hdgfrp2 cryptic exon  (New England Biolabs)


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    New England Biolabs hdgfrp2 cryptic exon
    Monarch Plasmid Miniprep Kit
    Monarch Plasmid Miniprep Kit 250 preps
    https://www.bioz.com/result/hdgfrp2 cryptic exon/product/New England Biolabs
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    hdgfrp2 cryptic exon - by Bioz Stars, 2021-02
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    Images

    1) Product Images from "Cryptic Exon Incorporation Occurs in Alzheimer’s Brain lacking TDP-43 Inclusion but Exhibiting Nuclear Clearance of TDP-43"

    Article Title: Cryptic Exon Incorporation Occurs in Alzheimer’s Brain lacking TDP-43 Inclusion but Exhibiting Nuclear Clearance of TDP-43

    Journal: Acta neuropathologica

    doi: 10.1007/s00401-017-1701-2

    Further validation of cryptic exon incorporation in AD brain tissue. ( A ) Diagram of RT-PCR detection strategy to amplify across the cryptic exon splice junction of HDGFRP2 . ( B ) Sequencing confirmed 263 bp HDGFRP2 RT-PCR products were detected in the same cases that showed cryptic exon incorporation of GPSM2 and ATG4B . Similarly, GPSM2 and ATG4B negative cases did not display HDGFRP2 RT-PCR fragment (the band as outlined in case #4 was confirmed to be negative by sequencing). +: inclusions seen in both amygdala and dentate gyrus of hippocampus; ─*: inclusions only seen in amygdala; ─: no solid inclusions. C, control. HIP, hippocampus.
    Figure Legend Snippet: Further validation of cryptic exon incorporation in AD brain tissue. ( A ) Diagram of RT-PCR detection strategy to amplify across the cryptic exon splice junction of HDGFRP2 . ( B ) Sequencing confirmed 263 bp HDGFRP2 RT-PCR products were detected in the same cases that showed cryptic exon incorporation of GPSM2 and ATG4B . Similarly, GPSM2 and ATG4B negative cases did not display HDGFRP2 RT-PCR fragment (the band as outlined in case #4 was confirmed to be negative by sequencing). +: inclusions seen in both amygdala and dentate gyrus of hippocampus; ─*: inclusions only seen in amygdala; ─: no solid inclusions. C, control. HIP, hippocampus.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing

    2) Product Images from "The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein"

    Article Title: The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879437

    The expression of type I IFN and pro-inflammatory genes was significantly reduced in B cells infected with strain UK661 compared to strain F52/70 in vitro . DT40 Cells were infected at an MOI of 0.1 with either the UK661 or F52/70 IBDV strains, or mock-infected with media alone and RNA was extracted from the cells at the indicated time points post-infection. RNA was reverse transcribed and amplified by qPCR using specific primer sets. The CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (A). The log 2 fold change in host-cell gene expression was also determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (B-G). Data subsequently passed a Shapiro-Wilk normality test before being analysed by a one-way ANOVA and a Tukey’s multiple comparison test (*P
    Figure Legend Snippet: The expression of type I IFN and pro-inflammatory genes was significantly reduced in B cells infected with strain UK661 compared to strain F52/70 in vitro . DT40 Cells were infected at an MOI of 0.1 with either the UK661 or F52/70 IBDV strains, or mock-infected with media alone and RNA was extracted from the cells at the indicated time points post-infection. RNA was reverse transcribed and amplified by qPCR using specific primer sets. The CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (A). The log 2 fold change in host-cell gene expression was also determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted (B-G). Data subsequently passed a Shapiro-Wilk normality test before being analysed by a one-way ANOVA and a Tukey’s multiple comparison test (*P

    Techniques Used: Expressing, Infection, In Vitro, Amplification, Real-time Polymerase Chain Reaction

    The UK661 strain was more virulent than the F52/70 strain, but both strains replicated to the same peak titre in vivo . Birds were checked twice daily by two independent observers for clinical signs and a Kaplan Meier survival curve plotted of mock- (black), F52/70- (pink) and UK661- (grey) inoculated birds that reached their humane end points (clinical score of 11) (A). Clinical signs were quantified by a scoring system and divided into mild (1-7) and moderate (8-11). Each bird was assigned a clinical score at the indicated time points post-infection (B). Six birds per group were humanely culled at 24 and 48 hours post-infection (hpi), one F52/70 and three UK661-infected birds reached their humane end-points at 54 hpi and the remaining birds were culled at 72 hpi. The bursa of Fabricius was harvested at necropsy and the log 10 fold change in viral RNA copies/g tissue determined by RT-qPCR (C). The infectious titre was determined by titration onto DT40 cells in the method described by Reed Muench. Virus titres were expressed as log 10 TCID 50 /g of tissue (D). The horizontal lines are the mean values. Data passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The UK661 strain was more virulent than the F52/70 strain, but both strains replicated to the same peak titre in vivo . Birds were checked twice daily by two independent observers for clinical signs and a Kaplan Meier survival curve plotted of mock- (black), F52/70- (pink) and UK661- (grey) inoculated birds that reached their humane end points (clinical score of 11) (A). Clinical signs were quantified by a scoring system and divided into mild (1-7) and moderate (8-11). Each bird was assigned a clinical score at the indicated time points post-infection (B). Six birds per group were humanely culled at 24 and 48 hours post-infection (hpi), one F52/70 and three UK661-infected birds reached their humane end-points at 54 hpi and the remaining birds were culled at 72 hpi. The bursa of Fabricius was harvested at necropsy and the log 10 fold change in viral RNA copies/g tissue determined by RT-qPCR (C). The infectious titre was determined by titration onto DT40 cells in the method described by Reed Muench. Virus titres were expressed as log 10 TCID 50 /g of tissue (D). The horizontal lines are the mean values. Data passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vivo, Infection, Quantitative RT-PCR, Titration, Two Tailed Test

    The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . Birds were inoculated with 1.8×10 3 TCID 50 of the PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses, and the bursa of Fabricius was harvested at necropsy from 6 birds per group at 2, 4 and 14 days post-inoculation. RNA was extracted prior to reverse transcription to cDNA and qPCR amplification with virus-specific primers. CT values were normalised to a housekeeping gene and expressed as log 10 fold change viral RNA relative to mock-infected samples as per the ΔΔCT method. The data passed a Shapiro-Wilk normality test before being analysed using a two-way ANOVA (not significant) (A). At 2 and 4 days post-inoculation, cDNA was amplified by qPCR for a panel genes: IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F). The CT values were normalised to the housekeeping gene RPLPO and expressed relative to mock-infected samples using the ΔΔCT method. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . Birds were inoculated with 1.8×10 3 TCID 50 of the PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses, and the bursa of Fabricius was harvested at necropsy from 6 birds per group at 2, 4 and 14 days post-inoculation. RNA was extracted prior to reverse transcription to cDNA and qPCR amplification with virus-specific primers. CT values were normalised to a housekeeping gene and expressed as log 10 fold change viral RNA relative to mock-infected samples as per the ΔΔCT method. The data passed a Shapiro-Wilk normality test before being analysed using a two-way ANOVA (not significant) (A). At 2 and 4 days post-inoculation, cDNA was amplified by qPCR for a panel genes: IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F). The CT values were normalised to the housekeeping gene RPLPO and expressed relative to mock-infected samples using the ΔΔCT method. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Amplification, Infection, Two Tailed Test

    The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . DF-1 cells were infected with PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses at an MOI of 1, before RNA was extracted at the indicated time points post-infection and reverse transcribed. Virus specific primers were used to amplify the cDNA by quantitative PCR, the CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression was determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. A Kruskal-Wallis test was performed with a Dunn’s multiple comparison test where no significant difference was found at any time point between the three viruses (A). A panel of genes, IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F), were amplified by quantitative PCR using specific primer sets for target genes, before the CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The ability of the UK661 VP4 protein to antagonise type I IFN responses was reduced in the context of the whole virus in vitro . DF-1 cells were infected with PBG98, PBG98-VP4 UK661 and PBG98-VP4 F52/70 viruses at an MOI of 1, before RNA was extracted at the indicated time points post-infection and reverse transcribed. Virus specific primers were used to amplify the cDNA by quantitative PCR, the CT values were normalised to the housekeeping gene RPLPO and the log 10 fold change in virus gene expression was determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. A Kruskal-Wallis test was performed with a Dunn’s multiple comparison test where no significant difference was found at any time point between the three viruses (A). A panel of genes, IFNα (B), IFNβ (C), Mx1 (D), IL-1β (E), and IL-8 (F), were amplified by quantitative PCR using specific primer sets for target genes, before the CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected controls in a ΔΔCT analysis and plotted. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: In Vitro, Infection, Real-time Polymerase Chain Reaction, Expressing, Amplification, Two Tailed Test

    The expression of type I IFN and pro-inflammatory genes was significantly reduced in BF tissue harvested from birds infected with strain UK661 compared to strain F52/70 in vivo . The bursa of Fabricius was harvested from mock and infected birds at necropsy and RNA extracted. RNA was reverse transcribed and amplified by quantitative PCR using specific primer sets for target genes. The CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted for individual birds. The horizontal lines are the mean values. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P
    Figure Legend Snippet: The expression of type I IFN and pro-inflammatory genes was significantly reduced in BF tissue harvested from birds infected with strain UK661 compared to strain F52/70 in vivo . The bursa of Fabricius was harvested from mock and infected birds at necropsy and RNA extracted. RNA was reverse transcribed and amplified by quantitative PCR using specific primer sets for target genes. The CT values were normalised to the housekeeping gene RPLPO and the log 2 fold change in gene expression determined for the infected samples relative to the mock-infected samples in a ΔΔCT analysis and plotted for individual birds. The horizontal lines are the mean values. Data are representative of at least three replicate experiments and passed a Shapiro-Wilk normality test before analysis using a two-tailed unpaired Student’s t-test (*P

    Techniques Used: Expressing, Infection, In Vivo, Amplification, Real-time Polymerase Chain Reaction, Two Tailed Test

    3) Product Images from "CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti"

    Article Title: CAPA neuropeptides and their receptor form an anti-diuretic hormone signaling system in the human disease vector, Aedes aegypti

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-58731-y

    RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. ( A ) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. ( B ) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In ( A ), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p
    Figure Legend Snippet: RNA interference (RNAi) of CAPAr abolishes anti-diuretic activity of CAPA neuropeptide on adult female A. aegypti MTs. ( A ) Verification of significant knockdown ( > 75%) of CAPAr transcript in MTs of four-day old adult female A. aegypti by RNAi achieved through injection of dsCAPAr on day one post-eclosion. ( B ) Functional consequences of CAPAr knockdown demonstrating loss of anti-diuretic hormone activity by Aedae CAPA-1 against Drome DH 31 -stimulated fluid secretion by MTs. In ( A ), knockdown of CAPAr transcript was analyzed by one-tailed t-test (* denotes significant knockdown, p

    Techniques Used: Activity Assay, Injection, Functional Assay, One-tailed Test

    4) Product Images from "Evolution of a General RNA-Cleaving FANA Enzyme"

    Article Title: Evolution of a General RNA-Cleaving FANA Enzyme

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07611-1

    FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)
    Figure Legend Snippet: FANA transcription and reverse transcription in vitro. a Constitutional structures for 2’-deoxyribonucleic acid (DNA) and 2’-fluoroarabino nucleic acid (FANA). b FANA transcription activity for wild-type archaeal DNA polymerases (exo−) from 9°N, DV, Kod, and Tgo (left panel). Samples were analyzed after 15 and 30 min at 55 °C. FANA reverse transcriptase activity of Bst DNA polymerase LF, 2.0, 3.0, and LF* (right panel). LF* denotes wild-type Bst DNA polymerase, large fragment, expressed and purified from E. coli . Samples were analyzed after 30 min at 50 °C. All samples were resolved on denaturing PAGE and visualized using a LI-COR Odyssey CLx. c Fidelity profile observed for FANA replication using Tgo and Bst LF* polymerases. The mutation profile reveals a mutation rate of 8 × 10 -4 and an overall fidelity of ~99.9%. d Catalytic rates observed for FANA synthesis with Tgo (left panel) and reverse transcription with Bst LF* (right panel)

    Techniques Used: In Vitro, Activity Assay, Purification, Polyacrylamide Gel Electrophoresis, Mutagenesis

    5) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Predicted ORFs encoded in the fosmid G7 nucleotide sequence. A , fosmid G7 was sequenced on the PacBio RSII platform. ORFs were predicted with MetaGeneMark and classified into three categories based on their homology to proteins from the nonredundant protein database (NCBI). Black , proteins with a known annotated function; gray , “hypothetical proteins” with no annotated function; orange , proteins involved in saccharide utilization. B , database annotations for all 40 ORFs.
    Figure Legend Snippet: Predicted ORFs encoded in the fosmid G7 nucleotide sequence. A , fosmid G7 was sequenced on the PacBio RSII platform. ORFs were predicted with MetaGeneMark and classified into three categories based on their homology to proteins from the nonredundant protein database (NCBI). Black , proteins with a known annotated function; gray , “hypothetical proteins” with no annotated function; orange , proteins involved in saccharide utilization. B , database annotations for all 40 ORFs.

