hdgfrp2 cryptic exon  (New England Biolabs)


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    Monarch Plasmid Miniprep Kit
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    Monarch Plasmid Miniprep Kit 250 preps
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    Structured Review

    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
    Average 91 stars, based on 1739 article reviews
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    hdgfrp2 cryptic exon - by Bioz Stars, 2020-05
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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 "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

    3) 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:

    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 "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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) Product Images from "Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes"

    Article Title: Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00186

    UV–vis absorption and second derivative (D 2 ) spectra (Panel A) and RR spectra in the high frequency region (Panel B) of the coproheme complexes with WT and the Q187A, M149A/Q187A, and M149A mutants. The band wavelengths and wavenumbers assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively. The spectra have been shifted along the ordinate axis to allow better visualization. The 450–700 nm region of the spectra in Panel A is expanded 9-fold. Experimental conditions of the RR spectra: 406.7 nm excitation wavelength, laser power at the sample of 5 mW, average of 9 spectra with a 90 min integration time (WT), 14 spectra with a 140 min integration time (Q187A), 10 spectra with a 100 min integration time (M149A/Q187A), and 4 spectra with a 40 min integration time (M149A).
    Figure Legend Snippet: UV–vis absorption and second derivative (D 2 ) spectra (Panel A) and RR spectra in the high frequency region (Panel B) of the coproheme complexes with WT and the Q187A, M149A/Q187A, and M149A mutants. The band wavelengths and wavenumbers assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively. The spectra have been shifted along the ordinate axis to allow better visualization. The 450–700 nm region of the spectra in Panel A is expanded 9-fold. Experimental conditions of the RR spectra: 406.7 nm excitation wavelength, laser power at the sample of 5 mW, average of 9 spectra with a 90 min integration time (WT), 14 spectra with a 140 min integration time (Q187A), 10 spectra with a 100 min integration time (M149A/Q187A), and 4 spectra with a 40 min integration time (M149A).

    Techniques Used:

    UV–vis (panel A) and RR (panel B) spectra in the low (left) and high (right) frequency regions of the 12 CO adducts of the coproheme complexes of Mb, WT, M149A, M149A/Q187A, Q187A, and coproheme. The frequencies of the ν(FeC), δ(FeCO), and ν(CO) modes are indicated in red. The spectra have been shifted along the ordinate axis to allow better visualization. Panel A: the 480–700 nm region is expanded 10-fold. Panel B: experimental conditions: Mb and coproheme: λ exc 406.7 nm, laser power at the sample 5 mW, average of 4 spectra with 40 min integration time and 10 spectra with 100 min integration time in the low and high frequency regions, respectively (Mb), average of 6 spectra with 60 min integration time and 12 spectra with 120 min integration time in the low and high frequency regions, respectively (coproheme); WT and its mutants, λ exc 413.1 nm, laser power at the sample 1–3 mW; average of 28 spectra with 280 min integration time and 22 spectra with 220 min integration time in the low and high frequency regions, respectively (WT), average of 6 spectra with 60 min integration time and 18 spectra with 180 min integration time in the low and high frequency regions, respectively (M149A), average of 6 spectra with 60 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively (M149A/Q187A), and average of 9 spectra with 90 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively (Q187A).
    Figure Legend Snippet: UV–vis (panel A) and RR (panel B) spectra in the low (left) and high (right) frequency regions of the 12 CO adducts of the coproheme complexes of Mb, WT, M149A, M149A/Q187A, Q187A, and coproheme. The frequencies of the ν(FeC), δ(FeCO), and ν(CO) modes are indicated in red. The spectra have been shifted along the ordinate axis to allow better visualization. Panel A: the 480–700 nm region is expanded 10-fold. Panel B: experimental conditions: Mb and coproheme: λ exc 406.7 nm, laser power at the sample 5 mW, average of 4 spectra with 40 min integration time and 10 spectra with 100 min integration time in the low and high frequency regions, respectively (Mb), average of 6 spectra with 60 min integration time and 12 spectra with 120 min integration time in the low and high frequency regions, respectively (coproheme); WT and its mutants, λ exc 413.1 nm, laser power at the sample 1–3 mW; average of 28 spectra with 280 min integration time and 22 spectra with 220 min integration time in the low and high frequency regions, respectively (WT), average of 6 spectra with 60 min integration time and 18 spectra with 180 min integration time in the low and high frequency regions, respectively (M149A), average of 6 spectra with 60 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively (M149A/Q187A), and average of 9 spectra with 90 min integration time and 15 spectra with 150 min integration time in the low and high frequency regions, respectively (Q187A).

