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
    Category:
    Other Kits
    Applications:
    DNA Manipulation
    Size:
    250 preps
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    Structured Review

    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, 2021-07
    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 "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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) Product Images from "Dynamic topology of double-stranded telomeric DNA studied by single-molecule manipulation in vitro"

    Article Title: Dynamic topology of double-stranded telomeric DNA studied by single-molecule manipulation in vitro

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa479

    ( A ) Schematics of the construction of the recombinant plasmid. Short (TTAGGG) 5 (blue fragment) and (CCCTAA) 5 (red fragment) DNAs were extended by EM-PCR and then inserted into the T-Vector. The recombinant plasmid that contains double-stranded telomeric DNA can be obtained after overnight transfection and incubation. ( B ) Agarose gel analysis of the extension products produced from (TTAGGG) 5 and (CCCTAA) 5 during EM-PCR. Lane 1: 4 cycles; lane 2: 7 cycles; lane 3: 10 cycles; lane 4: 14 cycles; lane 5: 17 cycles; and lane 6: 20 cycles. ( C ) The T-Vector and the recombinant plasmid were double-digested by BspQI and BstAPI restriction endonucleases. The products were analyzed by agarose gel as shown in lane 1 (T-Vector) and lane 2 (recombinant, pUC18-T2AG3), respectively. ( D ) AFM imaging of telomeric-sequence-containing DNA (∼1200 bp) on mica.
    Figure Legend Snippet: ( A ) Schematics of the construction of the recombinant plasmid. Short (TTAGGG) 5 (blue fragment) and (CCCTAA) 5 (red fragment) DNAs were extended by EM-PCR and then inserted into the T-Vector. The recombinant plasmid that contains double-stranded telomeric DNA can be obtained after overnight transfection and incubation. ( B ) Agarose gel analysis of the extension products produced from (TTAGGG) 5 and (CCCTAA) 5 during EM-PCR. Lane 1: 4 cycles; lane 2: 7 cycles; lane 3: 10 cycles; lane 4: 14 cycles; lane 5: 17 cycles; and lane 6: 20 cycles. ( C ) The T-Vector and the recombinant plasmid were double-digested by BspQI and BstAPI restriction endonucleases. The products were analyzed by agarose gel as shown in lane 1 (T-Vector) and lane 2 (recombinant, pUC18-T2AG3), respectively. ( D ) AFM imaging of telomeric-sequence-containing DNA (∼1200 bp) on mica.

    Techniques Used: Recombinant, Plasmid Preparation, Polymerase Chain Reaction, Transfection, Incubation, Agarose Gel Electrophoresis, Produced, Imaging, Sequencing

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) Product Images from "CRISPR-Cas12a-assisted PCR tagging of mammalian genes"

    Article Title: CRISPR-Cas12a-assisted PCR tagging of mammalian genes

    Journal: bioRxiv

    doi: 10.1101/473876

    PCR Strategy PCR is performed with M1 and M2 tagging oligos and a template cassette that contains the tag. The M1 and M2 tagging oligos provide the homology arms (HA, ∼55 to 90 nt in length) for targeted integration. The M2 tagging oligo additionally provides the direct repeat and a protospacer sequence (orange) for a Cas12a endonuclease. The template cassette contains the desired tag and additional features, such as a selection marker. It also contains the U6 Pol III promoter for driving crRNA expression. PCR yields a linear DNA fragment (PCR cassette) that contains homology arms to the target locus and a functional crRNA gene to cleave the locus.
    Figure Legend Snippet: PCR Strategy PCR is performed with M1 and M2 tagging oligos and a template cassette that contains the tag. The M1 and M2 tagging oligos provide the homology arms (HA, ∼55 to 90 nt in length) for targeted integration. The M2 tagging oligo additionally provides the direct repeat and a protospacer sequence (orange) for a Cas12a endonuclease. The template cassette contains the desired tag and additional features, such as a selection marker. It also contains the U6 Pol III promoter for driving crRNA expression. PCR yields a linear DNA fragment (PCR cassette) that contains homology arms to the target locus and a functional crRNA gene to cleave the locus.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Selection, Marker, Expressing, Functional Assay

    Tagging efficiency as a function of different parameters. ( a ) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5’-HA and 3’-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 days after transfection as described before. Data from three replicates is shown. Error bars indicate SD. ( b ) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: Homology arms (90-bp and 55-bp homology, for HR; A ), blunt ended arms without homology to the target locus (blunt; B ), Hga I cut ( D ) and uncut ends ( C ). Cutting with the type IIS restriction enzyme Hga I results in 5-nt 3’-overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated three days later as described in (a) using HEK293T cells. Data from three replicates is shown. Error bars indicate SD. ( c ) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were ‘cartridge’ purified except for the ones denoted with ‘PAGE’, which were size selected using polyacrylamide gel electrophoresis. Tagging efficiency was estimated three days after transfection as described before using HEK293T cells. Data from three replicates is shown. Error bars indicate SD.
    Figure Legend Snippet: Tagging efficiency as a function of different parameters. ( a ) Length of homology arms. M1 and M2 tagging oligos containing the indicated sequence lengths of homology arm (5’-HA and 3’-HA, respectively) to the destination locus were used for PCR tagging of the HNRNPA1 locus in HEK293T cells. Tagging efficiency was estimated 3 days after transfection as described before. Data from three replicates is shown. Error bars indicate SD. ( b ) PCR cassettes containing various types of ends to direct the choice of DNA repair pathway: Homology arms (90-bp and 55-bp homology, for HR; A ), blunt ended arms without homology to the target locus (blunt; B ), Hga I cut ( D ) and uncut ends ( C ). Cutting with the type IIS restriction enzyme Hga I results in 5-nt 3’-overhangs that are complementary to the overhangs generated by the crRNA directed Cas12a-cleavage of the destination locus. Tagging efficiency was estimated three days later as described in (a) using HEK293T cells. Data from three replicates is shown. Error bars indicate SD. ( c ) Use of modified and/or purified oligos. M1/M2 tagging oligos with the indicated number of phosphorothioate bonds and/or biotin as indicated were used for generation of PCR cassettes. All oligos were ‘cartridge’ purified except for the ones denoted with ‘PAGE’, which were size selected using polyacrylamide gel electrophoresis. Tagging efficiency was estimated three days after transfection as described before using HEK293T cells. Data from three replicates is shown. Error bars indicate SD.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Transfection, Generated, Modification, Purification, Polyacrylamide Gel Electrophoresis