    Techniques Used: Sequencing

    6) Product Images from "Filter paper-based spin column method for cost-efficient DNA or RNA purification"

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0203011

    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.
    Figure Legend Snippet: The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Techniques Used: Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Gel Extraction, Plasmid Preparation

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

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

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

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

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

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

    Techniques Used: Sequencing, Polymerase Chain Reaction

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

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

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

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

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

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

    Techniques Used: Sequencing, Polymerase Chain Reaction

    9) Product Images from "lncRNA CASC2 downregulation participates in rheumatoid arthritis, and CASC2 overexpression promotes the apoptosis of fibroblast-like synoviocytes by downregulating IL-17"

    Article Title: lncRNA CASC2 downregulation participates in rheumatoid arthritis, and CASC2 overexpression promotes the apoptosis of fibroblast-like synoviocytes by downregulating IL-17

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2020.11018

    lncRNA CASC2 is downregulated, while IL-17 is upregulated in the plasma of RA patients. (A) Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. qPCR results showed that, compared with the healthy controls, plasma levels of lncRNA CASC2 were significantly decreased in patients with RA. (B) In contrast, ELISA results showed that plasma IL-17 was upregulated in the plasma of RA patients when compared to that noted in the plasma of healthy controls (*P
    Figure Legend Snippet: lncRNA CASC2 is downregulated, while IL-17 is upregulated in the plasma of RA patients. (A) Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. qPCR results showed that, compared with the healthy controls, plasma levels of lncRNA CASC2 were significantly decreased in patients with RA. (B) In contrast, ELISA results showed that plasma IL-17 was upregulated in the plasma of RA patients when compared to that noted in the plasma of healthy controls (*P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    lncRNA CASC2 is a potential upstream inhibitor of IL-17 in HFLSs. (A) Overexpression of lncRNA CASC2 inhibited IL-17 expression in HFLSs, while (B) treatment with IL-17 did not significantly affect the expression of lncRNA CASC2 (*P
    Figure Legend Snippet: lncRNA CASC2 is a potential upstream inhibitor of IL-17 in HFLSs. (A) Overexpression of lncRNA CASC2 inhibited IL-17 expression in HFLSs, while (B) treatment with IL-17 did not significantly affect the expression of lncRNA CASC2 (*P

    Techniques Used: Over Expression, Expressing

    Altered plasma levels of lncRNA CASC2 and IL-17 differentiate RA patients from healthy controls. ROC curve analysis showed that altered plasma levels of (A) lncRNA CASC2 and (B) IL-17 were able to differentiate RA patients from healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.
    Figure Legend Snippet: Altered plasma levels of lncRNA CASC2 and IL-17 differentiate RA patients from healthy controls. ROC curve analysis showed that altered plasma levels of (A) lncRNA CASC2 and (B) IL-17 were able to differentiate RA patients from healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.

    Techniques Used:

    lncRNA CASC2 overexpression promotes while IL-17 inhibits the apoptosis of HFLSs. (A and B) Overexpression of lncRNA CASC2 was reached after transfection in HFLSs isolated from 2 RA patients. (C and D) Overexpression of lncRNA CASC2 led to a significant promotion of the percentage of apoptosis in HFLSs isolated from 2 RA patients. In contrast, treatment with IL-17 at a dose of 10 ng/ml significantly inhibited the apoptosis of HFLSs. In addition, IL-17 treatment attenuated the promoting effect of lncRNA CASC2 overexpression on cell apoptosis (*P
    Figure Legend Snippet: lncRNA CASC2 overexpression promotes while IL-17 inhibits the apoptosis of HFLSs. (A and B) Overexpression of lncRNA CASC2 was reached after transfection in HFLSs isolated from 2 RA patients. (C and D) Overexpression of lncRNA CASC2 led to a significant promotion of the percentage of apoptosis in HFLSs isolated from 2 RA patients. In contrast, treatment with IL-17 at a dose of 10 ng/ml significantly inhibited the apoptosis of HFLSs. In addition, IL-17 treatment attenuated the promoting effect of lncRNA CASC2 overexpression on cell apoptosis (*P

    Techniques Used: Over Expression, Transfection, Isolation

    Plasma levels of lncRNA CASC2 and IL-17 are significantly and inversely correlated in both RA patients and healthy controls. Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. Pearson's correlation coefficient showed that plasma levels of lncRNA CASC2 and IL-17 were significantly and inversely correlated in both (A) RA patients and (B) healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.
    Figure Legend Snippet: Plasma levels of lncRNA CASC2 and IL-17 are significantly and inversely correlated in both RA patients and healthy controls. Relative expression of lncRNA CASC2 was normalized to the patients with the lowest expression level. Pearson's correlation coefficient showed that plasma levels of lncRNA CASC2 and IL-17 were significantly and inversely correlated in both (A) RA patients and (B) healthy controls. CASC2, lncRNA cancer susceptibility candidate 2; IL-17, interleukin-17; RA, rheumatoid arthritis.

    Techniques Used: Expressing

    10) Product Images from "Ommochrome pathway genes kynurenine 3-hydroxylase and cardinal participate in eye pigmentation in Plutella xylostella"

    Article Title: Ommochrome pathway genes kynurenine 3-hydroxylase and cardinal participate in eye pigmentation in Plutella xylostella

    Journal: BMC Molecular and Cell Biology

    doi: 10.1186/s12860-020-00308-8

    Gene structure ( a ) and phylogenetic analysis ( b ) of Pxkmo and Pxcardinal a Gene structure of Pxkmo (upper) and Pxcardinal (lower). Predicted splicing patterns are indicated with black boxes (exons) and broken lines (introns). Red boxes and blue dashed lines are used to mark the relative positions of transmembrane structures and conserved domains in translated proteins, respectively. sgRNAs binding sites are indicated with black arrows. b Phylogenetic trees of kmo (upper) and cardinal (lower) proteins across insect species (with Maximum Likelihood Method, 1000 bootstraps). Species name (left) and accession number (right) are listed at the terminal of tree branches. Tree scales are provided to indicate the branch length
    Figure Legend Snippet: Gene structure ( a ) and phylogenetic analysis ( b ) of Pxkmo and Pxcardinal a Gene structure of Pxkmo (upper) and Pxcardinal (lower). Predicted splicing patterns are indicated with black boxes (exons) and broken lines (introns). Red boxes and blue dashed lines are used to mark the relative positions of transmembrane structures and conserved domains in translated proteins, respectively. sgRNAs binding sites are indicated with black arrows. b Phylogenetic trees of kmo (upper) and cardinal (lower) proteins across insect species (with Maximum Likelihood Method, 1000 bootstraps). Species name (left) and accession number (right) are listed at the terminal of tree branches. Tree scales are provided to indicate the branch length

    Techniques Used: Binding Assay

    Phenotypes and genotypes of Pxkmo and Pxcardinal mutants. a and h : Wildtype phenotype. b and i : Pxkmo yellow-eye mutant phenotype. c and j : Pxkmo red-eye mutant phenotype. d - g and K-M: Pxcardinal mutant phenotypes. For Pxcardinal newly eclosed adults showed yellow compound eyes ( d and k ), which started to change on Day 2 or Day 3 post eclosion and gradually turned red (in the order of D to G, or K to M), although a small minority of individuals did not change. h - m : Red dashed circles indicate the positions of ocelli on increased magnification images. o : Pxkmo mutant genotypes. Two sgRNA targets are shown with red color. p : Pxcardinal mutant genotypes. Two sgRNA targets are marked with blue color. The protospacer adjacent motif (PAM) sites are underlined. Abbreviation: YE: yellow-eye; RE: red-eye. Scale bar = 0.2 mm
    Figure Legend Snippet: Phenotypes and genotypes of Pxkmo and Pxcardinal mutants. a and h : Wildtype phenotype. b and i : Pxkmo yellow-eye mutant phenotype. c and j : Pxkmo red-eye mutant phenotype. d - g and K-M: Pxcardinal mutant phenotypes. For Pxcardinal newly eclosed adults showed yellow compound eyes ( d and k ), which started to change on Day 2 or Day 3 post eclosion and gradually turned red (in the order of D to G, or K to M), although a small minority of individuals did not change. h - m : Red dashed circles indicate the positions of ocelli on increased magnification images. o : Pxkmo mutant genotypes. Two sgRNA targets are shown with red color. p : Pxcardinal mutant genotypes. Two sgRNA targets are marked with blue color. The protospacer adjacent motif (PAM) sites are underlined. Abbreviation: YE: yellow-eye; RE: red-eye. Scale bar = 0.2 mm

    Techniques Used: Mutagenesis

    Representative phenotypes of G 0 mosaics. Pupae (a-e) and adults (A’-E’). A and A’: wildtype (WT) control. B, B′, C and C′: Pxkmo mosaic mutants. D, D’, E and E’: Pxcardinal G 0 mosaic mutants. Differences of pupal body color were due to differences in developmental stages, while patchy eye pigmentation was linked to gene editing. Scale bar = 0.5 mm
    Figure Legend Snippet: Representative phenotypes of G 0 mosaics. Pupae (a-e) and adults (A’-E’). A and A’: wildtype (WT) control. B, B′, C and C′: Pxkmo mosaic mutants. D, D’, E and E’: Pxcardinal G 0 mosaic mutants. Differences of pupal body color were due to differences in developmental stages, while patchy eye pigmentation was linked to gene editing. Scale bar = 0.5 mm

    Techniques Used:

    The 4th instar larvae and pupae of Pxkmo and Pxcardinal yellow-eye knock-out lines. a - c : larval body including head and partial thorax. d - g : dissected larval heads. h - j : dissected larval testes. a , d and h : wildtype larva. b , e and i : Pxkmo larva. c, f and J: Pxcardinal larva. White dash rectangles are used to indicate dorsal protothorax of larvae. Dissected brains are highlighted with white dashed lines while colorless ocelli are indicated with red dashed circles. a, b, c and g : dorsal view of larval heads. d - f : lateral view of larval heads. K: wildtype pupa. l : Pxkmo pupa. m : Pxcardinal pupa. Scale bar = 0.5 mm
    Figure Legend Snippet: The 4th instar larvae and pupae of Pxkmo and Pxcardinal yellow-eye knock-out lines. a - c : larval body including head and partial thorax. d - g : dissected larval heads. h - j : dissected larval testes. a , d and h : wildtype larva. b , e and i : Pxkmo larva. c, f and J: Pxcardinal larva. White dash rectangles are used to indicate dorsal protothorax of larvae. Dissected brains are highlighted with white dashed lines while colorless ocelli are indicated with red dashed circles. a, b, c and g : dorsal view of larval heads. d - f : lateral view of larval heads. K: wildtype pupa. l : Pxkmo pupa. m : Pxcardinal pupa. Scale bar = 0.5 mm