    Techniques Used:

    17) Product Images from "Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes"

    Article Title: Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00186

    UV–vis absorption and second derivative (D 2 ) spectra of coproheme and the coproheme complexes with LmChdC WT, its M149A mutant, and Mb. The band wavelengths assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively, (see text). The spectra have been shifted along the ordinate axis to allow better visualization. The 450–700 nm region of coproheme and the coproheme complexes spectra is expanded 20- and 9-fold, respectively. The excitation wavelengths used for the RR experiments are also shown in light violet (the 356.4 nm line) and in violet (the 406.7 nm line).
    Figure Legend Snippet: UV–vis absorption and second derivative (D 2 ) spectra of coproheme and the coproheme complexes with LmChdC WT, its M149A mutant, and Mb. The band wavelengths assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively, (see text). The spectra have been shifted along the ordinate axis to allow better visualization. The 450–700 nm region of coproheme and the coproheme complexes spectra is expanded 20- and 9-fold, respectively. The excitation wavelengths used for the RR experiments are also shown in light violet (the 356.4 nm line) and in violet (the 406.7 nm line).

    Techniques Used: Mutagenesis

    High frequency region RR spectra obtained at room temperature, with the 356.4 (Panel A) and 406.7 nm (Panel B) excitation wavelengths, of coproheme, and the coproheme complexes of LmChdC WT, its M149A mutant, and Mb. The band wavenumbers assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively (see text). The spectra have been shifted along the ordinate axis to allow better visualization. Experimental conditions: (A) laser power at the sample 5 mW, average of 10 spectra with 120 min integration time (Coproheme); laser power at the sample 2 mW; average of 7 spectra with 70 min integration time (WT), 8 spectra with 80 min integration time (M149A), and 24 spectra with 240 min integration time (Mb). (B) laser power at the sample 5 mW; average of 2 spectra with 10 min integration time with 1800 grating (Coproheme), 9 spectra with 90 min integration time (WT), 4 spectra with 40 min integration time (M149A), and 6 spectra with 60 min integration time (Mb).
    Figure Legend Snippet: High frequency region RR spectra obtained at room temperature, with the 356.4 (Panel A) and 406.7 nm (Panel B) excitation wavelengths, of coproheme, and the coproheme complexes of LmChdC WT, its M149A mutant, and Mb. The band wavenumbers assigned to 5cHS, 5cQS, 6cHS, and 6cLS species are indicated in orange, olive green, blue, and magenta, respectively (see text). The spectra have been shifted along the ordinate axis to allow better visualization. Experimental conditions: (A) laser power at the sample 5 mW, average of 10 spectra with 120 min integration time (Coproheme); laser power at the sample 2 mW; average of 7 spectra with 70 min integration time (WT), 8 spectra with 80 min integration time (M149A), and 24 spectra with 240 min integration time (Mb). (B) laser power at the sample 5 mW; average of 2 spectra with 10 min integration time with 1800 grating (Coproheme), 9 spectra with 90 min integration time (WT), 4 spectra with 40 min integration time (M149A), and 6 spectra with 60 min integration time (Mb).

    Techniques Used: Mutagenesis

    Cross-linking of heme b to LmChdC mediated by excess hydrogen peroxide. Mass spectrometric analysis of the entire protein of LmChdC WT (apo-form 31 977.2 Da; cross-linked 32 590.5 Da, black) and LmChdC M149A (31 917.0 Da, red). The green line with its label shows the mass difference between apo-LmChd WT and holo-LmChdC WT. The Coproheme-LmChdC complexes were titrated with H 2 O 2 up to a 2-fold excess; subsequently, the mass spectroscopic measurements were performed on heme b -LmChdC WT and heme b -LmChdC M149A.
    Figure Legend Snippet: Cross-linking of heme b to LmChdC mediated by excess hydrogen peroxide. Mass spectrometric analysis of the entire protein of LmChdC WT (apo-form 31 977.2 Da; cross-linked 32 590.5 Da, black) and LmChdC M149A (31 917.0 Da, red). The green line with its label shows the mass difference between apo-LmChd WT and holo-LmChdC WT. The Coproheme-LmChdC complexes were titrated with H 2 O 2 up to a 2-fold excess; subsequently, the mass spectroscopic measurements were performed on heme b -LmChdC WT and heme b -LmChdC M149A.