    Endogenous C-terminal gene tagging in mammalian cells using PCR tagging. ( a ) For tag insertion before the STOP codon of an ORF, two ‘Gene specific tagging oligos’ (termed M1 and M2) are designed using an online tool ( www.pcr-tagging.com ). A ‘Tagging PCR’ with a generic ‘Template plasmid’ generates the gene-specific ‘PCR cassette’. The ‘Template plasmid’ provides the tag (e.g. a fluorescent protein), a possible selection marker and a Pol III promoter. For gene tagging the ‘PCR cassette’ is transfected into the target cell together with a helper plasmid containing a Cas12a endonuclease gene. This leads to insertion of the PCR cassette into the chromosome, which yields a fusion of the tag (e.g. GFP) with the target gene. ( b ) Tagging Principle: The PCR cassette contains a crRNA sequence that is expressed inside the cell via an U6 promoter (Pol III promoter). The crRNA directs Cas12a (which is expressed from the helper plasmid) to the target locus close to the insertion site. Stimulated by the DSB the linear PCR cassette is then inserted into the genome. The homology arm of the M1 tagging oligo thereby directs in frame fusion of the tag with the target ORF, leading to the expression of a tagged protein from the target locus. Integration leads to destruction of the crRNA target site, thus preventing re-cleavage of the modified locus. ( c ) Efficiency of C-terminal mNeonGreen-tagging for 16 organelle specific genes. For each gene, specific M1/M2 tagging oligos were used to amplify an mNeonGreen containing template plasmid. The resulting PCR cassettes were transfected in HEK293T cells. HOECHST staining of live cells and analysis by fluorescence microscopy was performed three days after transfection. Fractions of cells exhibiting the expected localization or diffuse cytoplasmic green fluorescence are shown. For information on selected genes, see Table S1. Data from one representative experiment is shown. ( d ) Representative images from HEK293T cells 3 days after transfection. mNeonGreen fluorescence and HOECHST staining (DNA) is shown. In addition to the expected localization, cells showing diffuse cytoplasmic fluorescence (arrows) are detected. ( e ) Tagging is specific for the crRNA and guided by the homology arms (HAs). Efficiency of control transfections (see Fig. S2 for representative examples). * in this transfection indicates that a matching combination of crRNA and HAs was used, but the crRNA was expressed from a different PCR fragment. ** indicates that in this case a PCR cassette was used where the crRNA (for CANX) lead to cleavage of a different gene than the one specified by the HAs (HNRNPA1). A small fraction of cells (
    Figure Legend Snippet: Endogenous C-terminal gene tagging in mammalian cells using PCR tagging. ( a ) For tag insertion before the STOP codon of an ORF, two ‘Gene specific tagging oligos’ (termed M1 and M2) are designed using an online tool ( www.pcr-tagging.com ). A ‘Tagging PCR’ with a generic ‘Template plasmid’ generates the gene-specific ‘PCR cassette’. The ‘Template plasmid’ provides the tag (e.g. a fluorescent protein), a possible selection marker and a Pol III promoter. For gene tagging the ‘PCR cassette’ is transfected into the target cell together with a helper plasmid containing a Cas12a endonuclease gene. This leads to insertion of the PCR cassette into the chromosome, which yields a fusion of the tag (e.g. GFP) with the target gene. ( b ) Tagging Principle: The PCR cassette contains a crRNA sequence that is expressed inside the cell via an U6 promoter (Pol III promoter). The crRNA directs Cas12a (which is expressed from the helper plasmid) to the target locus close to the insertion site. Stimulated by the DSB the linear PCR cassette is then inserted into the genome. The homology arm of the M1 tagging oligo thereby directs in frame fusion of the tag with the target ORF, leading to the expression of a tagged protein from the target locus. Integration leads to destruction of the crRNA target site, thus preventing re-cleavage of the modified locus. ( c ) Efficiency of C-terminal mNeonGreen-tagging for 16 organelle specific genes. For each gene, specific M1/M2 tagging oligos were used to amplify an mNeonGreen containing template plasmid. The resulting PCR cassettes were transfected in HEK293T cells. HOECHST staining of live cells and analysis by fluorescence microscopy was performed three days after transfection. Fractions of cells exhibiting the expected localization or diffuse cytoplasmic green fluorescence are shown. For information on selected genes, see Table S1. Data from one representative experiment is shown. ( d ) Representative images from HEK293T cells 3 days after transfection. mNeonGreen fluorescence and HOECHST staining (DNA) is shown. In addition to the expected localization, cells showing diffuse cytoplasmic fluorescence (arrows) are detected. ( e ) Tagging is specific for the crRNA and guided by the homology arms (HAs). Efficiency of control transfections (see Fig. S2 for representative examples). * in this transfection indicates that a matching combination of crRNA and HAs was used, but the crRNA was expressed from a different PCR fragment. ** indicates that in this case a PCR cassette was used where the crRNA (for CANX) lead to cleavage of a different gene than the one specified by the HAs (HNRNPA1). A small fraction of cells (

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Selection, Marker, Transfection, Sequencing, Expressing, Modification, Staining, Fluorescence, Microscopy

    Exploring transfection parameters ( a ) Impact of transfected amounts of DNA on tagging efficiency using HEK293T cells. Transfected amounts of PCR cassette and Cas12a plasmid as indicated. Always 1 µg of DNA was transfected using lipofectamine. pUC18 was used as neutral DNA. Tagging efficiency was determined three days later by HOECHST staining and live cell imaging. Data from one representative experiment is shown. ( b ) HEK293T cells were transfected for 4 hours or overnight using Lipofectamine 2000 or transfected using electroporation, as indicated. Tagging efficiency was determined three days later as described in (a). Data from one representative experiment is shown. ( c ) HEK293T cells were transfected in duplicates by electroporation with Cas12a protein, Cas12a-encoding mRNA or Cas12-encoding plasmid. For protein-based expression 100 ng of PCR cassette while for mRNA and plasmid 1.5 µg PCR cassette were electroporated. Error bars indicate range between the technical duplicates.
    Figure Legend Snippet: Exploring transfection parameters ( a ) Impact of transfected amounts of DNA on tagging efficiency using HEK293T cells. Transfected amounts of PCR cassette and Cas12a plasmid as indicated. Always 1 µg of DNA was transfected using lipofectamine. pUC18 was used as neutral DNA. Tagging efficiency was determined three days later by HOECHST staining and live cell imaging. Data from one representative experiment is shown. ( b ) HEK293T cells were transfected for 4 hours or overnight using Lipofectamine 2000 or transfected using electroporation, as indicated. Tagging efficiency was determined three days later as described in (a). Data from one representative experiment is shown. ( c ) HEK293T cells were transfected in duplicates by electroporation with Cas12a protein, Cas12a-encoding mRNA or Cas12-encoding plasmid. For protein-based expression 100 ng of PCR cassette while for mRNA and plasmid 1.5 µg PCR cassette were electroporated. Error bars indicate range between the technical duplicates.

    Techniques Used: Transfection, Polymerase Chain Reaction, Plasmid Preparation, Staining, Live Cell Imaging, Electroporation, Expressing

    Analysis of clones from a CANX-mNeonGreen tagging experiment. PCR analysis of single clones using primer for PCR of characteristic fragments indicative for correctly inserted fragments. Primer that anneal to chromosomal DNA were chosen to reside outside of the sequences that are contained in the homology arms for recombination. For western blot analysis antibodies specific to mNeonGreen or to Calnexin were used.
    Figure Legend Snippet: Analysis of clones from a CANX-mNeonGreen tagging experiment. PCR analysis of single clones using primer for PCR of characteristic fragments indicative for correctly inserted fragments. Primer that anneal to chromosomal DNA were chosen to reside outside of the sequences that are contained in the homology arms for recombination. For western blot analysis antibodies specific to mNeonGreen or to Calnexin were used.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Western Blot

    24) Product Images from "Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development"