    Techniques Used: Knock-Out

    11) Product Images from "Correction of a Factor VIII genomic inversion with designer-recombinases"

    Article Title: Correction of a Factor VIII genomic inversion with designer-recombinases

    Journal: bioRxiv

    doi: 10.1101/2020.11.02.328013

    Activity of the D7 heterodimer in human cells. a) Schematic overview of the integrated reporter construct in HEK293T cells. Site-specific recombination excises the spleen focus forming virus (SFFV) promoter-driven puromycin resistance gene (PURO), which leads to expression of the red-fluorescent mCherry gene. b) Depiction of the mRNAs that were transfected for transient expression of the recombinase dimer. tagBFP mRNA was used to monitor transfection efficiencies. NLS, nuclear localization signal. c) Fluorescent and brightfield images of transfected HEK293T loxF8 reporter cells with indicated recombinases. Note that mCherry expression is only visible in cells receiving both monomers. 200 μm white scale bars are indicated. d) Quantification of the recombination efficiencies 48 h after transfection of HEK293T loxF8 reporter cells with indicated recombinases, analyzed by FACS (n=3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Recombination efficiencies were calculated based on the presented formula. e) Schematic overview of a fraction of the F8 gene displaying the PCR primers used to detect the orientation of the loxF8 locus. Exons are displayed in magenta and the repeated regions int1h-1 and int1h-2 are shown in grey. Primer binding sites are indicated with arrows. The transcription start site is depicted by a black arrow. f) Gel image of PCR products generated using indicated primer combinations to detect the orientation of the loxF8 locus with and without treatment with the D7 heterodimer. Band sizes of the marker lane (M) are indicated.
    Figure Legend Snippet: Activity of the D7 heterodimer in human cells. a) Schematic overview of the integrated reporter construct in HEK293T cells. Site-specific recombination excises the spleen focus forming virus (SFFV) promoter-driven puromycin resistance gene (PURO), which leads to expression of the red-fluorescent mCherry gene. b) Depiction of the mRNAs that were transfected for transient expression of the recombinase dimer. tagBFP mRNA was used to monitor transfection efficiencies. NLS, nuclear localization signal. c) Fluorescent and brightfield images of transfected HEK293T loxF8 reporter cells with indicated recombinases. Note that mCherry expression is only visible in cells receiving both monomers. 200 μm white scale bars are indicated. d) Quantification of the recombination efficiencies 48 h after transfection of HEK293T loxF8 reporter cells with indicated recombinases, analyzed by FACS (n=3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Recombination efficiencies were calculated based on the presented formula. e) Schematic overview of a fraction of the F8 gene displaying the PCR primers used to detect the orientation of the loxF8 locus. Exons are displayed in magenta and the repeated regions int1h-1 and int1h-2 are shown in grey. Primer binding sites are indicated with arrows. The transcription start site is depicted by a black arrow. f) Gel image of PCR products generated using indicated primer combinations to detect the orientation of the loxF8 locus with and without treatment with the D7 heterodimer. Band sizes of the marker lane (M) are indicated.

    Techniques Used: Activity Assay, Construct, Expressing, Transfection, FACS, Standard Deviation, Polymerase Chain Reaction, Binding Assay, Generated, Marker

    Strategy to evaluate recombinases in mammalian cells. a) Schematic representation of the generation of the HEK293T loxF8 reporter cell line. Important steps are shown on the time-line. b) Representative FACS plot and gating strategy to evaluate the transfection efficiency measured by tagBFP+ cells (transfection of tagBFP mRNA only). c) Representative FACS plot and gating strategy of HEK293T loxF8 reporter cells that were transfected with tagBFP and indicated recombinase mRNAs. Upon successful site-specific recombination, cells express mCherry. tagBFP+ and mCherry+ were used to calculate the recombination efficiencies (see formula in Fig. 3 ). Parts of the figure are created with BioRender.com .
    Figure Legend Snippet: Strategy to evaluate recombinases in mammalian cells. a) Schematic representation of the generation of the HEK293T loxF8 reporter cell line. Important steps are shown on the time-line. b) Representative FACS plot and gating strategy to evaluate the transfection efficiency measured by tagBFP+ cells (transfection of tagBFP mRNA only). c) Representative FACS plot and gating strategy of HEK293T loxF8 reporter cells that were transfected with tagBFP and indicated recombinase mRNAs. Upon successful site-specific recombination, cells express mCherry. tagBFP+ and mCherry+ were used to calculate the recombination efficiencies (see formula in Fig. 3 ). Parts of the figure are created with BioRender.com .

    Techniques Used: FACS, Transfection

    12) Product Images from "Correction of a Factor VIII genomic inversion with designer-recombinases"

    Article Title: Correction of a Factor VIII genomic inversion with designer-recombinases

    Journal: bioRxiv

    doi: 10.1101/2020.11.02.328013

    Activity of the D7 heterodimer in human cells. a) Schematic overview of the integrated reporter construct in HEK293T cells. Site-specific recombination excises the spleen focus forming virus (SFFV) promoter-driven puromycin resistance gene (PURO), which leads to expression of the red-fluorescent mCherry gene. b) Depiction of the mRNAs that were transfected for transient expression of the recombinase dimer. tagBFP mRNA was used to monitor transfection efficiencies. NLS, nuclear localization signal. c) Fluorescent and brightfield images of transfected HEK293T loxF8 reporter cells with indicated recombinases. Note that mCherry expression is only visible in cells receiving both monomers. 200 μm white scale bars are indicated. d) Quantification of the recombination efficiencies 48 h after transfection of HEK293T loxF8 reporter cells with indicated recombinases, analyzed by FACS (n=3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Recombination efficiencies were calculated based on the presented formula. e) Schematic overview of a fraction of the F8 gene displaying the PCR primers used to detect the orientation of the loxF8 locus. Exons are displayed in magenta and the repeated regions int1h-1 and int1h-2 are shown in grey. Primer binding sites are indicated with arrows. The transcription start site is depicted by a black arrow. f) Gel image of PCR products generated using indicated primer combinations to detect the orientation of the loxF8 locus with and without treatment with the D7 heterodimer. Band sizes of the marker lane (M) are indicated.
    Figure Legend Snippet: Activity of the D7 heterodimer in human cells. a) Schematic overview of the integrated reporter construct in HEK293T cells. Site-specific recombination excises the spleen focus forming virus (SFFV) promoter-driven puromycin resistance gene (PURO), which leads to expression of the red-fluorescent mCherry gene. b) Depiction of the mRNAs that were transfected for transient expression of the recombinase dimer. tagBFP mRNA was used to monitor transfection efficiencies. NLS, nuclear localization signal. c) Fluorescent and brightfield images of transfected HEK293T loxF8 reporter cells with indicated recombinases. Note that mCherry expression is only visible in cells receiving both monomers. 200 μm white scale bars are indicated. d) Quantification of the recombination efficiencies 48 h after transfection of HEK293T loxF8 reporter cells with indicated recombinases, analyzed by FACS (n=3, biological replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Recombination efficiencies were calculated based on the presented formula. e) Schematic overview of a fraction of the F8 gene displaying the PCR primers used to detect the orientation of the loxF8 locus. Exons are displayed in magenta and the repeated regions int1h-1 and int1h-2 are shown in grey. Primer binding sites are indicated with arrows. The transcription start site is depicted by a black arrow. f) Gel image of PCR products generated using indicated primer combinations to detect the orientation of the loxF8 locus with and without treatment with the D7 heterodimer. Band sizes of the marker lane (M) are indicated.

    Techniques Used: Activity Assay, Construct, Expressing, Transfection, FACS, Standard Deviation, Polymerase Chain Reaction, Binding Assay, Generated, Marker

    Strategy to evaluate recombinases in mammalian cells. a) Schematic representation of the generation of the HEK293T loxF8 reporter cell line. Important steps are shown on the time-line. b) Representative FACS plot and gating strategy to evaluate the transfection efficiency measured by tagBFP+ cells (transfection of tagBFP mRNA only). c) Representative FACS plot and gating strategy of HEK293T loxF8 reporter cells that were transfected with tagBFP and indicated recombinase mRNAs. Upon successful site-specific recombination, cells express mCherry. tagBFP+ and mCherry+ were used to calculate the recombination efficiencies (see formula in Fig. 3 ). Parts of the figure are created with BioRender.com .
    Figure Legend Snippet: Strategy to evaluate recombinases in mammalian cells. a) Schematic representation of the generation of the HEK293T loxF8 reporter cell line. Important steps are shown on the time-line. b) Representative FACS plot and gating strategy to evaluate the transfection efficiency measured by tagBFP+ cells (transfection of tagBFP mRNA only). c) Representative FACS plot and gating strategy of HEK293T loxF8 reporter cells that were transfected with tagBFP and indicated recombinase mRNAs. Upon successful site-specific recombination, cells express mCherry. tagBFP+ and mCherry+ were used to calculate the recombination efficiencies (see formula in Fig. 3 ). Parts of the figure are created with BioRender.com .

    Techniques Used: FACS, Transfection

    Functional correction of the inverted F8 gene in differentiated endothelial cells. a) Schematic overview of the EC differentiation protocol. Important steps are highlighted on the timeline. b) Efficiency of transfection of mCherry mRNA to differentiated endothelial cells. Brightfield (BF) image, mCherry image and the merged image a shown. Images were taken 48 h post transfection. White bars indicated 50 μm scale bar. c) RecF8 expression corrects the int1h inversion. The arrangement of different primers around the first and second loxF8 site to detect the orientation of the full 140 kb fragment before and after RecF8 treatment are shown in the top panels. The lower panels show gels of PCR products obtained on genomic DNA from patient specific ECs with and without treatment with RecF8. The combination of the primers used for every PCR gel picture is shown below. d) qPCR-based quantification of the genomic inversion efficiencies in ECs after RecF8 treatment. The quantification of the inversion was extrapolated from a standard of defined ratios of WT and F8 patient iPSCs genomic DNA (1%, 5%, 10%, 25% and 50% normal orientation of the loxF8 locus, n=3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). e) qPCR-based quantification of Factor VIII mRNA transcript (amplification of exon1-exon2 boundary transcript). The relative expression of the Factor VIII mRNA was measured after treating patient specific ECs with indicated amounts of RecF8 mRNA (n=3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Factor VIII mRNA expression in untreated WT ECs was used for normalization. The Factor VIII mRNA expression was normalized against ß-actin expression. f) Sequencing read of Factor VIII cDNA of RecF8 treated patient-derived ECs. Note that the exon1-exon2 boundary can only occur, if the inversion is corrected to the WT orientation, the gene is transcribed and the pre-mRNA spliced correctly. Parts of the figure were created with BioRender.com .
    Figure Legend Snippet: Functional correction of the inverted F8 gene in differentiated endothelial cells. a) Schematic overview of the EC differentiation protocol. Important steps are highlighted on the timeline. b) Efficiency of transfection of mCherry mRNA to differentiated endothelial cells. Brightfield (BF) image, mCherry image and the merged image a shown. Images were taken 48 h post transfection. White bars indicated 50 μm scale bar. c) RecF8 expression corrects the int1h inversion. The arrangement of different primers around the first and second loxF8 site to detect the orientation of the full 140 kb fragment before and after RecF8 treatment are shown in the top panels. The lower panels show gels of PCR products obtained on genomic DNA from patient specific ECs with and without treatment with RecF8. The combination of the primers used for every PCR gel picture is shown below. d) qPCR-based quantification of the genomic inversion efficiencies in ECs after RecF8 treatment. The quantification of the inversion was extrapolated from a standard of defined ratios of WT and F8 patient iPSCs genomic DNA (1%, 5%, 10%, 25% and 50% normal orientation of the loxF8 locus, n=3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). e) qPCR-based quantification of Factor VIII mRNA transcript (amplification of exon1-exon2 boundary transcript). The relative expression of the Factor VIII mRNA was measured after treating patient specific ECs with indicated amounts of RecF8 mRNA (n=3, replicates are shown as dots). Error bars represent standard deviation of the mean (SD). Factor VIII mRNA expression in untreated WT ECs was used for normalization. The Factor VIII mRNA expression was normalized against ß-actin expression. f) Sequencing read of Factor VIII cDNA of RecF8 treated patient-derived ECs. Note that the exon1-exon2 boundary can only occur, if the inversion is corrected to the WT orientation, the gene is transcribed and the pre-mRNA spliced correctly. Parts of the figure were created with BioRender.com .