    Techniques Used:

    Heme coordination for the coproheme complexes of LmChdC, its M149A mutant, and Mb determined by RR and EPR spectroscopy at room and low temperatures..
    Figure Legend Snippet: Heme coordination for the coproheme complexes of LmChdC, its M149A mutant, and Mb determined by RR and EPR spectroscopy at room and low temperatures..

    Techniques Used: Mutagenesis, Electron Paramagnetic Resonance, Spectroscopy

    18) 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

    19) Product Images from "Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies"

    Article Title: Genome Assembly and Annotation of the Trichoplusia ni Tni-FNL Insect Cell Line Enabled by Long-Read Technologies

    Journal: Genes

    doi: 10.3390/genes10020079

    Functional annotation of the Tni-FNL ( Trichoplusia ni ), B. mori and D. melanogaster results comparison. The circos plot describes the shared cellular components, molecular functions and biological processes among the three species.
    Figure Legend Snippet: Functional annotation of the Tni-FNL ( Trichoplusia ni ), B. mori and D. melanogaster results comparison. The circos plot describes the shared cellular components, molecular functions and biological processes among the three species.

    Techniques Used: Functional Assay

    20) Product Images from "Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food"

    Article Title: Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION – Promising Strategies for Multiplex Identification of Viable Pathogens in Food

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00514

    RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24, and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from five time points. (C) qPCR for 0–72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from five time points. (E) The melting curve analysis of RT-qPCR for 0–72 h RNA.
    Figure Legend Snippet: RT-qPCR and qPCR of E. coli O157:H7 from 0, 4, 8, 24, and 72 h growth in BHI. (A) The growth curve of E. coli O157:H7 at 0, 4, 8, 24, 72 h in BHI. The initial concentration was 3-log CFU/mL. (B) RT-qPCR for RNA collected from five time points. (C) qPCR for 0–72 h RNA as the negative control (NC) for DNA contamination – no DNA contamination was found in those samples. (D) qPCR for DNA collected from five time points. (E) The melting curve analysis of RT-qPCR for 0–72 h RNA.

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Concentration Assay, Negative Control

    Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.
    Figure Legend Snippet: Taxonomic and genus level bacterial classification of MinION R9.4 Rev D multiplex RT-PCR amplicon sequencing. (A) Taxonomy tree of BHI 334 4-h sample generated by EPI2ME. (B) Taxonomy tree of LJE 334 4-h sample generated by EPI2ME.

    Techniques Used: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Generated

    21) 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

    22) Product Images from "Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice"

    Article Title: Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23830-4

    Detection of genomic Bordetella DNA by PCR. ( a ) A PCR product of 318 bp was amplified with B. pseudohinzii -specific primers from DNA of faeces (upper panel) and larynx (lower panel) from infected (lanes 1–4) and non-infected (lanes 5–8) mice, as classified by MALDI-TOF MS. DNA isolated from cultured B. pseudohinzii (strain 3227) served as a positive control (lane 9). Control runs without template were negative (lane 10). M: 100 bp molecular weight marker. ( b ) Trachea and lung taken were taken from 6 animals which were positive for B. pseudohinzii .
    Figure Legend Snippet: Detection of genomic Bordetella DNA by PCR. ( a ) A PCR product of 318 bp was amplified with B. pseudohinzii -specific primers from DNA of faeces (upper panel) and larynx (lower panel) from infected (lanes 1–4) and non-infected (lanes 5–8) mice, as classified by MALDI-TOF MS. DNA isolated from cultured B. pseudohinzii (strain 3227) served as a positive control (lane 9). Control runs without template were negative (lane 10). M: 100 bp molecular weight marker. ( b ) Trachea and lung taken were taken from 6 animals which were positive for B. pseudohinzii .