    Article Title: Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development

    Journal: bioRxiv

    doi: 10.1101/2021.01.27.428341

    The published Fzd2 tm1Eem flox allele is a complex genomic alteration. A. Maps detailing our working model of the Fzd2 tm1Eem flox allele. A wild-type Fzd2 gene structure is shown, with 5’UTR in purple, CDS in blue, and 3’UTR in white. The Fzd2 tm1Eem flox allele contains a duplication of Fzd2, resulting in two copies (blue). There is 10 kb of exogenous DNA (bacterial gDNA and targeting plasmid PL253 sequence, light green) in between the two copies of Fzd2. “Fzd2 #1” contains the 3’ loxP insertion but not 5’ loxP . “Fzd2 #2” contains 5’ loxP insertion as well as a 92bp deletion (orange) downstream of the 5’ loxP insertion site and immediately upstream of the Fzd2 start sequence. “Fzd2 #2” has a shortened 3’ UTR that is connected to an inverted 100 kb sequence downstream of the Fzd2 gene (refer to Supplemental Figure 1A for more details). The sequence between the two Fzd2 copies is of unknown length and denoted by a black curved lined (refer to Supplemental Figure 1B for more details). Because we cannot confirm the orientations of the two loxP sites within the two Fzd2 copies due to the long sequence between the two Fzd2 copies, we hypothesize cre-driven recombination results in continuous sequence inversion between the two loxP sites due to the opposite loxP orientations (refer to Discussion for more details). B . The IGV coverage tracks of Fzd2 tm1Eem flox/flox and homozygous global mutant WGS reads were merged to show no Fzd2 or other genomic DNA deletion after Cre exposure. C . Real-time quantitative PCR on genomic DNA shows a 50% decrease in Fzd2 gene content in Fzd2 tm1 . 1Nat heterozygotes and no Fzd2 gene content in homozygous mutants (primer targeting the N-terminal region). The Fzd2 tm1Eem display a ∼2-fold increase in Fzd2 gene content in flox/flox and ∼ homozygous mutant (Mut) samples, which supports Fzd2 being duplicated and loxP sites being in opposite orientations. D . FZD2 flow cytometric analysis of E14.5 limb buds from wild-type (WT) or mice homozygous for a germline cre-mediated recombination of the Fzd2 tm1Eem allele (Mut). Cells were gated to discard debris and doublets, and live cells were determined by DAPI exclusion. FZD2 median fluorescent intensity (MFI) was compared between WT and Mut. WT MFI = 25954; Mut MFI = 12680 E . Limb micromass cultures from homozygous flox Fzd2 tm1Eem mice were infected with Ad5CMV:eGFP (control) or Ad5CMV:Cre-eGFP at an MOI of 100 and assessed by qRT-PCR for Fzd2 mRNA expression.
    Figure Legend Snippet: The published Fzd2 tm1Eem flox allele is a complex genomic alteration. A. Maps detailing our working model of the Fzd2 tm1Eem flox allele. A wild-type Fzd2 gene structure is shown, with 5’UTR in purple, CDS in blue, and 3’UTR in white. The Fzd2 tm1Eem flox allele contains a duplication of Fzd2, resulting in two copies (blue). There is 10 kb of exogenous DNA (bacterial gDNA and targeting plasmid PL253 sequence, light green) in between the two copies of Fzd2. “Fzd2 #1” contains the 3’ loxP insertion but not 5’ loxP . “Fzd2 #2” contains 5’ loxP insertion as well as a 92bp deletion (orange) downstream of the 5’ loxP insertion site and immediately upstream of the Fzd2 start sequence. “Fzd2 #2” has a shortened 3’ UTR that is connected to an inverted 100 kb sequence downstream of the Fzd2 gene (refer to Supplemental Figure 1A for more details). The sequence between the two Fzd2 copies is of unknown length and denoted by a black curved lined (refer to Supplemental Figure 1B for more details). Because we cannot confirm the orientations of the two loxP sites within the two Fzd2 copies due to the long sequence between the two Fzd2 copies, we hypothesize cre-driven recombination results in continuous sequence inversion between the two loxP sites due to the opposite loxP orientations (refer to Discussion for more details). B . The IGV coverage tracks of Fzd2 tm1Eem flox/flox and homozygous global mutant WGS reads were merged to show no Fzd2 or other genomic DNA deletion after Cre exposure. C . Real-time quantitative PCR on genomic DNA shows a 50% decrease in Fzd2 gene content in Fzd2 tm1 . 1Nat heterozygotes and no Fzd2 gene content in homozygous mutants (primer targeting the N-terminal region). The Fzd2 tm1Eem display a ∼2-fold increase in Fzd2 gene content in flox/flox and ∼ homozygous mutant (Mut) samples, which supports Fzd2 being duplicated and loxP sites being in opposite orientations. D . FZD2 flow cytometric analysis of E14.5 limb buds from wild-type (WT) or mice homozygous for a germline cre-mediated recombination of the Fzd2 tm1Eem allele (Mut). Cells were gated to discard debris and doublets, and live cells were determined by DAPI exclusion. FZD2 median fluorescent intensity (MFI) was compared between WT and Mut. WT MFI = 25954; Mut MFI = 12680 E . Limb micromass cultures from homozygous flox Fzd2 tm1Eem mice were infected with Ad5CMV:eGFP (control) or Ad5CMV:Cre-eGFP at an MOI of 100 and assessed by qRT-PCR for Fzd2 mRNA expression.

    Techniques Used: Plasmid Preparation, Sequencing, Mutagenesis, Real-time Polymerase Chain Reaction, Mouse Assay, Infection, Quantitative RT-PCR, Expressing

    25) 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

    26) Product Images from "An improved method for high-throughput quantification of autophagy in mammalian cells"

    Article Title: An improved method for high-throughput quantification of autophagy in mammalian cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-68607-w

    Assessing autophagy during viral infection and overexpression/knockout approaches. ( A ), HeLa GL cells were infected with influenza A virus (IAV, left panel, MOI 5), encephalomyocarditis virus (EMCV, middle panel, MOI 10) or measles virus (MeV, right panel, MOI 5). Cells were harvested, saponin treated, fixed, and the eGFP-LC3B fluorescence analyzed by flow cytometry at the indicated time points post infection. Treatment with Chloroquine (1 µM, 4 h) and Rapamycin (1 µM, 4 h) served as controls. Data are shown as mean(n = 3–6) ± SEM. ( B ), HEK293T GL cells were transiently transfected with an empty vector or a TRIM32-FLAG expressing construct. Cells were saponin treated, fixed, and stained with anti-FLAG antibodies (APC) (left and middle panel). eGFP-LC3B MFI of the transfected cell population was quantified for the TRIM32-FLAG sample and background (= vector) subtracted (right panel). Data are shown as mean(n = 3) ± SEM. ( C ), Agarose gel depicting the sgRNA amplicon amplified by PCR from genomic DNA isolated from saponin treated Jurkat GL cells that were either mock-infected or transduced with the Human CRISPR Knockout Pooled Library (GeCKO v2) ( D ) Exemplary distribution of high/low/medium autophagosome content (= eGFP MFI) in Jurkat eGFP-LC3B cells 10 days after transduction with the Human CRISPR Knockout Pooled Library (GeCKO v2) as assessed by FACS sorting ( E ) Scatter Plot (control population ‘input’ vs low autophagy population ‘low’) of aggregated counts of sgRNAs targeting indicated ATG proteins. Each dot is derived from 6 individual sgRNAs targeting the same gene. The dotted red line indicates an equal abundance of sgRNA counts in both populations. Statistical significance was assessed using unpaired t-test ( A ) or one-way ANOVA ( B ). ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Uncropped agarose gel in Supplementary Fig. 5.
    Figure Legend Snippet: Assessing autophagy during viral infection and overexpression/knockout approaches. ( A ), HeLa GL cells were infected with influenza A virus (IAV, left panel, MOI 5), encephalomyocarditis virus (EMCV, middle panel, MOI 10) or measles virus (MeV, right panel, MOI 5). Cells were harvested, saponin treated, fixed, and the eGFP-LC3B fluorescence analyzed by flow cytometry at the indicated time points post infection. Treatment with Chloroquine (1 µM, 4 h) and Rapamycin (1 µM, 4 h) served as controls. Data are shown as mean(n = 3–6) ± SEM. ( B ), HEK293T GL cells were transiently transfected with an empty vector or a TRIM32-FLAG expressing construct. Cells were saponin treated, fixed, and stained with anti-FLAG antibodies (APC) (left and middle panel). eGFP-LC3B MFI of the transfected cell population was quantified for the TRIM32-FLAG sample and background (= vector) subtracted (right panel). Data are shown as mean(n = 3) ± SEM. ( C ), Agarose gel depicting the sgRNA amplicon amplified by PCR from genomic DNA isolated from saponin treated Jurkat GL cells that were either mock-infected or transduced with the Human CRISPR Knockout Pooled Library (GeCKO v2) ( D ) Exemplary distribution of high/low/medium autophagosome content (= eGFP MFI) in Jurkat eGFP-LC3B cells 10 days after transduction with the Human CRISPR Knockout Pooled Library (GeCKO v2) as assessed by FACS sorting ( E ) Scatter Plot (control population ‘input’ vs low autophagy population ‘low’) of aggregated counts of sgRNAs targeting indicated ATG proteins. Each dot is derived from 6 individual sgRNAs targeting the same gene. The dotted red line indicates an equal abundance of sgRNA counts in both populations. Statistical significance was assessed using unpaired t-test ( A ) or one-way ANOVA ( B ). ns = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Uncropped agarose gel in Supplementary Fig. 5.