    Techniques Used: Functional Assay, Transfection, Expressing, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Standard Deviation, Amplification, Sequencing, Derivative Assay

    13) Product Images from "Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals"

    Article Title: Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals

    Journal: bioRxiv

    doi: 10.1101/808642

    A Cell-based System to Study the Effect of DSBs on the Epigenome, Related to Figure 1 (A) RNA-seq volcano plots (Cre vs. ICE) of cells from the same or different litters before 4-OHT treatment. (B) Slot blots to compare global 5mC levels. Methylene blue (MB) staining showed total genomic DNA used in each sample. (C) Genomic distribution of I- Ppo I canonical sites and all 90 CpG sites of the mouse epigenetic clock. (D) Percent non-mutated sequences of ∼100,000 random sites in post-treated ICE cells assessed by deep sequencing ( > 50x). (E and F) Immunostaining of DNA damage markers γH2AX and 53BP1 in post-treated ICE cells with and without exposure to the DNA damaging agents (ETS, etoposide; CPT, camptothecin; H 2 O 2 , hydrogen peroxide). Two-tailed Student’s t test. Scale bar, 10 µm. (G) Immunostaining of Lamin B1 in post-treated ICE cells. Data are mean (n=3) ± SD. *p
    Figure Legend Snippet: A Cell-based System to Study the Effect of DSBs on the Epigenome, Related to Figure 1 (A) RNA-seq volcano plots (Cre vs. ICE) of cells from the same or different litters before 4-OHT treatment. (B) Slot blots to compare global 5mC levels. Methylene blue (MB) staining showed total genomic DNA used in each sample. (C) Genomic distribution of I- Ppo I canonical sites and all 90 CpG sites of the mouse epigenetic clock. (D) Percent non-mutated sequences of ∼100,000 random sites in post-treated ICE cells assessed by deep sequencing ( > 50x). (E and F) Immunostaining of DNA damage markers γH2AX and 53BP1 in post-treated ICE cells with and without exposure to the DNA damaging agents (ETS, etoposide; CPT, camptothecin; H 2 O 2 , hydrogen peroxide). Two-tailed Student’s t test. Scale bar, 10 µm. (G) Immunostaining of Lamin B1 in post-treated ICE cells. Data are mean (n=3) ± SD. *p

    Techniques Used: RNA Sequencing Assay, Staining, Sequencing, Immunostaining, Two Tailed Test

    14) Product Images from "Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells"

    Article Title: Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01705-y

    CTCs isolated from prostate cancer patient blood contain intact RNA for RNA sequencing and Droplet Digital PCR after whole blood preservation. a RIN values of CTCs isolated from fresh and cold-stored blood (total 15 blood draws from 8 independent patients). b Scaled heatmap of log2-transformed normalized reads (RPMs) for four prostate cancer patients. For patient #3, data are available from three different draw dates stored for different durations (24, 48, and 72 h; denoted #3.1, #3.2, and #3.3, respectively). Statistical analysis comparing fresh vs. cold-stored blood (6 blood draws) found no significant differences in the mean RPMs for any of the 40 genes depicted in b , except for KRT18 ( p = 0.037, paired t -test). c The mRNA copy number of AR-V7 detected by Droplet Digital PCR (expressed as per mL of blood processed). Box-and-whiskers plots show median, interquartile range, maxima, and minima
    Figure Legend Snippet: CTCs isolated from prostate cancer patient blood contain intact RNA for RNA sequencing and Droplet Digital PCR after whole blood preservation. a RIN values of CTCs isolated from fresh and cold-stored blood (total 15 blood draws from 8 independent patients). b Scaled heatmap of log2-transformed normalized reads (RPMs) for four prostate cancer patients. For patient #3, data are available from three different draw dates stored for different durations (24, 48, and 72 h; denoted #3.1, #3.2, and #3.3, respectively). Statistical analysis comparing fresh vs. cold-stored blood (6 blood draws) found no significant differences in the mean RPMs for any of the 40 genes depicted in b , except for KRT18 ( p = 0.037, paired t -test). c The mRNA copy number of AR-V7 detected by Droplet Digital PCR (expressed as per mL of blood processed). Box-and-whiskers plots show median, interquartile range, maxima, and minima

    Techniques Used: Isolation, RNA Sequencing Assay, Digital PCR, Preserving, Transformation Assay

    15) Product Images from "Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase"

    Article Title: Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17443-7

    Direct visualization of RPA-facilitated processive fork unwinding by individual CMG molecules. a A 10-kb fragment of λ DNA was ligated to a short fork DNA substrate at one end and to a digoxigenin-modified DNA fragment on the opposite end. The 5′ tail of the forked end contained a biotin to attach the 10-kb substrate to biotin-functionalized glass through biotin-streptavidin binding. The digoxigenin-modified end was coupled to anti-digoxigenin-coated microsphere to stretch DNA molecules by buffer flow, and to subsequently attach this end to the surface (details are described in the Methods section). The DNA substrate was labeled with Cy3 at the 3′ dT 40 ssDNA tail near the fork junction. LD655-labeled CMG was bound to the dT 40 ssDNA in the presence of ATPγS. b A sample stretched 10-kb linear DNA stained with a fluorescent dsDNA intercalator Sytox Orange. Average length of stretched DNA molecules were determined by measuring the end-to-end distance of 717 individual DNA molecules ( n = 2 independent experiments). c After binding CMG on the surface-immobilized DNA, EGFP-RPA, and ATP was introduced to initiate unwinding. While CMG unwinds DNA at the fork, EGFP-RPA binds both strands of unwound DNA. d Kymograph showing a representative 10-kb DNA being unwound entirely by a single CMG complex. EGFP-RPA (left panel), LD655-labeled CMG (center panel), and 3′ Cy3 (right panel) are imaged during unwinding under near-TIRF conditions. Images were acquired in the absence of buffer flow. e Histogram of CMG-catalyzed DNA-unwinding rates on stretched DNA (74 individual DNA molecules analyzed from two independent experiments). Source data are provided as a Source Data file.
    Figure Legend Snippet: Direct visualization of RPA-facilitated processive fork unwinding by individual CMG molecules. a A 10-kb fragment of λ DNA was ligated to a short fork DNA substrate at one end and to a digoxigenin-modified DNA fragment on the opposite end. The 5′ tail of the forked end contained a biotin to attach the 10-kb substrate to biotin-functionalized glass through biotin-streptavidin binding. The digoxigenin-modified end was coupled to anti-digoxigenin-coated microsphere to stretch DNA molecules by buffer flow, and to subsequently attach this end to the surface (details are described in the Methods section). The DNA substrate was labeled with Cy3 at the 3′ dT 40 ssDNA tail near the fork junction. LD655-labeled CMG was bound to the dT 40 ssDNA in the presence of ATPγS. b A sample stretched 10-kb linear DNA stained with a fluorescent dsDNA intercalator Sytox Orange. Average length of stretched DNA molecules were determined by measuring the end-to-end distance of 717 individual DNA molecules ( n = 2 independent experiments). c After binding CMG on the surface-immobilized DNA, EGFP-RPA, and ATP was introduced to initiate unwinding. While CMG unwinds DNA at the fork, EGFP-RPA binds both strands of unwound DNA. d Kymograph showing a representative 10-kb DNA being unwound entirely by a single CMG complex. EGFP-RPA (left panel), LD655-labeled CMG (center panel), and 3′ Cy3 (right panel) are imaged during unwinding under near-TIRF conditions. Images were acquired in the absence of buffer flow. e Histogram of CMG-catalyzed DNA-unwinding rates on stretched DNA (74 individual DNA molecules analyzed from two independent experiments). Source data are provided as a Source Data file.

    Techniques Used: Recombinase Polymerase Amplification, Modification, Binding Assay, Labeling, Staining

    16) Product Images from "TOR coordinates nucleotide availability with ribosome biogenesis in plants"

    Article Title: TOR coordinates nucleotide availability with ribosome biogenesis in plants

    Journal: bioRxiv

    doi: 10.1101/2020.01.30.927418

    Cytosolic PRS (PRS4) drives plant development and TOR activity in N. benthamiana . (A) Silencing PRS4 drastically reduces TOR activity. S6K-pT449 levels reflect TOR activity, because S6K-T449 is a direct substrate of TOR ( Xiong and Sheen, 2012 ). S6K-pT449 and total S6K levels were assayed by Western blots in knockdown TRV:: NbPRS4 plants or mock controls (representative images shown here). Quantification of band densities confirmed that S6K-pT449 levels decrease ∼5-fold in TRV:: NbPRS4 knockdowns, but total S6K levels are not affected in TRV:: NbPRS4 . (B) PRS4 is required for shoot development. There are fewer leaves in TRV:: NbPRS4 knockdowns, and the leaves are misshapen and small. We observed individual-to-individual variation in phenotypic severity after silencing PRS4 by VIGS; a representative of the “moderate” TRV:: NbPRS4 phenotype and of the “severe” TRV:: NbPRS4 phenotype are shown. Outlines of leaf shapes are shown, including all leaves with silenced PRS4 expression (i.e., only leaves above the primary infected leaf), with the oldest leaf on the left and the youngest leaf on the right. (C) Silencing PRS4 impairs cell expansion and cell division. Epidermal pavement cell shape was not dramatically altered in TRV:: NbPRS4 knockdowns, but epidermal pavement cell size was significantly lower. This difference in cell size is insufficient to account for the decrease in total leaf area shown in panel B; therefore, there are also fewer epidermal pavement cells in TRV:: NbPRS4 . (D) We did not observe clear effects of silencing PRS4 on vegetative shoot apical meristem morphology.
    Figure Legend Snippet: Cytosolic PRS (PRS4) drives plant development and TOR activity in N. benthamiana . (A) Silencing PRS4 drastically reduces TOR activity. S6K-pT449 levels reflect TOR activity, because S6K-T449 is a direct substrate of TOR ( Xiong and Sheen, 2012 ). S6K-pT449 and total S6K levels were assayed by Western blots in knockdown TRV:: NbPRS4 plants or mock controls (representative images shown here). Quantification of band densities confirmed that S6K-pT449 levels decrease ∼5-fold in TRV:: NbPRS4 knockdowns, but total S6K levels are not affected in TRV:: NbPRS4 . (B) PRS4 is required for shoot development. There are fewer leaves in TRV:: NbPRS4 knockdowns, and the leaves are misshapen and small. We observed individual-to-individual variation in phenotypic severity after silencing PRS4 by VIGS; a representative of the “moderate” TRV:: NbPRS4 phenotype and of the “severe” TRV:: NbPRS4 phenotype are shown. Outlines of leaf shapes are shown, including all leaves with silenced PRS4 expression (i.e., only leaves above the primary infected leaf), with the oldest leaf on the left and the youngest leaf on the right. (C) Silencing PRS4 impairs cell expansion and cell division. Epidermal pavement cell shape was not dramatically altered in TRV:: NbPRS4 knockdowns, but epidermal pavement cell size was significantly lower. This difference in cell size is insufficient to account for the decrease in total leaf area shown in panel B; therefore, there are also fewer epidermal pavement cells in TRV:: NbPRS4 . (D) We did not observe clear effects of silencing PRS4 on vegetative shoot apical meristem morphology.