    Techniques Used: Polymerase Chain Reaction, Amplification, Infection, Mouse Assay, Mass Spectrometry, Isolation, Cell Culture, Positive Control, Molecular Weight, Marker

    23) Product Images from "Selection of an Efficient AAV Vector for Robust CNS Transgene Expression"

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.10.007

    iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.
    Figure Legend Snippet: iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.

    Techniques Used: Selection, Expressing, Construct, Crocin Bleaching Assay, Clone Assay, Injection, Transgenic Assay, Functional Assay, Transduction, Isolation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, DNA Sequencing

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007533

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

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

    25) Product Images from "Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy"

    Article Title: Characterisation of resistance mechanisms developed by basal cell carcinoma cells in response to repeated cycles of Photodynamic Therapy

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41313-y

    Localisation and expression of E-cadherin. ( A ) Immunofluorescence images of E-cadherin expression in green and DNA staining (DAPI) in blue. A secondary antibody control (AlexaFluor 488) for P cells is included. In CSZ, polygonal population is indicated with an asterisk and spindled cells with and an arrow. Scale bar: 40 µm. ( B ) A representative experiment of E-cadherin expression by Western Blot and densitometry graphics corresponding to three independent experiments are showed (Mean ± SD). Load control: β-actin. Separate gels where used for cell line and tumour cells. ROD (Relative optic density). ( C ) mRNA levels resulting from RT-PCR analysis. Relative data to their corresponding P or P T population are presented in the graph. (* P ≤ 0.05; ** P ≤ 0.01).
    Figure Legend Snippet: Localisation and expression of E-cadherin. ( A ) Immunofluorescence images of E-cadherin expression in green and DNA staining (DAPI) in blue. A secondary antibody control (AlexaFluor 488) for P cells is included. In CSZ, polygonal population is indicated with an asterisk and spindled cells with and an arrow. Scale bar: 40 µm. ( B ) A representative experiment of E-cadherin expression by Western Blot and densitometry graphics corresponding to three independent experiments are showed (Mean ± SD). Load control: β-actin. Separate gels where used for cell line and tumour cells. ROD (Relative optic density). ( C ) mRNA levels resulting from RT-PCR analysis. Relative data to their corresponding P or P T population are presented in the graph. (* P ≤ 0.05; ** P ≤ 0.01).

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz851

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

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

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

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

    27) Product Images from "Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis"

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02487

    Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.
    Figure Legend Snippet: Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Sequencing, Incubation, Imaging, Plasmid Preparation

    Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.
    Figure Legend Snippet: Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.

    Techniques Used: Sequencing, Incubation, Staining, RNA Sequencing Assay

    Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.
    Figure Legend Snippet: Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.

    Techniques Used: Sequencing, Incubation, Staining

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

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

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

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

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

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

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

    29) 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

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

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

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

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

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

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

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

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

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

    Techniques Used: Sequencing, Polymerase Chain Reaction

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

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

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

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

    Techniques Used: Sequencing, Polymerase Chain Reaction

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

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

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky067

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

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

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

    Techniques Used: Sequencing, Polymerase Chain Reaction

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

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

    34) Product Images from "Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti"

    Article Title: Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2019.00266

    Distinct localization of LGR1 immunoreactivity in late-staged spermatids of adult A. aegypti testes. (A,C,E,G) Testes were stained with anti-LGR1 (green), (B,C) anti-gamma tubulin (red) as a marker for the centriole adjunct ( Dallai et al., 2016 ), (F,G) MitoTracker (red) to detect mitochondrial derivatives in flagella, and nuclei (blue). (C) LGR1 immunoreactivity co-localizes with gamma-tubulin at the centriole adjunct. (D) Transmission electron microscopy (TEM) image showing the centriole adjunct (arrow) is positioned between the nucleus (n) and flagella (f) of spermatids. (G) LGR1 immunoreactivity is not found associated with the mitochondrial derivatives in the flagella of late-staged spermatids, comprised of (H) two mitochondrial derivatives (m) and an axoneme (a). Early staged spermatids (est), late-staged spermatids (Lst), cyst cell nuclei ( ∗ ). Scale bars: 10 μm in panels A–C , 5 μm in panel D , 20 μm in panels E–G and 200 nm in panel H .
    Figure Legend Snippet: Distinct localization of LGR1 immunoreactivity in late-staged spermatids of adult A. aegypti testes. (A,C,E,G) Testes were stained with anti-LGR1 (green), (B,C) anti-gamma tubulin (red) as a marker for the centriole adjunct ( Dallai et al., 2016 ), (F,G) MitoTracker (red) to detect mitochondrial derivatives in flagella, and nuclei (blue). (C) LGR1 immunoreactivity co-localizes with gamma-tubulin at the centriole adjunct. (D) Transmission electron microscopy (TEM) image showing the centriole adjunct (arrow) is positioned between the nucleus (n) and flagella (f) of spermatids. (G) LGR1 immunoreactivity is not found associated with the mitochondrial derivatives in the flagella of late-staged spermatids, comprised of (H) two mitochondrial derivatives (m) and an axoneme (a). Early staged spermatids (est), late-staged spermatids (Lst), cyst cell nuclei ( ∗ ). Scale bars: 10 μm in panels A–C , 5 μm in panel D , 20 μm in panels E–G and 200 nm in panel H .