    Techniques Used: Infection, Over Expression, Knock-Out, Fluorescence, Flow Cytometry, Transfection, Plasmid Preparation, Expressing, Construct, Staining, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Isolation, Transduction, CRISPR, FACS, Derivative Assay

    27) 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

    28) 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

    29) Product Images from "An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola"

    Article Title: An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-79120-5

    Generation of marker-free mutants with the CRISPR/Cas9 technique. ( a ) Strategies for CRISPR/Cas9-assisted inactivation of pks1 . Three protospacers (PS) for inducing DSBs in different regions of pks1 were combined with the sgRNA backbone, cas9 , a hygR cassette as well as the AMA1 sequence (Table S1 ). PS- and PS-adjacent motifs (PAM) are shown. ( b ) Random mutation of pks1 via non-homologous end joining (NHEJ). WT:A95 protoplasts were transformed with the circular cas9/pks1 -sgRNA-containing AMA plasmids (Table S3 ). 31 mel− mutants were studied. MEA was inoculated with cell suspensions and incubated for 11 days. ∆ p/ ∆ p − ∆ pks1/∆phd1 . Sequencing of PS-spanning regions revealed point mutations and short deletions at the Cas9 sites (green triangles) resulting in truncated proteins. Mutants H2.7 and H2.6 contain in-frame deletions (Fig. S5 ). ( c ) Increase of editing efficiency by addition of single-stranded DNA oligonucleotides. WT:A95 protoplasts were co-transformed with cas9 -sgRNA-containing AMA plasmids and single-stranded 80-bp-long DNA oligonucleotides (+ O) that covered the Cas9 cutting sites and comprised mutations (Fig. S6 ). Numbers of differentially pigmented colonies on the transformation plates were quantified from four independent experiments (Table S6 ). Representative plates for pks1 are shown on the left. The PS-spanning regions of chosen mutants were amplified by PCR and sequenced to detect the mutations at the Cas9 cutting sites (Fig. S6 ). MEA was inoculated with cell suspensions and incubated for 8 days.
    Figure Legend Snippet: Generation of marker-free mutants with the CRISPR/Cas9 technique. ( a ) Strategies for CRISPR/Cas9-assisted inactivation of pks1 . Three protospacers (PS) for inducing DSBs in different regions of pks1 were combined with the sgRNA backbone, cas9 , a hygR cassette as well as the AMA1 sequence (Table S1 ). PS- and PS-adjacent motifs (PAM) are shown. ( b ) Random mutation of pks1 via non-homologous end joining (NHEJ). WT:A95 protoplasts were transformed with the circular cas9/pks1 -sgRNA-containing AMA plasmids (Table S3 ). 31 mel− mutants were studied. MEA was inoculated with cell suspensions and incubated for 11 days. ∆ p/ ∆ p − ∆ pks1/∆phd1 . Sequencing of PS-spanning regions revealed point mutations and short deletions at the Cas9 sites (green triangles) resulting in truncated proteins. Mutants H2.7 and H2.6 contain in-frame deletions (Fig. S5 ). ( c ) Increase of editing efficiency by addition of single-stranded DNA oligonucleotides. WT:A95 protoplasts were co-transformed with cas9 -sgRNA-containing AMA plasmids and single-stranded 80-bp-long DNA oligonucleotides (+ O) that covered the Cas9 cutting sites and comprised mutations (Fig. S6 ). Numbers of differentially pigmented colonies on the transformation plates were quantified from four independent experiments (Table S6 ). Representative plates for pks1 are shown on the left. The PS-spanning regions of chosen mutants were amplified by PCR and sequenced to detect the mutations at the Cas9 cutting sites (Fig. S6 ). MEA was inoculated with cell suspensions and incubated for 8 days.

    Techniques Used: Marker, CRISPR, Sequencing, Mutagenesis, Non-Homologous End Joining, Transformation Assay, Microelectrode Array, Incubation, Amplification, Polymerase Chain Reaction

    30) Product Images from "A comprehensive analysis of e-CAS cell line reveals they are mouse macrophages"

    Article Title: A comprehensive analysis of e-CAS cell line reveals they are mouse macrophages

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26512-3

    PCR reactions for equine TLR s. Reactions performed with ( A ), DNA sample obtained from e-CAS cells and ( B ), DNA sample obtained from eqT8888 cells. L: Ladder. The picture has been cropped for clarity and conciseness. A picture of the full-length gel is presented in Supplementary Fig. S1 .
    Figure Legend Snippet: PCR reactions for equine TLR s. Reactions performed with ( A ), DNA sample obtained from e-CAS cells and ( B ), DNA sample obtained from eqT8888 cells. L: Ladder. The picture has been cropped for clarity and conciseness. A picture of the full-length gel is presented in Supplementary Fig. S1 .

    Techniques Used: Polymerase Chain Reaction

    31) Product Images from "High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus"

    Article Title: High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus

    Journal: Genetics

    doi: 10.1534/genetics.120.303269

    Oncopeltus CRISPR/Cas9 mutagenesis workflow. (A) (i) Genomic structure of Of-w ; exon boundaries are based on transcriptome data and the alignment of a cDNA sequenced in this study to the genome; however, exon 4 was not found in the genome. The dsRNA target region (519 bp) used in this study is shown as an orange bar (exons only). (ii) The target location gRNAs (A–C) used in this study are in red. (iii) The primers used to PCR-amplify exon 2 for our two genotyping assays are shown as black arrows. The amplicon is 513 bp and includes some intronic regions surrounding the exon. Stars mark the predicted loci of Cas9 cleavage and thus likely mutation; the predicted Surveyor cleavage product sizes (in base pairs) for each gRNA are shown above the strands. (B) Gels showing representative results from the Surveyor digest and the heteroduplex mobility assays used to genotype G1s. (i) In the Surveyor assay, PCR products were digested with Surveyor nuclease, resulting in cleavage in samples derived from heterozygous individuals. All gRNA A individuals screened here (lanes 1–7) show the expected cleavage products (∼100 and 413 bp), as do all gRNA B individuals (lanes 8–9; ∼277 and 236 bp, asterisks), identifying all individuals shown here as heterozygotes. (ii) In the heteroduplex mobility assay, 5 μl of PCR product was electrophoresed on a 4% agarose gel for > 5 hr at 80 V, allowing visualization of heteroduplexes, which migrate more slowly than homoduplexes, in samples from heterozygotes (lanes 11–14); thus, samples from homozygous individuals (WT) should produce only the 513-bp band (lane 10). (C) Sequenced mutant alleles induced by (i) gRNA-A/Cas9 and (ii) gRNA-B/Cas9. Red and blue lettering indicate the PAM site and insertions, respectively. (i) The mutant allele from individual A6-17 has a 16-bp insertion (blue) that adds a premature stop codon (red box); the allele from individual A12-10 has a 15-bp insertion (blue) and 1-bp deletion that induces a frameshift. (ii) The allele from individual B4-4 has a 7-bp deletion, resulting in a frameshift; the allele from individual B5-16 is complex, showing substitutions (blue) replacing sequence (underlined) 5′ and 3′ of the PAM site (red), including mutation of the splicing donor site (black box). cDNA, complementary DNA; CRISPR, clustered regularly interspaced short palindromic repeats; dsRNA, double-stranded RNA; gRNA, guide RNA; PAM, protospacer-adjacent motif; WT, wild-type.
    Figure Legend Snippet: Oncopeltus CRISPR/Cas9 mutagenesis workflow. (A) (i) Genomic structure of Of-w ; exon boundaries are based on transcriptome data and the alignment of a cDNA sequenced in this study to the genome; however, exon 4 was not found in the genome. The dsRNA target region (519 bp) used in this study is shown as an orange bar (exons only). (ii) The target location gRNAs (A–C) used in this study are in red. (iii) The primers used to PCR-amplify exon 2 for our two genotyping assays are shown as black arrows. The amplicon is 513 bp and includes some intronic regions surrounding the exon. Stars mark the predicted loci of Cas9 cleavage and thus likely mutation; the predicted Surveyor cleavage product sizes (in base pairs) for each gRNA are shown above the strands. (B) Gels showing representative results from the Surveyor digest and the heteroduplex mobility assays used to genotype G1s. (i) In the Surveyor assay, PCR products were digested with Surveyor nuclease, resulting in cleavage in samples derived from heterozygous individuals. All gRNA A individuals screened here (lanes 1–7) show the expected cleavage products (∼100 and 413 bp), as do all gRNA B individuals (lanes 8–9; ∼277 and 236 bp, asterisks), identifying all individuals shown here as heterozygotes. (ii) In the heteroduplex mobility assay, 5 μl of PCR product was electrophoresed on a 4% agarose gel for > 5 hr at 80 V, allowing visualization of heteroduplexes, which migrate more slowly than homoduplexes, in samples from heterozygotes (lanes 11–14); thus, samples from homozygous individuals (WT) should produce only the 513-bp band (lane 10). (C) Sequenced mutant alleles induced by (i) gRNA-A/Cas9 and (ii) gRNA-B/Cas9. Red and blue lettering indicate the PAM site and insertions, respectively. (i) The mutant allele from individual A6-17 has a 16-bp insertion (blue) that adds a premature stop codon (red box); the allele from individual A12-10 has a 15-bp insertion (blue) and 1-bp deletion that induces a frameshift. (ii) The allele from individual B4-4 has a 7-bp deletion, resulting in a frameshift; the allele from individual B5-16 is complex, showing substitutions (blue) replacing sequence (underlined) 5′ and 3′ of the PAM site (red), including mutation of the splicing donor site (black box). cDNA, complementary DNA; CRISPR, clustered regularly interspaced short palindromic repeats; dsRNA, double-stranded RNA; gRNA, guide RNA; PAM, protospacer-adjacent motif; WT, wild-type.