    Techniques Used: Activity Assay, Western Blot, Expressing, Infection

    Silencing key genes in nucleotide biosynthesis inhibits TOR activity. (A) Nucleotide biosynthesis is necessary for normal shoot development and physiology. Silencing genes downstream of PRS4 in nucleotide biosynthesis in N. benthamiana reduced leaf number and size, disrupted leaf shape, and caused chlorosis, similar to the phenotypes observed in TRV:: NbPRS4 plants. Each gene was silenced in at least six plants per experiment, and the entire experiment was replicated three times; representative individuals of each silenced gene are shown. (B) Silencing nucleotide biosynthesis genes lowers TOR activity. S6K-pT449 levels are strongly reduced in silenced plants compared to mock-infected controls, and the S6K-pT449/S6K ratios are consistently lower.
    Figure Legend Snippet: Silencing key genes in nucleotide biosynthesis inhibits TOR activity. (A) Nucleotide biosynthesis is necessary for normal shoot development and physiology. Silencing genes downstream of PRS4 in nucleotide biosynthesis in N. benthamiana reduced leaf number and size, disrupted leaf shape, and caused chlorosis, similar to the phenotypes observed in TRV:: NbPRS4 plants. Each gene was silenced in at least six plants per experiment, and the entire experiment was replicated three times; representative individuals of each silenced gene are shown. (B) Silencing nucleotide biosynthesis genes lowers TOR activity. S6K-pT449 levels are strongly reduced in silenced plants compared to mock-infected controls, and the S6K-pT449/S6K ratios are consistently lower.

    Techniques Used: Activity Assay, Infection

    Silencing PRS4 reprograms the transcriptome to repress ribosome biogenesis. (A) Scatterplots of gene expression changes in N. benthamiana after VIGS. 4,986 genes were significantly differentially expressed between TRV:: NbPRS4 knockdowns with severe phenotypes and mock plants (top panel), but only 489 genes were significantly differentially expressed between TRV:: NbPRS4 knockdowns with severe versus moderate phenotypes (middle panel). Principal component analysis demonstrates that the mock-treated transcriptomes are readily distinguished from the TRV:: NbPRS4 knockdowns, but that the TRV:: NbPRS4 transcriptomes from plants with severe or moderate phenotypes are not grouped separately. (B) MapMan functional analysis of DEGs in TRV:: NbPRS4 revealed 48 significantly-affected categories ( p adj .
    Figure Legend Snippet: Silencing PRS4 reprograms the transcriptome to repress ribosome biogenesis. (A) Scatterplots of gene expression changes in N. benthamiana after VIGS. 4,986 genes were significantly differentially expressed between TRV:: NbPRS4 knockdowns with severe phenotypes and mock plants (top panel), but only 489 genes were significantly differentially expressed between TRV:: NbPRS4 knockdowns with severe versus moderate phenotypes (middle panel). Principal component analysis demonstrates that the mock-treated transcriptomes are readily distinguished from the TRV:: NbPRS4 knockdowns, but that the TRV:: NbPRS4 transcriptomes from plants with severe or moderate phenotypes are not grouped separately. (B) MapMan functional analysis of DEGs in TRV:: NbPRS4 revealed 48 significantly-affected categories ( p adj .

    Techniques Used: Expressing, Functional Assay

    17) Product Images from "lncRNA MEG8 Upregulates miR-770-5p Through Methylation and Promotes Cell Apoptosis in Diabetic Nephropathy"

    Article Title: lncRNA MEG8 Upregulates miR-770-5p Through Methylation and Promotes Cell Apoptosis in Diabetic Nephropathy

    Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

    doi: 10.2147/DMSO.S255183

    MEG8 upregulated the expression of miR-770-5p in CIHP-1 cells by reducing the methylation of miR-770-5p. To explore the interaction between MEG8 and miR-770-5p in DN, CIHP-1 cells were treated with 40 nM D-glucose for 48 h to mimic DN, followed by transfections of MEG8 expression vector or miR-770-5p mimic. Transfections were confirmed by RT-qPCR ( A ). The effects of MEG8 expression vector transfection on the expression of miR-770-5p ( B ), and the effects of overexpression of miR-770-5p on MEG8 ( C ) were also analyzed by RT-qPCR. MSP was performed to analyze the effects of overexpression of MEG8 on the methylation of miR-770-5p. In MSP, MSP primers were designed to clone methylated miR-770-5p gene and non-MSP primers were used to clone un-methylated miR-770-5p gene in cells transfected with MEG8 expression vector or empty pcDNA3.1 vector. PCR products were subjected to 1% agarose gel electrophoresis, followed by ethidium bromide staining ( D ). Mean ± SD values of 3 replicates were presented. U, un-methylation; M, methylation; * p
    Figure Legend Snippet: MEG8 upregulated the expression of miR-770-5p in CIHP-1 cells by reducing the methylation of miR-770-5p. To explore the interaction between MEG8 and miR-770-5p in DN, CIHP-1 cells were treated with 40 nM D-glucose for 48 h to mimic DN, followed by transfections of MEG8 expression vector or miR-770-5p mimic. Transfections were confirmed by RT-qPCR ( A ). The effects of MEG8 expression vector transfection on the expression of miR-770-5p ( B ), and the effects of overexpression of miR-770-5p on MEG8 ( C ) were also analyzed by RT-qPCR. MSP was performed to analyze the effects of overexpression of MEG8 on the methylation of miR-770-5p. In MSP, MSP primers were designed to clone methylated miR-770-5p gene and non-MSP primers were used to clone un-methylated miR-770-5p gene in cells transfected with MEG8 expression vector or empty pcDNA3.1 vector. PCR products were subjected to 1% agarose gel electrophoresis, followed by ethidium bromide staining ( D ). Mean ± SD values of 3 replicates were presented. U, un-methylation; M, methylation; * p

    Techniques Used: Expressing, Methylation, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Glucose-inducible MEG8 and miR-770-5p promoted glucose-mediated apoptosis of CIHP-1 cells. CIHP-1 cells were cultivated in medium containing 10, 20 and 40 mM D-glucose for 48 h, followed by determining the expression of MEG8 ( A ) and miR-770-5p ( B ) by RT-qPCR. Cell apoptosis assay was performed to explore the role of MEG8 and miR-770-5p in regulate the apoptosis of CIHP-1 cells ( C ). Mean ± SD values of 3 replicates were presented. * p
    Figure Legend Snippet: Glucose-inducible MEG8 and miR-770-5p promoted glucose-mediated apoptosis of CIHP-1 cells. CIHP-1 cells were cultivated in medium containing 10, 20 and 40 mM D-glucose for 48 h, followed by determining the expression of MEG8 ( A ) and miR-770-5p ( B ) by RT-qPCR. Cell apoptosis assay was performed to explore the role of MEG8 and miR-770-5p in regulate the apoptosis of CIHP-1 cells ( C ). Mean ± SD values of 3 replicates were presented. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Apoptosis Assay

    18) Product Images from "lncRNA NR4A1AS Upregulates miR-221 Through Demethylation to Promote Cell Proliferation in Oral Squamous Cell Carcinoma"

    Article Title: lncRNA NR4A1AS Upregulates miR-221 Through Demethylation to Promote Cell Proliferation in Oral Squamous Cell Carcinoma

    Journal: Cancer Management and Research

    doi: 10.2147/CMAR.S241769

    Overexpression of NR4A1AS led to the upregulation of miR-221 by demethylation of miR-221 in OSCC cells. SCC25 and SCC090 cells were transfected with NR4A1AS expression vector or miR-221 mimic. Overexpression of NR4A1AS and miR-221 was confirmed by RT-qPCR at 48 h post-transfection ( A ). The regulation of overexpression of NR4A1AS on miR-221 was evaluated by RT-qPCR at 48 h post-transfection ( B ). The effect of overexpression of miR-221 on NR4A1AS was detected by RT-qPCR at 48 h post-transfection ( C ). MSP was performed to analyze the effects of overexpression of NR4A1AS on miR-221 demethylation ( D ). All experiments were repeated 3 times and mean values were presented and compared. * p
    Figure Legend Snippet: Overexpression of NR4A1AS led to the upregulation of miR-221 by demethylation of miR-221 in OSCC cells. SCC25 and SCC090 cells were transfected with NR4A1AS expression vector or miR-221 mimic. Overexpression of NR4A1AS and miR-221 was confirmed by RT-qPCR at 48 h post-transfection ( A ). The regulation of overexpression of NR4A1AS on miR-221 was evaluated by RT-qPCR at 48 h post-transfection ( B ). The effect of overexpression of miR-221 on NR4A1AS was detected by RT-qPCR at 48 h post-transfection ( C ). MSP was performed to analyze the effects of overexpression of NR4A1AS on miR-221 demethylation ( D ). All experiments were repeated 3 times and mean values were presented and compared. * p

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR

    Overexpression of NR4A1AS or/and miR-221 promote the proliferation of OSCC cells. The effects of overexpression of NR4A1AS or/and miR-221 on the proliferation of SCC25 and SCC090 cells were determined by CCK-8 assay. Overexpression of NR4A1AS could significantly increase the proliferation rate of OSCC cells. MiR-221 also had the same effect on the proliferation of OSCC cells, and there was no difference observed between C and NC groups. All experiments were repeated 3 times and mean values were presented and compared. * p
    Figure Legend Snippet: Overexpression of NR4A1AS or/and miR-221 promote the proliferation of OSCC cells. The effects of overexpression of NR4A1AS or/and miR-221 on the proliferation of SCC25 and SCC090 cells were determined by CCK-8 assay. Overexpression of NR4A1AS could significantly increase the proliferation rate of OSCC cells. MiR-221 also had the same effect on the proliferation of OSCC cells, and there was no difference observed between C and NC groups. All experiments were repeated 3 times and mean values were presented and compared. * p

    Techniques Used: Over Expression, CCK-8 Assay

    19) Product Images from "In vitro Type II Restriction of Bacteriophage DNA With Modified Pyrimidines"

    Article Title: In vitro Type II Restriction of Bacteriophage DNA With Modified Pyrimidines

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.604618

    Restriction of phage SP8 (5hmdU) DNA. Example of 10 restriction digestions analyzed in 1% agarose gel (A) . NEBcutter predicted cleavage patterns in the absence of T modification (B) . Enzyme recognition sequences and restriction result (C) .
    Figure Legend Snippet: Restriction of phage SP8 (5hmdU) DNA. Example of 10 restriction digestions analyzed in 1% agarose gel (A) . NEBcutter predicted cleavage patterns in the absence of T modification (B) . Enzyme recognition sequences and restriction result (C) .

    Techniques Used: Agarose Gel Electrophoresis, Modification

    Restriction digestion of SP8 gDNA following treatment with 5hmdU DNA kinase. Top panel, restriction of phosphorylated SP8 DNA (SP8- p ). Bottom panel, control: restriction of non- p phage SP8 DNA. The phosphorylated DNA appeared largely resistant to restrictions by Nco I (CCA TG G), Alw NI (CAGN 3 C TG ), Hpy CH4V (ACG Tn ), Nde I (CATA TG ), Bbv CI (CC TC AGC), Bcc I (CTA TC ), Msl I (CAYN 4 R TG ), Nla III (CA TG ), Pvu II (CAGC TG ), Pml I (CACG TG ), Nsp I (RCA TG Y), and Nsi I (A TG CA Tn ) digestion; and partially resistant to Eco RV ( nG ATATCn), Mly I ( nG AGTCn), Mbo I ( nG ATCn), Hpy 188I ( TCNG A), Sap I ( nG C TC T TC ) digestion. Phosphorylation of 5hmdU had no inhibitory effect on Aat II ( nG ACG TC ) and Hin fI ( nG AN TC ) digestions. The TG, TC, nG, and Tn dinucleotides in the restriction sites are underlined.
    Figure Legend Snippet: Restriction digestion of SP8 gDNA following treatment with 5hmdU DNA kinase. Top panel, restriction of phosphorylated SP8 DNA (SP8- p ). Bottom panel, control: restriction of non- p phage SP8 DNA. The phosphorylated DNA appeared largely resistant to restrictions by Nco I (CCA TG G), Alw NI (CAGN 3 C TG ), Hpy CH4V (ACG Tn ), Nde I (CATA TG ), Bbv CI (CC TC AGC), Bcc I (CTA TC ), Msl I (CAYN 4 R TG ), Nla III (CA TG ), Pvu II (CAGC TG ), Pml I (CACG TG ), Nsp I (RCA TG Y), and Nsi I (A TG CA Tn ) digestion; and partially resistant to Eco RV ( nG ATATCn), Mly I ( nG AGTCn), Mbo I ( nG ATCn), Hpy 188I ( TCNG A), Sap I ( nG C TC T TC ) digestion. Phosphorylation of 5hmdU had no inhibitory effect on Aat II ( nG ACG TC ) and Hin fI ( nG AN TC ) digestions. The TG, TC, nG, and Tn dinucleotides in the restriction sites are underlined.