    Techniques Used: Staining, Marker, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

    LGR1 knockdown efficiency in adult A. aegypti . Second instar larvae were fed with Escherichia coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector). After feeding, LGR1 transcript knockdown efficiency was determined in panel (A) whole 4th-instar larvae and one-day old adults by quantitative RT-PCR. LGR1 immunoreactivity was examined in control and LGR1 knockdown treatments within (B–F) the testes of virgin one-day adult males (LGR1, green; nuclei, blue). (A) LGR1 transcript levels of 4th instar larvae and newly emerged adults fed with LGR1 dsRNA were significantly reduced compared to control dsRNA-fed mosquitoes. (B) Quantification of LGR1 immunoreactivity from control and dsLGR1-treated larvae confirms downregulation. Representative confocal images demonstrate reduced LGR1 immunofluorescence in cysts (dotted outline) containing late-staged spermatids (Lst) when comparing (C,E) control and (D,F) dsLGR1-treated mosquitoes. All data are presented as mean ± SEM ( n = 3 in panel A , n = 11–13 in panel B ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗ P
    Figure Legend Snippet: LGR1 knockdown efficiency in adult A. aegypti . Second instar larvae were fed with Escherichia coli expressing LGR1 dsRNA or control dsRNA (empty L4440 vector). After feeding, LGR1 transcript knockdown efficiency was determined in panel (A) whole 4th-instar larvae and one-day old adults by quantitative RT-PCR. LGR1 immunoreactivity was examined in control and LGR1 knockdown treatments within (B–F) the testes of virgin one-day adult males (LGR1, green; nuclei, blue). (A) LGR1 transcript levels of 4th instar larvae and newly emerged adults fed with LGR1 dsRNA were significantly reduced compared to control dsRNA-fed mosquitoes. (B) Quantification of LGR1 immunoreactivity from control and dsLGR1-treated larvae confirms downregulation. Representative confocal images demonstrate reduced LGR1 immunofluorescence in cysts (dotted outline) containing late-staged spermatids (Lst) when comparing (C,E) control and (D,F) dsLGR1-treated mosquitoes. All data are presented as mean ± SEM ( n = 3 in panel A , n = 11–13 in panel B ) with asterisks representing significant differences between control and dsLGR1-treated mosquitoes as determined using an unpaired t -test: ∗ P

    Techniques Used: Expressing, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence

    35) Product Images from "A selective class of inhibitors for the CLC-Ka chloride ion channel"

    Article Title: A selective class of inhibitors for the CLC-Ka chloride ion channel