    Techniques Used: CRISPR, Mutagenesis, Polymerase Chain Reaction, Amplification, Derivative Assay, Agarose Gel Electrophoresis, Sequencing

    32) 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

    33) 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

    34) Product Images from "Engineering Maize rayado fino virus for virus‐induced gene silencing. Engineering Maize rayado fino virus for virus‐induced gene silencing"

    Article Title: Engineering Maize rayado fino virus for virus‐induced gene silencing. Engineering Maize rayado fino virus for virus‐induced gene silencing

    Journal: Plant Direct

    doi: 10.1002/pld3.224

    Utility of the MRFV VIGS system on different maize inbred lines. (a) MRFV symptoms and PDS photobleaching induced by MRFV‐PDS 120 on B73, Mo17, and Va35 maize inbred lines compared to plants inoculated with MRFV‐WT or noninoculated healthy controls (HC). (b) RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of B73, Mo17, and Va35 plants 30 dpi. Noninoculated plants (HC) and water (H 2 O) were included as negative controls; MRFV‐PDS 120 plasmid (PL) served as a PCR‐positive control (not shown). M: 100 bp DNA marker. (c) Northern blot analysis of VIGS induced by MRFV‐PDS 120 on B73, Mo17, and Va35 maize inbred lines. Blot hybridizations were as described for Figure 3 . PDS mRNA and siRNA levels in MRFV‐PDS 120 ‐infected plants are shown (lanes 1–2; 7–8; and 13–14) compared to MRFV infected (lanes 3–4; 9–10; and 15–16) and healthy plants (lanes 5–6; 11–12; and 17–18). The relative levels of PDS mRNA were determined as described in Figure 3
    Figure Legend Snippet: Utility of the MRFV VIGS system on different maize inbred lines. (a) MRFV symptoms and PDS photobleaching induced by MRFV‐PDS 120 on B73, Mo17, and Va35 maize inbred lines compared to plants inoculated with MRFV‐WT or noninoculated healthy controls (HC). (b) RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of B73, Mo17, and Va35 plants 30 dpi. Noninoculated plants (HC) and water (H 2 O) were included as negative controls; MRFV‐PDS 120 plasmid (PL) served as a PCR‐positive control (not shown). M: 100 bp DNA marker. (c) Northern blot analysis of VIGS induced by MRFV‐PDS 120 on B73, Mo17, and Va35 maize inbred lines. Blot hybridizations were as described for Figure 3 . PDS mRNA and siRNA levels in MRFV‐PDS 120 ‐infected plants are shown (lanes 1–2; 7–8; and 13–14) compared to MRFV infected (lanes 3–4; 9–10; and 15–16) and healthy plants (lanes 5–6; 11–12; and 17–18). The relative levels of PDS mRNA were determined as described in Figure 3

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Polymerase Chain Reaction, Positive Control, Marker, Northern Blot, Infection

    (a) Transmissibility of MRFV‐PDS 120 by D. maidis ( Dm ) and RT‐PCR analysis (primers sm151 and 152) of insert stability. Replicated experiments showing virus symptoms and chlorophyll photobleaching phenotype induced by D. maidis‐ transmitted MRFV‐PDS 120 at 30 dpi compared to MRFV‐WT and noninoculated plants (i, iii and v); and corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants (ii, iv and vi). Healthy plants (HC) and water (H 2 O) were included as negative controls, and the MRFV‐PDS 120 plasmid (PL) as a positive control. M: 100 bp DNA marker. (b) Stability of MRFV‐PDS 120 through D. maidis ( Dm ) passaging. (i) Virus symptoms and chlorophyll photo bleaching phenotype induced by MRFV‐PDS 120 at 30 dpi after passage 2 acquisition and transmission by D. maidis , compared to MRFV‐WT and noninoculated plants, and (ii) corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants . The controls and DNA marker were included as described above. (iii) Virus symptoms and chlorophyll photobleaching phenotype induced by MRFV‐PDS 120 at 30 dpi after passage three acquisition and transmission by D. maidis , compared to MRFV‐WT and noninoculated plants, and (iv) corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants . The controls were included as described above
    Figure Legend Snippet: (a) Transmissibility of MRFV‐PDS 120 by D. maidis ( Dm ) and RT‐PCR analysis (primers sm151 and 152) of insert stability. Replicated experiments showing virus symptoms and chlorophyll photobleaching phenotype induced by D. maidis‐ transmitted MRFV‐PDS 120 at 30 dpi compared to MRFV‐WT and noninoculated plants (i, iii and v); and corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants (ii, iv and vi). Healthy plants (HC) and water (H 2 O) were included as negative controls, and the MRFV‐PDS 120 plasmid (PL) as a positive control. M: 100 bp DNA marker. (b) Stability of MRFV‐PDS 120 through D. maidis ( Dm ) passaging. (i) Virus symptoms and chlorophyll photo bleaching phenotype induced by MRFV‐PDS 120 at 30 dpi after passage 2 acquisition and transmission by D. maidis , compared to MRFV‐WT and noninoculated plants, and (ii) corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants . The controls and DNA marker were included as described above. (iii) Virus symptoms and chlorophyll photobleaching phenotype induced by MRFV‐PDS 120 at 30 dpi after passage three acquisition and transmission by D. maidis , compared to MRFV‐WT and noninoculated plants, and (iv) corresponding RT‐PCR assays of MRFV‐PDS 120 and MRFV‐WT in systemic leaves of infected plants . The controls were included as described above

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Positive Control, Marker, Passaging, Transmission Assay

    Testing of the stability of MRFV‐PDS 120 using serial passage inoculations with crude plant sap. (a) MRFV‐PDS 120 symptoms and chlorophyll photobleaching phenotype at 30 dpi for each passage, compared to MRFV‐WT and noninoculated plants (HC). (b) RT‐PCR analysis (primers sm151 and sm152) of the integrity of MRFV‐PDS 120 at each of the four passages. All the MRFV‐PDS 120 ‐infected plants obtained for each passage were RT‐PCR assayed both at 15 dpi (not shown) and at 60 dpi (representative gel shown here). Healthy plants (HC) and water (H 2 O) were included as negative controls, and MRFV‐PDS 120 plasmid (PL) as a positive control. M: 100 bp DNA marker.
    Figure Legend Snippet: Testing of the stability of MRFV‐PDS 120 using serial passage inoculations with crude plant sap. (a) MRFV‐PDS 120 symptoms and chlorophyll photobleaching phenotype at 30 dpi for each passage, compared to MRFV‐WT and noninoculated plants (HC). (b) RT‐PCR analysis (primers sm151 and sm152) of the integrity of MRFV‐PDS 120 at each of the four passages. All the MRFV‐PDS 120 ‐infected plants obtained for each passage were RT‐PCR assayed both at 15 dpi (not shown) and at 60 dpi (representative gel shown here). Healthy plants (HC) and water (H 2 O) were included as negative controls, and MRFV‐PDS 120 plasmid (PL) as a positive control. M: 100 bp DNA marker.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Plasmid Preparation, Positive Control, Marker