    Techniques Used:

    20) Product Images from "A low-bias and sensitive small RNA library preparation method using randomized splint ligation"

    Article Title: A low-bias and sensitive small RNA library preparation method using randomized splint ligation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa480

    Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.
    Figure Legend Snippet: Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.

    Techniques Used: Ligation, Synthesized, Polymerase Chain Reaction

    21) Product Images from "Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump"

    Article Title: Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0243082

    Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value
    Figure Legend Snippet: Effect of chlorhexidine and resveratrol alone or in combination on expression of efflux pump and efflux pump regulator genes in A . baumannii strain L14. RT-qPCR assay of adeB (a), adeR (b), adeS (c), adeJ (d), adeG (e), abeS (f), and aceI (g) expression in the presence of MHB control, DMSO control, either chlorhexidine (8 mg/L) or resveratrol (128 mg/L) and in the combination of chlorhexidine with resveratrol. Relative number of transcripts of each gene was normalized to 16S rRNA expression in each condition and calculated using the 2 -ΔΔct method compared to the expression level in MHB control. The relative number of transcripts of each gene was plotted with error bars representing standard deviations. All experiments were performed in triplicate. p- values were calculated using ANOVA (#, p -value

    Techniques Used: Expressing, Quantitative RT-PCR

    22) Product Images from "Staphylococcus aureus Cas9 is a multiple-turnover enzyme"

    Article Title: Staphylococcus aureus Cas9 is a multiple-turnover enzyme

    Journal: RNA

    doi: 10.1261/rna.067355.118

    S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA
    Figure Legend Snippet: S. pyogenes Cas9 binds sgRNA with a higher affinity than SauCas9 and both form active, sgRNA-dependent complexes with comparable K 1/2 for sgRNA

    Techniques Used:

    23) 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

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

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

    Journal: 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

    25) 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

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

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

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2020.00400

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

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

    27) 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

    28) Product Images from "Efficient Genome Editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 System for Analyzing Magnetotactic Behavior"

    Article Title: Efficient Genome Editing of Magnetospirillum magneticum AMB-1 by CRISPR-Cas9 System for Analyzing Magnetotactic Behavior

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01569

    CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.
    Figure Legend Snippet: CRISPR-Cas9-assisted genome editing in M. magneticum AMB-1 cells. (A) Strategy for deletion of the amb0994 gene by CRISPR-Cas9 assisted HDR in M. magneticum AMB-1 cells. An sgRNA transcripts guide Cas9 nuclease to introduce DSBs at ends of amb0994 gene, while a codelivered editing template repairs the gap via HR. Kan is kanamycin. Gm is gentamycin. (B) Schematic of RNA-guided Cas9 nuclease uses for editing of the AMB-1 amb0994 . An sgRNA consisting of 20 nt sequence (black bar) guide the Cas9 nuclease (orange) to target and cleavage the genomic DNA. Cleavage sites are indicated by red arrows for ~3 bp upstream of PAM. (C,D) PCR evaluation of amb0994 deletion from five colonies (1–5) with WT control. (E) Six fragments within MAI were amplified to evaluate the maintenance of genomic MAI during deletion.

    Techniques Used: CRISPR, Introduce, Sequencing, Polymerase Chain Reaction, Amplification

    29) Product Images from "Ablation of Mature miR-183 Leads to Retinal Dysfunction in Mice"

    Article Title: Ablation of Mature miR-183 Leads to Retinal Dysfunction in Mice

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.61.3.12

    Complete deletion of miR-183 by CRISPR/Cas9-mediated genome engineering technology in the mouse germ line. ( A ) Location of mouse miR-183 in the genomic organization and map of the Cas9 protein bound to the single guide RNAs. The Cas9 nuclease from S pyogenes (in aquamarine ) targets the 22-nucleotide sequences in miR-183 ( red ) by a single guide RNA consisting of a 22-nt guide sequence ( red ) and a scaffold ( purple ). The guide sequence pairs with the DNA target ( red bar on the top strand ), directly upstream of a requisite 5′-NGG adjacent motif (PAM; pink ). tel, telomeric; cent, centromeric. ( B ) Genotyping strategy for the WT mice and mutants (miR-183 −/− ) by DNA sequencing. The sequencing samples of the pair-wise sequence comparison between WT, heterozygous (miR-183 +/− ), and miR-183 −/- mice are shown. WT DNA was used as a negative control for sequencing in parallel. Blue shade, the seed sequence of miR-183. Red dotted line , the 53-bp deletion including the miR-183 seed sequence of miR-183 −/− mice. ( C ) qRT-PCR analysis ( n = 3) of miR-183 expression at P30 in the retinas of the WT, homozygous (miR-183 −/− ), and heterozygous (miR-183 +/− ) mice, respectively. Relative expression levels of microRNAs were normalized to the level of U6. * P
    Figure Legend Snippet: Complete deletion of miR-183 by CRISPR/Cas9-mediated genome engineering technology in the mouse germ line. ( A ) Location of mouse miR-183 in the genomic organization and map of the Cas9 protein bound to the single guide RNAs. The Cas9 nuclease from S pyogenes (in aquamarine ) targets the 22-nucleotide sequences in miR-183 ( red ) by a single guide RNA consisting of a 22-nt guide sequence ( red ) and a scaffold ( purple ). The guide sequence pairs with the DNA target ( red bar on the top strand ), directly upstream of a requisite 5′-NGG adjacent motif (PAM; pink ). tel, telomeric; cent, centromeric. ( B ) Genotyping strategy for the WT mice and mutants (miR-183 −/− ) by DNA sequencing. The sequencing samples of the pair-wise sequence comparison between WT, heterozygous (miR-183 +/− ), and miR-183 −/- mice are shown. WT DNA was used as a negative control for sequencing in parallel. Blue shade, the seed sequence of miR-183. Red dotted line , the 53-bp deletion including the miR-183 seed sequence of miR-183 −/− mice. ( C ) qRT-PCR analysis ( n = 3) of miR-183 expression at P30 in the retinas of the WT, homozygous (miR-183 −/− ), and heterozygous (miR-183 +/− ) mice, respectively. Relative expression levels of microRNAs were normalized to the level of U6. * P

    Techniques Used: CRISPR, Sequencing, Mouse Assay, DNA Sequencing, Negative Control, Quantitative RT-PCR, Expressing

    30) Product Images from "Reduced Stability and pH-Dependent Activity of a Common Obesity-Linked PCSK1 Polymorphism, N221D"

    Article Title: Reduced Stability and pH-Dependent Activity of a Common Obesity-Linked PCSK1 Polymorphism, N221D

    Journal: Endocrinology

    doi: 10.1210/en.2019-00418

    PC1/3 N221D mice do not show increased body weight or alterations in fat, lean, and fluid mass. (a) DNA sequencing chromatograms showing the successful A661G nucleotide substitution. DNA from a WT mouse (left panel) has a single A peak at position 661, whereas a heterozygous mouse (center panel) has a peak for both the A and G alleles, and a homozygous mouse (right panel) has a single G peak. The position of the edited residue is underlined in the sequence and marked by an arrow in the sequence chromatogram. (b) Body weight. There was no significant change in body weight between WT and N221D mutant mice during 16 wk. Data are presented as the mean ± SD (n = 7 to 13). Means are shown offset by 5% from each other on the x -axis to provide visual clarity. (c) Total fat mass and (d) lean mass. (e) Fluid mass, as analyzed by NMR. (f–h) Body mass measurements shown as a percentage of total body mass. There were no significant differences between genotypes for any of the parameters. Bar graphs represent the average of 7 to 13 different mice ± SD. Individual mouse values are represented by symbols; data were analyzed using a two-way ANOVA with a Tukey posttest.
    Figure Legend Snippet: PC1/3 N221D mice do not show increased body weight or alterations in fat, lean, and fluid mass. (a) DNA sequencing chromatograms showing the successful A661G nucleotide substitution. DNA from a WT mouse (left panel) has a single A peak at position 661, whereas a heterozygous mouse (center panel) has a peak for both the A and G alleles, and a homozygous mouse (right panel) has a single G peak. The position of the edited residue is underlined in the sequence and marked by an arrow in the sequence chromatogram. (b) Body weight. There was no significant change in body weight between WT and N221D mutant mice during 16 wk. Data are presented as the mean ± SD (n = 7 to 13). Means are shown offset by 5% from each other on the x -axis to provide visual clarity. (c) Total fat mass and (d) lean mass. (e) Fluid mass, as analyzed by NMR. (f–h) Body mass measurements shown as a percentage of total body mass. There were no significant differences between genotypes for any of the parameters. Bar graphs represent the average of 7 to 13 different mice ± SD. Individual mouse values are represented by symbols; data were analyzed using a two-way ANOVA with a Tukey posttest.

    Techniques Used: Mouse Assay, DNA Sequencing, Sequencing, Mutagenesis, Nuclear Magnetic Resonance

    31) Product Images from "A novel allele of ASY3 is associated with greater meiotic stability in autotetraploid Arabidopsis lyrata"

    Article Title: A novel allele of ASY3 is associated with greater meiotic stability in autotetraploid Arabidopsis lyrata

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008900

    Structural variants of the three major A . lyrata ASY3 alleles. (A) The serine-rich region (red box) with serines highlighted in bold in the non-duplicated (ND) ancestral diploid allele, absence in the deletion (DEL) diploid allele and tandemly duplicated (TD) in the derived tetraploid allele possessing two serine-rich regions (red boxes) as well as putative SUMO sites at K517, K531 and K556. (B) Major structural variation at the ASY3 locus suggesting that misalignment by DNA microhomology (highlighted yellow) and recombinational repair between the ancestral ND alleles may have led to the formation of the TD allele (blue = region of duplication). The outcome does not quite match the prediction as there are bases of unknown origin in the DEL allele (shaded in grey), but formation of these alleles may be independent events. The x’s highlighted in blue represent bases between the AGAGA sites as shown for A . lyrata 2n ND .
    Figure Legend Snippet: Structural variants of the three major A . lyrata ASY3 alleles. (A) The serine-rich region (red box) with serines highlighted in bold in the non-duplicated (ND) ancestral diploid allele, absence in the deletion (DEL) diploid allele and tandemly duplicated (TD) in the derived tetraploid allele possessing two serine-rich regions (red boxes) as well as putative SUMO sites at K517, K531 and K556. (B) Major structural variation at the ASY3 locus suggesting that misalignment by DNA microhomology (highlighted yellow) and recombinational repair between the ancestral ND alleles may have led to the formation of the TD allele (blue = region of duplication). The outcome does not quite match the prediction as there are bases of unknown origin in the DEL allele (shaded in grey), but formation of these alleles may be independent events. The x’s highlighted in blue represent bases between the AGAGA sites as shown for A . lyrata 2n ND .

