

    doi: 10.3390/microorganisms9081649

    Figure Lengend Snippet: Efficiencies of two- and three-steps epicPCR protocols using different blocking primers (BPs) concentrations. The epicPCRs were run on SXT/R391 carrying bacteria from the Meurthe River water. A and C indicate wells with amplification products from two-steps epicPCR protocols (fusion-PCR on polyacrylamide beads + nested PCR). In the A lane, fusion-PCR products from the first step were used as template DNA in the second step without dilution whereas these products were diluted ten times in line C to circumvent the possible presence of PCR inhibitors. B indicates wells loaded with amplification products resulting from a three-steps epicPCR protocol (fusion PCR on polyacrylamide beads + blocking PCR with BPs as sole primers + nested PCR). The expected size of the final nested-PCR product is around 350 bp (depending on the 16S rRNA gene fragment polymorphism). The conditions of epicPCR as used in Hultman et al. (2018) and that we determined to be the best in our conditions (epicPCR 2.0) are indicated by black arrows. The minus symbols indicate negative controls with epicPCRs run without polyacrylamide beads-template.

    Article Snippet: The PCR products were pooled, cleaned (Monarch PCR & DNA Cleanup Kit; New England Biolabs, Ipswich, MA, USA) and sequenced on Illumina Miseq (2 × 250) (Genewiz, South Plainfield, NJ, USA).

    Techniques: Blocking Assay, Amplification, Polymerase Chain Reaction, Nested PCR

    Control epicPCR amplifications targeting SXT/R391 ICEs performed on beads carrying E. coli MG1656::SXT MO10 as template. Each experiment was done in duplicates with a no-template DNA control, using either the Phusion DNA Polymerase GC or HF buffers. In the nested-PCR step, the use of blocking primers (BPs) has been done as depicted in Spencer et al. (2016), and usually performed so far. All these conditions are summarized in the table upper the gel. The expected size of the desired DNA fragment is 367 bp. Black arrows indicate epicPCR products obtained after performing fusion and nested PCRs using HF but not GC buffer.

    Journal: Microorganisms

    Article Title: EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

    doi: 10.3390/microorganisms9081649

    Figure Lengend Snippet: Control epicPCR amplifications targeting SXT/R391 ICEs performed on beads carrying E. coli MG1656::SXT MO10 as template. Each experiment was done in duplicates with a no-template DNA control, using either the Phusion DNA Polymerase GC or HF buffers. In the nested-PCR step, the use of blocking primers (BPs) has been done as depicted in Spencer et al. (2016), and usually performed so far. All these conditions are summarized in the table upper the gel. The expected size of the desired DNA fragment is 367 bp. Black arrows indicate epicPCR products obtained after performing fusion and nested PCRs using HF but not GC buffer.

    Article Snippet: The PCR products were pooled, cleaned (Monarch PCR & DNA Cleanup Kit; New England Biolabs, Ipswich, MA, USA) and sequenced on Illumina Miseq (2 × 250) (Genewiz, South Plainfield, NJ, USA).

    Techniques: Nested PCR, Blocking Assay