    Viability of MRFV HEL/POL junction as insertion site. (a) Location of primers (sm151 and sm152) used for RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT. The primers straddle the HEL/POL junction and amplify 1,164 bp in MRFV‐PDS 120 and 963bp in wild‐type MRFV. (b) Virus symptoms and the chlorophyll photobleaching phenotype induced by MRFV‐PDS 120 at 30 dpi, compared to leaves of plants inoculated with MRFV without insert and noninoculated leaves (HC). (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 days post‐inoculation. The three gels show RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT in inoculated plants in three replicated experiments, with RNA from noninoculated plants (HC) and water (H 2 O) included as negative control templates; and the MRFV‐PDS 120 plasmid (PL) serving as a positive control. Blank lanes in MRFV‐PDS 120 inoculations represent nonsymptomatic plants. M: 100 bp DNA marker. (d) RT‐PCR analysis of insert retention in MRFV‐PDS 120 at 60 days post‐inoculation. RT‐PCR was repeated at 60 dpi only for RT‐PCR positive (symptomatic/successfully inoculated) MRFV‐PDS 120 plants in (c) above, with controls and DNA marker included as described above. This was done for all the three replicated experiments. (e) RT‐PCR assays for virus accumulation in tassels and silks for a subset of MRFV‐PDS 120 symptomatic plants. Controls and DNA marker were included as described above. (f) Transmission electron micrographs of MRFV‐PDS 120 (left panel) and MRFV‐WT particles (right panel) in maize (Silver Queen) extract
    Figure Legend Snippet: Viability of MRFV HEL/POL junction as insertion site. (a) Location of primers (sm151 and sm152) used for RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT. The primers straddle the HEL/POL junction and amplify 1,164 bp in MRFV‐PDS 120 and 963bp in wild‐type MRFV. (b) Virus symptoms and the chlorophyll photobleaching phenotype induced by MRFV‐PDS 120 at 30 dpi, compared to leaves of plants inoculated with MRFV without insert and noninoculated leaves (HC). (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 days post‐inoculation. The three gels show RT‐PCR detection of MRFV‐PDS 120 and MRFV‐WT in inoculated plants in three replicated experiments, with RNA from noninoculated plants (HC) and water (H 2 O) included as negative control templates; and the MRFV‐PDS 120 plasmid (PL) serving as a positive control. Blank lanes in MRFV‐PDS 120 inoculations represent nonsymptomatic plants. M: 100 bp DNA marker. (d) RT‐PCR analysis of insert retention in MRFV‐PDS 120 at 60 days post‐inoculation. RT‐PCR was repeated at 60 dpi only for RT‐PCR positive (symptomatic/successfully inoculated) MRFV‐PDS 120 plants in (c) above, with controls and DNA marker included as described above. This was done for all the three replicated experiments. (e) RT‐PCR assays for virus accumulation in tassels and silks for a subset of MRFV‐PDS 120 symptomatic plants. Controls and DNA marker were included as described above. (f) Transmission electron micrographs of MRFV‐PDS 120 (left panel) and MRFV‐WT particles (right panel) in maize (Silver Queen) extract

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Negative Control, Plasmid Preparation, Positive Control, Marker, Transmission Assay

    Capacity of HEL/POL junction to hold larger inserts. (a) Similar to MRFV‐PDS 120, molecular assays for MRFV‐PDS 231 used primers sm151 and 152 for RT‐PCR detection, amplifying 1,275 bp in MRFV‐PDS 231 and 963bp in MRFV‐WT. (b) Virus symptoms and chlorophyll photobleaching phenotype induced by MRFV‐PDS 231 at 30 dpi, compared to MRFV‐WT and noninoculated plants. (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 dpi. The gel shows RT‐PCR detection of MRFV‐PDS 231 and MRFV‐WT in systemic leaves of inoculated plants. Healthy plants (HC) and water (H 2 O) were included as negative controls and the MRFV‐PDS 231 plasmid (PL) as a positive control. M: 100 bp DNA marker. (d) Northern blot analysis of photobleaching phenotype induced by MRFV‐PDS 231 was as described in Figure 3 . Levels of PDS mRNA and siRNAs from MRFV‐PDS 231 ‐infected leaves is shown (lanes 1–4) compared to MRFV‐infected (lanes 5–6) and healthy plants (lanes 7–8). The relative levels of PDS mRNA were determined as described in Figure 3
    Figure Legend Snippet: Capacity of HEL/POL junction to hold larger inserts. (a) Similar to MRFV‐PDS 120, molecular assays for MRFV‐PDS 231 used primers sm151 and 152 for RT‐PCR detection, amplifying 1,275 bp in MRFV‐PDS 231 and 963bp in MRFV‐WT. (b) Virus symptoms and chlorophyll photobleaching phenotype induced by MRFV‐PDS 231 at 30 dpi, compared to MRFV‐WT and noninoculated plants. (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 dpi. The gel shows RT‐PCR detection of MRFV‐PDS 231 and MRFV‐WT in systemic leaves of inoculated plants. Healthy plants (HC) and water (H 2 O) were included as negative controls and the MRFV‐PDS 231 plasmid (PL) as a positive control. M: 100 bp DNA marker. (d) Northern blot analysis of photobleaching phenotype induced by MRFV‐PDS 231 was as described in Figure 3 . Levels of PDS mRNA and siRNAs from MRFV‐PDS 231 ‐infected leaves is shown (lanes 1–4) compared to MRFV‐infected (lanes 5–6) and healthy plants (lanes 7–8). The relative levels of PDS mRNA were determined as described in Figure 3

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Marker, Northern Blot, Infection

    Efficacy of MRFV VIGS system in silencing ZmlspH gene. (a) Molecular assays for MRFV‐LSP 210 used primers sm151 and 152 for RT‐PCR detection, amplifying 1,254 bp in MRFV‐LSP 210 and 963bp in MRFV‐WT. (b) MRFV symptoms and yellow (albino) VIGS phenotype induced by MRFV‐LSP 210 at 30 dpi, compared to plants inoculated with MRFV‐WT or noninoculated (HC). (c) RT‐PCR analysis of MRFV‐LSP 210 and MRFV‐WT accumulation in systemic leaves 30 dpi. Noninoculated plants (HC) were included as negative controls; MRFV‐LSP 210 plasmid (PL) served as a PCR‐positive control. M: 100 bp DNA marker. (d) Northern blot analysis of VIGS induced by MRFV‐LSP 210. RNA blots were hybridized with LSP‐specific RNA probes to detect LSP mRNAs or antisense siRNAs. rRNA: EtBr‐stained ribosomal RNA used as loading control. For small RNA gel, the major low molecular weight RNA species was EtBr‐stained for loading control prior to bloating of gel onto membrane. Levels of LSP mRNA and siRNAs from MRFV‐LSP 210 ‐infected plants (lanes 1–4) compared to MRFV‐infected (lanes 5–6) and healthy plants (lanes 7–8) are shown, with positions of 21‐ to 24‐nucleotide RNA size markers indicated by arrow heads. The relative levels of LSP mRNA were determined as described for PDS in Figure 3
    Figure Legend Snippet: Efficacy of MRFV VIGS system in silencing ZmlspH gene. (a) Molecular assays for MRFV‐LSP 210 used primers sm151 and 152 for RT‐PCR detection, amplifying 1,254 bp in MRFV‐LSP 210 and 963bp in MRFV‐WT. (b) MRFV symptoms and yellow (albino) VIGS phenotype induced by MRFV‐LSP 210 at 30 dpi, compared to plants inoculated with MRFV‐WT or noninoculated (HC). (c) RT‐PCR analysis of MRFV‐LSP 210 and MRFV‐WT accumulation in systemic leaves 30 dpi. Noninoculated plants (HC) were included as negative controls; MRFV‐LSP 210 plasmid (PL) served as a PCR‐positive control. M: 100 bp DNA marker. (d) Northern blot analysis of VIGS induced by MRFV‐LSP 210. RNA blots were hybridized with LSP‐specific RNA probes to detect LSP mRNAs or antisense siRNAs. rRNA: EtBr‐stained ribosomal RNA used as loading control. For small RNA gel, the major low molecular weight RNA species was EtBr‐stained for loading control prior to bloating of gel onto membrane. Levels of LSP mRNA and siRNAs from MRFV‐LSP 210 ‐infected plants (lanes 1–4) compared to MRFV‐infected (lanes 5–6) and healthy plants (lanes 7–8) are shown, with positions of 21‐ to 24‐nucleotide RNA size markers indicated by arrow heads. The relative levels of LSP mRNA were determined as described for PDS in Figure 3