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1720584115

    Selectivity of BIM1 among mammalian CLC homologs. Representative currents from Xenopus oocytes expressing CLC-1 ( A ) or CLC-2 ( B ) shown before and after application of 100 µM BIM1. ( C ) Summary inhibition data (± SEM) for 100 µM BIM1 on CLC-1 ( n = 8), CLC-2 ( n = 8), CLC-Ka ( n = 9), and CLC-Kb ( n = 6). Inhibition is reported for data at +60 mV (CLC-Ka, CLC-Kb, and CLC-1) or −120 mV (CLC-2). For CLC-1 and CLC-2, inhibition is not significantly different from zero ( P = 0.55 and P = 0.84, respectively).
    Figure Legend Snippet: Selectivity of BIM1 among mammalian CLC homologs. Representative currents from Xenopus oocytes expressing CLC-1 ( A ) or CLC-2 ( B ) shown before and after application of 100 µM BIM1. ( C ) Summary inhibition data (± SEM) for 100 µM BIM1 on CLC-1 ( n = 8), CLC-2 ( n = 8), CLC-Ka ( n = 9), and CLC-Kb ( n = 6). Inhibition is reported for data at +60 mV (CLC-Ka, CLC-Kb, and CLC-1) or −120 mV (CLC-2). For CLC-1 and CLC-2, inhibition is not significantly different from zero ( P = 0.55 and P = 0.84, respectively).

    Techniques Used: Expressing, Inhibition

    36) Product Images from "AID–RNA polymerase II transcription-dependent deamination of IgV DNA"

    Article Title: AID–RNA polymerase II transcription-dependent deamination of IgV DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz821

    Experimental protocol used to analyze AID-catalyzed dC deamination on IGHV3-23*01 during transcription by human Pol II. ( A ) Pol II ± DSIF elongation complexes were assembled on a DNA–RNA ‘scaffolded bubble’ substrate and preincubated with AID. Transcription was initiated by the addition rNTP substrates, and the elongation reaction was performed at 30°C (Methods). Following transcription, Exonuclease I (Exo I) was added to digest ssDNA. TS and NTS DNAs were separately barcoded and subjected next-generation sequencing analysis using Maximum Depth Sequencing (MDS) ( 39 ) to assess AID-mediated dC deamination. ( B ) Transcription in the presence of AID and DSIF was visualized as 32 P-labeled RNA primer elongation bands that extend for the full length of the IgV DNA (198 nt). A strong transcription pause region is located ∼11 nt downstream of the scaffold bubble, and is followed by six C residues on the TS, in which deaminations are observed to occur at as many as three contiguous C sites – see also Supplemental Figure S9. ( C ) Distribution of Pol II extended transcripts. Percentage (mean ± standard deviation) of scaffold bubble proximal transcripts (1–12 nt from the end of the scaffold bubble to a run of six consecutive Cs) and full-length transcript (198 nt) were quantified by GE Healthcare ImageQuant software. A sketch depicting the transcribed IgV substrate and the scaffold bubble containing a 20 nt RNA primer strand is shown at the top.
    Figure Legend Snippet: Experimental protocol used to analyze AID-catalyzed dC deamination on IGHV3-23*01 during transcription by human Pol II. ( A ) Pol II ± DSIF elongation complexes were assembled on a DNA–RNA ‘scaffolded bubble’ substrate and preincubated with AID. Transcription was initiated by the addition rNTP substrates, and the elongation reaction was performed at 30°C (Methods). Following transcription, Exonuclease I (Exo I) was added to digest ssDNA. TS and NTS DNAs were separately barcoded and subjected next-generation sequencing analysis using Maximum Depth Sequencing (MDS) ( 39 ) to assess AID-mediated dC deamination. ( B ) Transcription in the presence of AID and DSIF was visualized as 32 P-labeled RNA primer elongation bands that extend for the full length of the IgV DNA (198 nt). A strong transcription pause region is located ∼11 nt downstream of the scaffold bubble, and is followed by six C residues on the TS, in which deaminations are observed to occur at as many as three contiguous C sites – see also Supplemental Figure S9. ( C ) Distribution of Pol II extended transcripts. Percentage (mean ± standard deviation) of scaffold bubble proximal transcripts (1–12 nt from the end of the scaffold bubble to a run of six consecutive Cs) and full-length transcript (198 nt) were quantified by GE Healthcare ImageQuant software. A sketch depicting the transcribed IgV substrate and the scaffold bubble containing a 20 nt RNA primer strand is shown at the top.