    Techniques Used: Derivative Assay

    32) Product Images from "Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design"

    Article Title: Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0238592

    Golden Gate assembly assay schematic. (A) Hairpin DNA substrates containing a Type IIS recognition sequence (orange), randomized nucleotides at the Type IIS restriction site (NNNN), an internal 6-base random barcode (black), and a PacBio SMRTbell adapter sequence (blue) were synthesized. Golden Gate assembly of these substrates was carried out with T4 DNA ligase and a Type IIS restriction enzyme to produce circular assembly products. The assembly products were sequenced utilizing the PacBio Single-Molecule Real-Time sequencing platform. (B) For each sequenced assembly product, the overhang pair identity was extracted. The relative frequency of each overhang pair was determined and was represented as a frequency heat map (log-scaled). Overhangs are listed alphabetically left to right (AAAA, AAAC…TTTG, TTTT) and bottom to top such that the Watson–Crick pairings are shown on the diagonal represented above.
    Figure Legend Snippet: Golden Gate assembly assay schematic. (A) Hairpin DNA substrates containing a Type IIS recognition sequence (orange), randomized nucleotides at the Type IIS restriction site (NNNN), an internal 6-base random barcode (black), and a PacBio SMRTbell adapter sequence (blue) were synthesized. Golden Gate assembly of these substrates was carried out with T4 DNA ligase and a Type IIS restriction enzyme to produce circular assembly products. The assembly products were sequenced utilizing the PacBio Single-Molecule Real-Time sequencing platform. (B) For each sequenced assembly product, the overhang pair identity was extracted. The relative frequency of each overhang pair was determined and was represented as a frequency heat map (log-scaled). Overhangs are listed alphabetically left to right (AAAA, AAAC…TTTG, TTTT) and bottom to top such that the Watson–Crick pairings are shown on the diagonal represented above.

    Techniques Used: Sequencing, Synthesized

    Assembly bias with T4 DNA ligase and Type IIS restriction enzymes generating four-base overhangs. (A) The normalized overhang ligation frequencies for all 120 non-palindromic Watson-Crick pairs were plotted for DNA assembly reactions containing T4 DNA ligase and the indicated Type IIS restriction enzyme. (B-C) The most and least frequently observed overhang pairs and their relative frequency per 100,000 ligation events are shown. The overhangs are written in a 5′ to 3′ orientation. The overhang pairs are color-coded according to their frequency relative to the average in terms of the number of standard deviations.
    Figure Legend Snippet: Assembly bias with T4 DNA ligase and Type IIS restriction enzymes generating four-base overhangs. (A) The normalized overhang ligation frequencies for all 120 non-palindromic Watson-Crick pairs were plotted for DNA assembly reactions containing T4 DNA ligase and the indicated Type IIS restriction enzyme. (B-C) The most and least frequently observed overhang pairs and their relative frequency per 100,000 ligation events are shown. The overhangs are written in a 5′ to 3′ orientation. The overhang pairs are color-coded according to their frequency relative to the average in terms of the number of standard deviations.

    Techniques Used: Ligation

    High capacity Golden Gate assembly with T4 DNA ligase and SapI. (A) Schematic of the 13-fragment lac operon cassette test system. (B) Results of the assembly reactions. Four replicate experiments were carried out to quantify the number of colony-forming units harboring correct and incorrect assembly products per μL of E . coli outgrowth plated (0.002 μL of the assembly reaction). On average, 91% of the observed transformants harbored correctly assembled products. (C) Representative agar plate with blue and white colonies. Blue transformants harbor correct assembly constructs, and white transformants harbor inaccurate assembly products.
    Figure Legend Snippet: High capacity Golden Gate assembly with T4 DNA ligase and SapI. (A) Schematic of the 13-fragment lac operon cassette test system. (B) Results of the assembly reactions. Four replicate experiments were carried out to quantify the number of colony-forming units harboring correct and incorrect assembly products per μL of E . coli outgrowth plated (0.002 μL of the assembly reaction). On average, 91% of the observed transformants harbored correctly assembled products. (C) Representative agar plate with blue and white colonies. Blue transformants harbor correct assembly constructs, and white transformants harbor inaccurate assembly products.

    Techniques Used: Construct

    Nucleotide mismatches in assembly reactions with T4 DNA ligase and Type IIS restriction enzymes generating four-base overhangs. Mismatch frequencies for assembly reactions with T4 DNA ligase and BsaI-HFv2 (blue), BsmBI-v2 (orange), Esp3I (gray), or BbsI-HF (yellow) were grouped according to nucleotide mispair (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). The error bars depict the range between the maximum and minimum observed mismatch frequencies for two experimental replicates.
    Figure Legend Snippet: Nucleotide mismatches in assembly reactions with T4 DNA ligase and Type IIS restriction enzymes generating four-base overhangs. Mismatch frequencies for assembly reactions with T4 DNA ligase and BsaI-HFv2 (blue), BsmBI-v2 (orange), Esp3I (gray), or BbsI-HF (yellow) were grouped according to nucleotide mispair (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). The error bars depict the range between the maximum and minimum observed mismatch frequencies for two experimental replicates.

    Techniques Used:

    High capacity Golden Gate assembly with T4 DNA ligase and BsmBI-v2. (A) Schematic of the 35-fragment lac operon cassette test system (B) Results of the assembly reactions. Four replicate experiments were carried out to quantify the number of colony-forming units harboring correct and incorrect assembly products per 50 μL of E . coli outgrowth plated (0.1 μL of the assembly reaction). On average, 71% of the observed transformants harbored correctly assembled products. (C) Representative agar plate with blue and white colonies. Blue transformants harbor correct assembly constructs, and white transformants harbor inaccurate assembly products.
    Figure Legend Snippet: High capacity Golden Gate assembly with T4 DNA ligase and BsmBI-v2. (A) Schematic of the 35-fragment lac operon cassette test system (B) Results of the assembly reactions. Four replicate experiments were carried out to quantify the number of colony-forming units harboring correct and incorrect assembly products per 50 μL of E . coli outgrowth plated (0.1 μL of the assembly reaction). On average, 71% of the observed transformants harbored correctly assembled products. (C) Representative agar plate with blue and white colonies. Blue transformants harbor correct assembly constructs, and white transformants harbor inaccurate assembly products.

    Techniques Used: Construct

    Golden Gate assembly fidelity predictions as a function of the overhang pairs in the assembly reaction. (A) The GetSet tool was used to estimate the fidelity of assembly reactions containing up to 30 overhang pairs with T4 DNA ligase and SapI. (B) GetSet was used to estimate assembly fidelity for overhangs sets with up to 40 overhang pairs in an assembly reaction with T4 DNA ligase and BsmBI-v2. Overhang pairs were selected using Data-optimized Assembly Design (DAD; blue), traditional rules for overhang selection by hand (gray), or by random overhang selection of non-palindromic overhang pairs (orange). The error bars indicate estimated fidelity scores based on replicate data analysis (see S1 Text for details).
    Figure Legend Snippet: Golden Gate assembly fidelity predictions as a function of the overhang pairs in the assembly reaction. (A) The GetSet tool was used to estimate the fidelity of assembly reactions containing up to 30 overhang pairs with T4 DNA ligase and SapI. (B) GetSet was used to estimate assembly fidelity for overhangs sets with up to 40 overhang pairs in an assembly reaction with T4 DNA ligase and BsmBI-v2. Overhang pairs were selected using Data-optimized Assembly Design (DAD; blue), traditional rules for overhang selection by hand (gray), or by random overhang selection of non-palindromic overhang pairs (orange). The error bars indicate estimated fidelity scores based on replicate data analysis (see S1 Text for details).

    Techniques Used: Selection

    33) Product Images from "DNA mismatch repair controls the host innate response and cell fate after influenza virus infection"

    Article Title: DNA mismatch repair controls the host innate response and cell fate after influenza virus infection

    Journal: Nature microbiology

    doi: 10.1038/s41564-019-0509-3

    Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.
    Figure Legend Snippet: Loss of DNA MMR activity reduces the innate antiviral transcriptional response against influenza A virus. (a) NanoLuc reporter expression and (b) relative cell viability in H441 cells that have been treated with PBS or H2O2 (for 30 min). Data shown as mean ± SD, n=4 independent samples. (c) Fold change of Mx1 RNA levels in H441 cells following treatment with PBS or IFN-alpha +/− H2O2 treatment (for 30 min). Data shown as mean ± SD, n=4 independent samples. (d) Western blot for Mx1 in H441 cells following the specified treatments. Tubulin = loading control. (e) NanoLuc reporter expression and (f) relative cell viability in H441 cells following the specified treatments. Data shown as mean ± SD, n=4 independent samples. (g) Median fluorescent intensity of the ISRE-GFP reporter in 293T cells following the specified treatments. Data shown as mean ± SD, n=3 independent samples. (h) Model depicting the role of DNA MMR in preserving antiviral gene expression. (i) RNAseq data showing fold change of mRNA levels in H441 cells comparing PR8-infected cells transfected with non-targeting siRNA (black) or MSH2+MSH6 siRNA (blue) to mock-infected cells. Inset is a magnified view of all genes induced > 5-fold in PR8-infected cells treated with non-targeting siRNA. (j) Chart grouping all of the genes induced > 5-fold in PR8-infected cells based on the effect MMR knockdown has on their mRNA levels. (k) Heat map displaying the effect of MMR knockdown on ISG and antiviral genes from the group of genes displayed in j. (l-o) Fold induction of (l) IFI44L and (n) IFIT1 RNA levels after viral infection as well as the difference in infection-induced (m) IFI44L and (o) IFIT1 RNA levels (48 hpi) after knockdown of control or MMR genes. Data shown as mean ± SD, n=4 independent samples. Data are representative of at least three independent experiments. (p) Western blot of IFIT1 in H441 cells following the specified treatments. Tubulin = loading control. For all panels: p-values calculated using unpaired two-tailed t tests; representative of two independent experiments, unless otherwise indicated.

    Techniques Used: Activity Assay, Expressing, Western Blot, Preserving, Infection, Transfection, Two Tailed Test

    34) Product Images from "Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design"

    Article Title: Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0238592

    Nucleotide mismatches in assembly reactions with T4 DNA ligase and Type IIS restriction enzymes generating four-base overhangs. Mismatch frequencies for assembly reactions with T4 DNA ligase and BsaI-HFv2 (blue), BsmBI-v2 (orange), Esp3I (gray), or BbsI-HF (yellow) were grouped according to nucleotide mispair (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). The error bars depict the range between the maximum and minimum observed mismatch frequencies for two experimental replicates.
    Figure Legend Snippet: Nucleotide mismatches in assembly reactions with T4 DNA ligase and Type IIS restriction enzymes generating four-base overhangs. Mismatch frequencies for assembly reactions with T4 DNA ligase and BsaI-HFv2 (blue), BsmBI-v2 (orange), Esp3I (gray), or BbsI-HF (yellow) were grouped according to nucleotide mispair (A:A, A:C, A:G, C:C, C:T, G:G, G:T, T:T). The error bars depict the range between the maximum and minimum observed mismatch frequencies for two experimental replicates.