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Polymerase Chain Reaction, Positive Control, Marker, Northern Blot, Staining, Molecular Weight, Infection

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

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

    Journal: bioRxiv

    doi: 10.1101/731216

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

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

    36) Product Images from "Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe"

    Article Title: Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

    Journal: bioRxiv

    doi: 10.1101/2021.04.12.437332

    Upf1 association to specific genes is RNase sensitive A ) IGB screenshot of ChIP-chip enrichments of Upf1-HA in asynchronous culture (top row) and in S-phase culture (bottom row) over act1 gene and its flanking region. Genes and genomic features are shown below. B ) Top panel-schematic diagram of the act1 gene with CDS sequence (in grey); the PCR amplicons used for the ChIP assay are indicated by the dotted lines above (numbers correspond to the primer positions relative to start codon). Bottom panel-polyacrylamide gels showing radiolabelled PCR products produced by the act1 specific primer pairs (top bands) and by the pair specific for the intergenic region (bottom bands); using input DNA before ChIP (left panel) and using ChIP-enriched DNA from asynchronous (middle panel) and S-phase culture (right panel). The relative enrichment of act1 DNA relative to intergenic sequence is expressed as a ratio of the intensity of the same fragments produced with the input DNA. C ) Independent qPCR quantification of Upf1-Flag ChIP signal on 4 specific regions of pma1 gene and 1 intergenic control in the absence and presence of RNase. N.T-non-tagged.
    Figure Legend Snippet: Upf1 association to specific genes is RNase sensitive A ) IGB screenshot of ChIP-chip enrichments of Upf1-HA in asynchronous culture (top row) and in S-phase culture (bottom row) over act1 gene and its flanking region. Genes and genomic features are shown below. B ) Top panel-schematic diagram of the act1 gene with CDS sequence (in grey); the PCR amplicons used for the ChIP assay are indicated by the dotted lines above (numbers correspond to the primer positions relative to start codon). Bottom panel-polyacrylamide gels showing radiolabelled PCR products produced by the act1 specific primer pairs (top bands) and by the pair specific for the intergenic region (bottom bands); using input DNA before ChIP (left panel) and using ChIP-enriched DNA from asynchronous (middle panel) and S-phase culture (right panel). The relative enrichment of act1 DNA relative to intergenic sequence is expressed as a ratio of the intensity of the same fragments produced with the input DNA. C ) Independent qPCR quantification of Upf1-Flag ChIP signal on 4 specific regions of pma1 gene and 1 intergenic control in the absence and presence of RNase. N.T-non-tagged.

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Polymerase Chain Reaction, Produced, Real-time Polymerase Chain Reaction

    Genome-wide association of Upf1 with protein coding genes A) IGB visualisation of Upf1-HA ChIP-chip enrichment in asynchronous culture (top track, shown in red) and in S-phase culture (bottom track, shown in sky blue) at a representative chromosomal region (270 kb) of S. pombe indicating enrichment of several specific genes, those discussed in the main text are labelled. Genes and genomic features are shown in black below. B ) Zoomed-in view of Upf1 enrichment over the entire pma1 gene (highlighted in dark blue) - 5’UTR (in grey), CDS (in purple) and 3’UTR (in grey) of pma1 are shown in the bottom row schematic. C ) Top panel-diagram of the pma1 gene (cDNA region in grey) and the positions of amplicons used for the PCR-ChIP assay are indicated by the dotted lines above (numbers correspond to the position of the primers relative to start codon). Bottom panel-polyacrylamide gels showing radiolabelled PCR products produced by the pma1 -specific primer pairs (top bands) and by the intergenic region specific primer (bottom bands, labelled Int.); using input DNA before ChIP (left panel) and using ChIP-enriched DNA from asynchronous culture without (middle panel) and with RNase pre-treatment of the chromatin (right panel). The relative enrichment of pma1 DNA relative to intergenic sequence is expressed as a ratio of the intensity of the same fragments produced with the input DNA. D ) PCR analysis as in C using input DNA before ChIP (left panel) and using ChIP-enriched DNA from S-phase culture without the RNase treatment (right panel). E ) Pie-charts showing the proportion of different classes of gene associated to Upf1 in asynchronous and S-phase culture of S. pombe. F ) ChIP-qPCR of Upf1-Flag enrichment over pma1 in three independent asynchronous cultures.
    Figure Legend Snippet: Genome-wide association of Upf1 with protein coding genes A) IGB visualisation of Upf1-HA ChIP-chip enrichment in asynchronous culture (top track, shown in red) and in S-phase culture (bottom track, shown in sky blue) at a representative chromosomal region (270 kb) of S. pombe indicating enrichment of several specific genes, those discussed in the main text are labelled. Genes and genomic features are shown in black below. B ) Zoomed-in view of Upf1 enrichment over the entire pma1 gene (highlighted in dark blue) - 5’UTR (in grey), CDS (in purple) and 3’UTR (in grey) of pma1 are shown in the bottom row schematic. C ) Top panel-diagram of the pma1 gene (cDNA region in grey) and the positions of amplicons used for the PCR-ChIP assay are indicated by the dotted lines above (numbers correspond to the position of the primers relative to start codon). Bottom panel-polyacrylamide gels showing radiolabelled PCR products produced by the pma1 -specific primer pairs (top bands) and by the intergenic region specific primer (bottom bands, labelled Int.); using input DNA before ChIP (left panel) and using ChIP-enriched DNA from asynchronous culture without (middle panel) and with RNase pre-treatment of the chromatin (right panel). The relative enrichment of pma1 DNA relative to intergenic sequence is expressed as a ratio of the intensity of the same fragments produced with the input DNA. D ) PCR analysis as in C using input DNA before ChIP (left panel) and using ChIP-enriched DNA from S-phase culture without the RNase treatment (right panel). E ) Pie-charts showing the proportion of different classes of gene associated to Upf1 in asynchronous and S-phase culture of S. pombe. F ) ChIP-qPCR of Upf1-Flag enrichment over pma1 in three independent asynchronous cultures.

    Techniques Used: GWAS, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Produced, Sequencing, Real-time Polymerase Chain Reaction

    37) Product Images from "Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA"

    Article Title: Identification of the First Gene Transfer Agent (GTA) Small Terminase in Rhodobacter capsulatus and Its Role in GTA Production and Packaging of DNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.01328-19

    RcGTA gp1 in vitro DNA binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp PCR amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.
    Figure Legend Snippet: RcGTA gp1 in vitro DNA binding. (A) Representative agarose gel (0.8%, wt/vol) showing the stated concentrations of gp1 protein binding to DNA in an electrophoretic mobility shift assay (EMSA). The locations of unbound and shifted DNA are annotated. Substrate DNA in the assay shown is a 1.4-kbp PCR amplification of an arbitrarily chosen region flanking the rcc01398 gene from R. capsulatus (amplified using rcc01398 forward and reverse primers [ Table 3 ]). Bioline HyperLadder 1kb DNA marker is shown for size comparison (lane M). (B) Quantification of EMSAs by band intensity analysis. Data shown are the average results of two EMSAs carried out independently in time and with different DNA substrates (flanking the rcc01397 and rcc01398 genes). Individual data points are plotted as well as the mean line.

    Techniques Used: In Vitro, Binding Assay, Agarose Gel Electrophoresis, Protein Binding, Electrophoretic Mobility Shift Assay, Polymerase Chain Reaction, Amplification, Marker

    38) 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

    39) 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

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    Article Snippet: In the second PCR, forward primer 5′-TGTCAGTCCACTTCACCAAG-3′ and reverse primer 5′-CTTTCCTTGATCTTGAGGTC-3′ were used. .. 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′. .. The results were compared with the sequence of NCBI Reference Sequence to identify mutations.