    Techniques Used: Next-Generation Sequencing, Sequencing, Labeling, Standard Deviation, Software

    A model for AID putative access to the NTS and TS during Pol II transcription. Sketch of how AID might interact with dC residues on both the TS or NTS to convert C → U. Pol II has been observed to pause and to backtrack during transcription elongation. We propose that paused or backtracked Pol II interacts with AID at the upstream edge of the transcription bubble where the TS and NTS strands exit the polymerase, as inferred from structural studies of Pol II elongation complexes. In this model, AID can interact with dC residues on both the TS or NTS to convert C → U. Structural and single-molecule resolution transcriptional data suggest that AID could have access to about a 15 nt region of transient ssDNA, corresponding to a stalled transcription bubble (∼10 nt) and perhaps an additional region of ssDNA resulting from a backtracked Pol II (∼5 nt).
    Figure Legend Snippet: A model for AID putative access to the NTS and TS during Pol II transcription. Sketch of how AID might interact with dC residues on both the TS or NTS to convert C → U. Pol II has been observed to pause and to backtrack during transcription elongation. We propose that paused or backtracked Pol II interacts with AID at the upstream edge of the transcription bubble where the TS and NTS strands exit the polymerase, as inferred from structural studies of Pol II elongation complexes. In this model, AID can interact with dC residues on both the TS or NTS to convert C → U. Structural and single-molecule resolution transcriptional data suggest that AID could have access to about a 15 nt region of transient ssDNA, corresponding to a stalled transcription bubble (∼10 nt) and perhaps an additional region of ssDNA resulting from a backtracked Pol II (∼5 nt).

    Techniques Used:

    37) Product Images from "Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice"

    Article Title: Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23830-4

    Detection of genomic Bordetella DNA by PCR. ( a ) A PCR product of 318 bp was amplified with B. pseudohinzii -specific primers from DNA of faeces (upper panel) and larynx (lower panel) from infected (lanes 1–4) and non-infected (lanes 5–8) mice, as classified by MALDI-TOF MS. DNA isolated from cultured B. pseudohinzii (strain 3227) served as a positive control (lane 9). Control runs without template were negative (lane 10). M: 100 bp molecular weight marker. ( b ) Trachea and lung taken were taken from 6 animals which were positive for B. pseudohinzii by PCR of faecal pellets, homogenized, and plated on selective agar plates. Number of colony forming units (CFU) is given per 100 mg of tissue. Individual data points shown; horizontal bar and whiskers indicate mean and standard error of the mean. Weight of tissue samples and CFU/organ are provided in Supplementary Table 1 .
    Figure Legend Snippet: Detection of genomic Bordetella DNA by PCR. ( a ) A PCR product of 318 bp was amplified with B. pseudohinzii -specific primers from DNA of faeces (upper panel) and larynx (lower panel) from infected (lanes 1–4) and non-infected (lanes 5–8) mice, as classified by MALDI-TOF MS. DNA isolated from cultured B. pseudohinzii (strain 3227) served as a positive control (lane 9). Control runs without template were negative (lane 10). M: 100 bp molecular weight marker. ( b ) Trachea and lung taken were taken from 6 animals which were positive for B. pseudohinzii by PCR of faecal pellets, homogenized, and plated on selective agar plates. Number of colony forming units (CFU) is given per 100 mg of tissue. Individual data points shown; horizontal bar and whiskers indicate mean and standard error of the mean. Weight of tissue samples and CFU/organ are provided in Supplementary Table 1 .

    Techniques Used: Polymerase Chain Reaction, Amplification, Infection, Mouse Assay, Mass Spectrometry, Isolation, Cell Culture, Positive Control, Molecular Weight, Marker

    38) 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

    39) 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

    Antisense dsRNA by-product formation is template 3′ sequence dependent. ( A ) dsRNA immunoblot using J2 antibody on IVT reactions with wild-type T7 at 37°C performed on modified 512B templates in which the 3′-terminal 25 bp sequence was moved to various positions within 512B template (512B–A to 512B–D). ( B ) dsRNA immunoblot and native gel analysis of modified 512B templates (512B–A to 512B–D) using TsT7-1 at 37°C versus 50°C. ( C ) A chimeric template was generated in which 26 bp of the CLuc 3′-end sequence was added to the 3′-end of 512B template (denoted 512B::CLuc). Native gel electrophoresis analyses and dsRNA immunoblot analysis of IVT reactions of the 512B::CLuc chimeric template compared to the original unmodified 512B template. Reactions were performed with TsT7-1 at either 37°C or 50°C for 1 h.
    Figure Legend Snippet: Antisense dsRNA by-product formation is template 3′ sequence dependent. ( A ) dsRNA immunoblot using J2 antibody on IVT reactions with wild-type T7 at 37°C performed on modified 512B templates in which the 3′-terminal 25 bp sequence was moved to various positions within 512B template (512B–A to 512B–D). ( B ) dsRNA immunoblot and native gel analysis of modified 512B templates (512B–A to 512B–D) using TsT7-1 at 37°C versus 50°C. ( C ) A chimeric template was generated in which 26 bp of the CLuc 3′-end sequence was added to the 3′-end of 512B template (denoted 512B::CLuc). Native gel electrophoresis analyses and dsRNA immunoblot analysis of IVT reactions of the 512B::CLuc chimeric template compared to the original unmodified 512B template. Reactions were performed with TsT7-1 at either 37°C or 50°C for 1 h.