    Techniques Used:

    35) 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

    36) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    37) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    38) Product Images from "The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation"

    Article Title: The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation

    Journal: bioRxiv

    doi: 10.1101/2021.02.14.429938

    Diverse Transcription-Regulatory Control of Stag1 in ES cells. (A, B) 5’ Rapid Amplification of cDNA ends (RACE) for SA1 in ES and EpiLC cells. Arrows indicate bands which were cloned and sequenced. In A, red star indicates SATS TSS and red arrow indicates canonical TSS. In B: red indicates full length Stag1 with both SATS and can TSSs; dark blue indicates alternatively spliced variants, skipping of various exons in 5’ region; light blue indicates the TSSs at e6, e7. (C) 3’ RACE for SA1 in ES cells. Arrows indicate bands which were cloned and sequenced. Red indicates canonical full-length end; green indicates end in i25. (D) Top, schematic of the Stag1 gene annotation in mm10 and the identified TSS and TTSs from RACE indicated. Bottom, aligned sequence clones from the PCR mini-screen and their predicted impact on the SA1 protein, right. Green arrows and red bars within the transcripts indicate start of the coding sequence and the TTS respectively. Shown also are the regions which code for the AT hook and SCD domains. (E) Percent Spliced In (PSI) calculations based on VAST-Tools analysis of RNA-seq from multiple 2i (blue) and FCS (red) datasets (see Methods for details of libraries). Data are shown relative to Neural Stem cell (NSC) frequencies to highlight the events that are ES-specific. (F) Genome topology at the Stag1 locus. Hi-C contact maps in ES (2i) and NS cells of the 900kb region on chromosome 9 containing the Stag1 topologically associated domain (TAD). TADs are denoted with a vertical line and as repressed (orange) or active (blue). Shown also are tracks for Genes, Nanog and CTCF ChIP-seq as well as a track indicating the directionality of CTCF binding sites (red, forward; blue, reverse). Aligned to the Gene track are also the SA1 transcripts discovered above where red represents the untranslated regions and blue the coding body. UMI-4C-seq viewpoints (asterisks on the ChIP tracks) are positioned to the leftmost CTCF site (‘CTCF bait’) and to the Nanog site 40 kb upstream of the Stag1 canonical TSS (‘Nanog bait’). For each bait, UMI information for each cell type is shown as well as the comparative plots where red represents an enrichment of contacts in ES compared to NS. (G) Top, cartoon depicting functional domains within Stag1 protein, including the AT-hook (aa 3-58); Stromalin conserved domain (SCD, aa 296-381) and the C-terminus. Bottom, the predicted Stag1 protein isoforms based on transcript analysis with estimated sizes for each isoform. Purple boxes in the 105kDa and 90kDa isoforms represent retained introns. (H) PONDR tracks as before shown for the N-terminal truncated (top) and C-terminal truncated (bottom) transcripts. (I) Chromatin immunoprecipitation of SA1 from ES cells. (J) WB analysis of SA1 isoforms in chromatin fractions from ES cells treated with siscr and siSA1. H3 serves as a fraction and loading control. (K) Chromatin immunoprecipitation for the v5 tag in SA1 NG-FKBP ES cells treated with DMSO-only or dTAG. Note, SA1 bands now run 42kDa higher due to the addition of the tag. See also Figure S3, Table S1 and Table S2.
    Figure Legend Snippet: Diverse Transcription-Regulatory Control of Stag1 in ES cells. (A, B) 5’ Rapid Amplification of cDNA ends (RACE) for SA1 in ES and EpiLC cells. Arrows indicate bands which were cloned and sequenced. In A, red star indicates SATS TSS and red arrow indicates canonical TSS. In B: red indicates full length Stag1 with both SATS and can TSSs; dark blue indicates alternatively spliced variants, skipping of various exons in 5’ region; light blue indicates the TSSs at e6, e7. (C) 3’ RACE for SA1 in ES cells. Arrows indicate bands which were cloned and sequenced. Red indicates canonical full-length end; green indicates end in i25. (D) Top, schematic of the Stag1 gene annotation in mm10 and the identified TSS and TTSs from RACE indicated. Bottom, aligned sequence clones from the PCR mini-screen and their predicted impact on the SA1 protein, right. Green arrows and red bars within the transcripts indicate start of the coding sequence and the TTS respectively. Shown also are the regions which code for the AT hook and SCD domains. (E) Percent Spliced In (PSI) calculations based on VAST-Tools analysis of RNA-seq from multiple 2i (blue) and FCS (red) datasets (see Methods for details of libraries). Data are shown relative to Neural Stem cell (NSC) frequencies to highlight the events that are ES-specific. (F) Genome topology at the Stag1 locus. Hi-C contact maps in ES (2i) and NS cells of the 900kb region on chromosome 9 containing the Stag1 topologically associated domain (TAD). TADs are denoted with a vertical line and as repressed (orange) or active (blue). Shown also are tracks for Genes, Nanog and CTCF ChIP-seq as well as a track indicating the directionality of CTCF binding sites (red, forward; blue, reverse). Aligned to the Gene track are also the SA1 transcripts discovered above where red represents the untranslated regions and blue the coding body. UMI-4C-seq viewpoints (asterisks on the ChIP tracks) are positioned to the leftmost CTCF site (‘CTCF bait’) and to the Nanog site 40 kb upstream of the Stag1 canonical TSS (‘Nanog bait’). For each bait, UMI information for each cell type is shown as well as the comparative plots where red represents an enrichment of contacts in ES compared to NS. (G) Top, cartoon depicting functional domains within Stag1 protein, including the AT-hook (aa 3-58); Stromalin conserved domain (SCD, aa 296-381) and the C-terminus. Bottom, the predicted Stag1 protein isoforms based on transcript analysis with estimated sizes for each isoform. Purple boxes in the 105kDa and 90kDa isoforms represent retained introns. (H) PONDR tracks as before shown for the N-terminal truncated (top) and C-terminal truncated (bottom) transcripts. (I) Chromatin immunoprecipitation of SA1 from ES cells. (J) WB analysis of SA1 isoforms in chromatin fractions from ES cells treated with siscr and siSA1. H3 serves as a fraction and loading control. (K) Chromatin immunoprecipitation for the v5 tag in SA1 NG-FKBP ES cells treated with DMSO-only or dTAG. Note, SA1 bands now run 42kDa higher due to the addition of the tag. See also Figure S3, Table S1 and Table S2.

    Techniques Used: Rapid Amplification of cDNA Ends, Clone Assay, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Hi-C, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, Western Blot

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

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

    Journal: Microbial Cell

    doi: 10.15698/mic2018.09.645

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

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

    40) Product Images from "Proteogenomic Identification of a Novel Protein-Encoding Gene in Bovine Herpesvirus 1 That Is Expressed during Productive Infection"

    Article Title: Proteogenomic Identification of a Novel Protein-Encoding Gene in Bovine Herpesvirus 1 That Is Expressed during Productive Infection

    Journal: Viruses

    doi: 10.3390/v10090499

    Primer walking for elucidation of ORF-A 3′ terminus. Strand-specific RT-PCR of BoHV-1.1-infected cell mRNA extracts was was performed on cDNA produced from BoHV-1 infected cells using 16 different reverse primers that anneal further down (3′) to the viral genome. Successful amplification allowed for capture of increasing lengths of the ORF-A transcript sequence. RT indicates the presence (+) or absence (-) of a retrotranscriptase step with mRNA as a template. ( a ) Amplicons were produced with primers 1–8 using a 1-min extension time during PCR. ( b ) Amplicons were produced using primers 9-16 with a 2-min extension time to account for the increase in amplicon length.
    Figure Legend Snippet: Primer walking for elucidation of ORF-A 3′ terminus. Strand-specific RT-PCR of BoHV-1.1-infected cell mRNA extracts was was performed on cDNA produced from BoHV-1 infected cells using 16 different reverse primers that anneal further down (3′) to the viral genome. Successful amplification allowed for capture of increasing lengths of the ORF-A transcript sequence. RT indicates the presence (+) or absence (-) of a retrotranscriptase step with mRNA as a template. ( a ) Amplicons were produced with primers 1–8 using a 1-min extension time during PCR. ( b ) Amplicons were produced using primers 9-16 with a 2-min extension time to account for the increase in amplicon length.

    Techniques Used: Chromosome Walking, Reverse Transcription Polymerase Chain Reaction, Infection, Produced, Amplification, Sequencing, Polymerase Chain Reaction

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  • 99
    New England Biolabs monarch plasmid miniprep kit
    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin <t>miniprep</t> kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.
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    New England Biolabs monarch pcr
    Principles of Darwin Assembly. Plasmid <t>DNA</t> (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 <t>PCR</t> 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).
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    New England Biolabs monarch gdna elution buffer
    A Cell-based System to Study the Effect of DSBs on the Epigenome, Related to Figure 1 (A) RNA-seq volcano plots (Cre vs. ICE) of cells from the same or different litters before 4-OHT treatment. (B) Slot blots to compare global 5mC levels. Methylene blue (MB) staining showed total genomic <t>DNA</t> used in each sample. (C) Genomic distribution of I- Ppo I canonical sites and all 90 CpG sites of the mouse epigenetic clock. (D) Percent non-mutated sequences of ∼100,000 random sites in post-treated ICE cells assessed by deep sequencing ( > 50x). (E and F) Immunostaining of DNA damage markers γH2AX and 53BP1 in post-treated ICE cells with and without exposure to the DNA damaging agents (ETS, etoposide; CPT, camptothecin; H 2 O 2 , hydrogen peroxide). Two-tailed Student’s t test. Scale bar, 10 µm. (G) Immunostaining of Lamin B1 in post-treated ICE cells. Data are mean (n=3) ± SD. *p
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    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Journal: PLoS ONE

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    doi: 10.1371/journal.pone.0203011

    Figure Lengend Snippet: The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Article Snippet: Alternatively, spin columns with a conical (V-shape) bottom and a drip opening, such as a miniprep column from Qiagen , and a recent version adopted in NEB Monarch plasmid miniprep kit, can be recharged by reloading filter paper discs with a diameter of 5/16 inch (~8 mm) ( ).

    Techniques: Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Gel Extraction, Plasmid Preparation

    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).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky067

    Figure Lengend 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).

    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).

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky067

    Figure Lengend 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).

    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).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gky067

    Figure Lengend 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.

    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).

    Techniques: Sequencing, Polymerase Chain Reaction

    A Cell-based System to Study the Effect of DSBs on the Epigenome, Related to Figure 1 (A) RNA-seq volcano plots (Cre vs. ICE) of cells from the same or different litters before 4-OHT treatment. (B) Slot blots to compare global 5mC levels. Methylene blue (MB) staining showed total genomic DNA used in each sample. (C) Genomic distribution of I- Ppo I canonical sites and all 90 CpG sites of the mouse epigenetic clock. (D) Percent non-mutated sequences of ∼100,000 random sites in post-treated ICE cells assessed by deep sequencing ( > 50x). (E and F) Immunostaining of DNA damage markers γH2AX and 53BP1 in post-treated ICE cells with and without exposure to the DNA damaging agents (ETS, etoposide; CPT, camptothecin; H 2 O 2 , hydrogen peroxide). Two-tailed Student’s t test. Scale bar, 10 µm. (G) Immunostaining of Lamin B1 in post-treated ICE cells. Data are mean (n=3) ± SD. *p

    Journal: bioRxiv

    Article Title: Erosion of the Epigenetic Landscape and Loss of Cellular Identity as a Cause of Aging in Mammals

    doi: 10.1101/808642

    Figure Lengend Snippet: A Cell-based System to Study the Effect of DSBs on the Epigenome, Related to Figure 1 (A) RNA-seq volcano plots (Cre vs. ICE) of cells from the same or different litters before 4-OHT treatment. (B) Slot blots to compare global 5mC levels. Methylene blue (MB) staining showed total genomic DNA used in each sample. (C) Genomic distribution of I- Ppo I canonical sites and all 90 CpG sites of the mouse epigenetic clock. (D) Percent non-mutated sequences of ∼100,000 random sites in post-treated ICE cells assessed by deep sequencing ( > 50x). (E and F) Immunostaining of DNA damage markers γH2AX and 53BP1 in post-treated ICE cells with and without exposure to the DNA damaging agents (ETS, etoposide; CPT, camptothecin; H 2 O 2 , hydrogen peroxide). Two-tailed Student’s t test. Scale bar, 10 µm. (G) Immunostaining of Lamin B1 in post-treated ICE cells. Data are mean (n=3) ± SD. *p

    Article Snippet: ChIP DNA was eluted by incubating at 65°C overnight in elution buffer containing 0.25% SDS, 1 mM EDTA, 10 mM Tris-HCl pH7.5.

    Techniques: RNA Sequencing Assay, Staining, Sequencing, Immunostaining, Two Tailed Test