    Agarose Gel Electrophoresis:

    Article Title: Bisulfite-free epigenomics and genomics of single cells through methylation-sensitive restriction
    Article Snippet: .. After PCR, product quality was assessed by agarose gel electrophoresis and the samples were cleaned up using the Monarch PCR and DNA Cleanup Kit (NEB), according to the manufacturer’s recommendations. .. Product quality was additionally assessed on a Bioanalyzer (Agilent) High-Sensitivity DNA chip for selected reactions.

    Purification:

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

    Article Title: Using weapons instead of perfume – chemical association strategies of the myrmecophilous bug Scolopostethus pacificus (Rhyparochromidae)
    Article Snippet: DNA extracts of the specimens were used in PCR reactions (initial denaturation 2 min at 95°C; 35 cycles of 0.5 min at 95°C; 1 min at 58°C, 1 min at 72°C; final extension for 5 min at 72°C and final indefinite hold at 4°C) using ITS2 primers [ supplementary Table S1 ; ] with GoTaq Green (12.5 µl master mix, 8.5 µl nuclease-free water, 2 ul 10 µM mixture of F/R primer, 2 µl template). .. A volume (5 µL) of the reaction was then checked for successful amplification on a 1% agarose with the 1 kb DNA Ladder from New England Biolabs and afterwards the remaining PCR product was purified using the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ipswich, MA). .. Purified products for S. pacificus and L. occidentale were sent for Sanger sequencing with Laragen Inc. (Culver City, CA).

    Article Title: Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex
    Article Snippet: A double-stranded DNA template for in vitro transcription of a 20-mer random RNA library was synthesized with a primer extension reaction in which 100 µl of a reaction mixture containing 1× Platinum SuperFi PCR Master Mix (Thermo Fisher Scientific, #12358-010), 1× SuperFi GC Enhancer (Thermo Fisher Scientific), 100 nM DNA template oligomer (5’-GAAATTAATACGACTCACTATAGGACGTGACACGACGTGCGCN20 GCGTACG TCGGACCTCAGGTCGACCATGGACGC-3’, where N20 is the DNA sequence encoding the 20-mer RNA sequence), 100 nM primer (5’-GCGTCCATGGTCGACCTGAGGTCC-3’), and nuclease-free water was incubated at 98°C for 130 s, at 50°C for 2 min, and then at 72°C for 10 min. .. The synthesized DNA template was purified with the use of a Monarch PCR & DNA Cleanup Kit (New England Biolabs, #T1030L). .. The random RNA library was then transcribed with the use of a MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific, #AM1354) in a reaction mixture containing 1× Reaction Buffer, 1× T7 Enzyme Mix, 7.5 mM ATP, 7.5 mM UTP, 7.5 mM GTP, 7.5 mM CTP, and 9.15 pmol of the DNA template.

    Article Title: Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing
    Article Snippet: The entire volume of a PCR reaction was loaded on 1% agarose for gel-electrophoresis. .. The gel bands were gel purified (Monarch, PCR & DNA Gel Cleanup Kit, NEB) and sent for sequencing using the above-mentioned FW and RV primers in independent reactions, respectively. ..

    Article Title: Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics
    Article Snippet: The Pfx PCR contained the following: 2.5 μL 10x Pfx buffer, 0.25 μL forward primer (100 uM), 0.25 μL reverse primer (100 uM), 0.75 μL DMSO (NEB), 0.25 μL Pfx Polymerase (Thermo), 20.5 water, and 0.5 μL template DNA (~20-50 ng/ul), in a total 25 μL reaction with the following program: 2 min at 95°C; 10 sec at 95°C; 30 sec at 58°C; 30s or 1 min at 68°C; cycled 9x; final extension of 5 min at 68°C. .. PCR reactions were purified with Monarch PCR & DNA Cleanup Kit (NEB, T1030L). .. For the hHBB-Fluc control mRNA, the DNA template was amplified from the pGL3-HBB plasmid using the primers KL588/KL589 which yielded a PCR product of 1,750 kb in length.

    Article Title: The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3
    Article Snippet: In the second PCR, forward primer 5′-TGTCAGTCCACTTCACCAAG-3′ and reverse primer 5′-CTTTCCTTGATCTTGAGGTC-3′ were used. .. 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′. .. The results were compared with the sequence of NCBI Reference Sequence to identify mutations.

    Amplification:

    Article Title: Using weapons instead of perfume – chemical association strategies of the myrmecophilous bug Scolopostethus pacificus (Rhyparochromidae)
    Article Snippet: DNA extracts of the specimens were used in PCR reactions (initial denaturation 2 min at 95°C; 35 cycles of 0.5 min at 95°C; 1 min at 58°C, 1 min at 72°C; final extension for 5 min at 72°C and final indefinite hold at 4°C) using ITS2 primers [ supplementary Table S1 ; ] with GoTaq Green (12.5 µl master mix, 8.5 µl nuclease-free water, 2 ul 10 µM mixture of F/R primer, 2 µl template). .. A volume (5 µL) of the reaction was then checked for successful amplification on a 1% agarose with the 1 kb DNA Ladder from New England Biolabs and afterwards the remaining PCR product was purified using the Monarch® PCR & DNA Cleanup Kit (New England Biolabs; Ipswich, MA). .. Purified products for S. pacificus and L. occidentale were sent for Sanger sequencing with Laragen Inc. (Culver City, CA).

    Synthesized:

    Article Title: Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex
    Article Snippet: A double-stranded DNA template for in vitro transcription of a 20-mer random RNA library was synthesized with a primer extension reaction in which 100 µl of a reaction mixture containing 1× Platinum SuperFi PCR Master Mix (Thermo Fisher Scientific, #12358-010), 1× SuperFi GC Enhancer (Thermo Fisher Scientific), 100 nM DNA template oligomer (5’-GAAATTAATACGACTCACTATAGGACGTGACACGACGTGCGCN20 GCGTACG TCGGACCTCAGGTCGACCATGGACGC-3’, where N20 is the DNA sequence encoding the 20-mer RNA sequence), 100 nM primer (5’-GCGTCCATGGTCGACCTGAGGTCC-3’), and nuclease-free water was incubated at 98°C for 130 s, at 50°C for 2 min, and then at 72°C for 10 min. .. The synthesized DNA template was purified with the use of a Monarch PCR & DNA Cleanup Kit (New England Biolabs, #T1030L). .. The random RNA library was then transcribed with the use of a MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific, #AM1354) in a reaction mixture containing 1× Reaction Buffer, 1× T7 Enzyme Mix, 7.5 mM ATP, 7.5 mM UTP, 7.5 mM GTP, 7.5 mM CTP, and 9.15 pmol of the DNA template.

    Sequencing:

    Article Title: Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing
    Article Snippet: The entire volume of a PCR reaction was loaded on 1% agarose for gel-electrophoresis. .. The gel bands were gel purified (Monarch, PCR & DNA Gel Cleanup Kit, NEB) and sent for sequencing using the above-mentioned FW and RV primers in independent reactions, respectively. ..

    Article Title: The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3
    Article Snippet: In the second PCR, forward primer 5′-TGTCAGTCCACTTCACCAAG-3′ and reverse primer 5′-CTTTCCTTGATCTTGAGGTC-3′ were used. .. 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′. .. The results were compared with the sequence of NCBI Reference Sequence to identify mutations.

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    New England Biolabs monarch pcr and dna cleanup kit
    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 <t>DNA</t> templates for full-length mRNAs were synthesized on the Codex platform and amplified in a pooled <t>PCR</t> 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.
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    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

    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

    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

    Journal: RNA Biology

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

    doi: 10.1080/15476286.2020.1813411

    Figure Lengend 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

    Article Snippet: The gel bands were gel purified (Monarch, PCR & DNA Gel Cleanup Kit, NEB) and sent for sequencing using the above-mentioned FW and RV primers in independent reactions, respectively.

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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0204265

    Figure Lengend 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.

    Article Snippet: The full volume of DNA was further cleaned up to remove any potential inhibitors using the Monarch PCR & DNA Cleanup Kit (5 μg) (New England BioLabs (NEB)).

    Techniques: Polymerase Chain Reaction, Sequencing