    Techniques Used: Sequencing, Modification, Generated, Nucleic Acid Electrophoresis

    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

    High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.
    Figure Legend Snippet: High-temperature IVT does not affect antisense dsRNA by-product formation. ( A ) Native gel electrophoresis analysis of IVT reactions on 512B DNA template using wild-type T7 (37°C) with/without RNase III treatment. ( B ) dsRNA immunoblot with J2 antibody on IVT reactions (crude and purified) with 512B template. ( C ) Native gel electrophoresis analyses and dsRNA immunoblot analysis of 512B IVT reactions conducted with TsT7-1 at 37°C versus 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Purification

    40) Product Images from "Selection of an Efficient AAV Vector for Robust CNS Transgene Expression"

    Article Title: Selection of an Efficient AAV Vector for Robust CNS Transgene Expression

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2019.10.007

    iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.
    Figure Legend Snippet: iTransduce Library for Selection of Novel AAV Capsids Capable of Efficient Transgene Expression in Target Tissue (A) Two-component system of the library construct. (1) Cre recombinase is driven by a minimal chicken β-actin (CBA) promoter. (2) p41 promoter-driven AAV9 capsid with random heptamer peptide is inserted between amino acids 588 and 589, cloned downstream of the Cre cassette. (B) Selection strategy. (Bi) The iTransduce library comprised of different peptide inserts expressed on the capsid (represented by different colors) is injected intravenously (i.v.) into an Ai9 transgenic mouse with a loxP-flanked STOP cassette upsteam of the tdTomato reporter gene, inserted into the Gt(ROSA)26Sor locus. AAV capsids able to enter the cell of interest but that do not functionally transduce the cell (no Cre expression) do not turn on tdTomato expression. Capsids that can mediate functional transduction (express Cre) will turn on tdTomato expression. (Bii) Cells are isolated from the organ of interest (e.g., brain), and transduced cells are sorted for tdTomato expression and optionally cell markers. (Biii) Capsid DNA is PCR amplified from the sorted cells, cloned back to the library vector, and repackaged for another round of selection. DNA sequencing analysis is utilized after each round to monitor the selection process.

    Techniques Used: Selection, Expressing, Construct, Crocin Bleaching Assay, Clone Assay, Injection, Transgenic Assay, Functional Assay, Transduction, Isolation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, DNA Sequencing

    Related Articles

    Isolation:

    Article Title: Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
    Article Snippet: .. After confirmation of the inserts´ identities and correct orientation by sequencing (Seqlab, Göttingen, Germany), plasmid DNA isolated from E . coli DH5α (Zippy Plasmid Miniprep kit, Epigenetics) was electroporated into E . coli BL21(DE3) expression host (New England Biolabs) as above. .. The cdt V-B deletion mutant (cdt V-ACΔB ) was constructed by inverse PCR using primer pair F-del-cdtB and R-del-cdtB ( ) and plasmid DNA from strain BL21(DE3)/pET23b(+)cdt V-ABC ( ) as a template.

    Purification:

    Article Title: High Nuclease Activity of Long Persisting Staphylococcus aureus Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing
    Article Snippet: .. For transformation, electro-competent cells of the CF isolate were mixed with 5 μl of the purified pCM28nuc plasmid (NEB Monarch Plasmid Miniprep Kit). .. Electroporation was executed by the Ec2 program of the BIORad MicroPulser Electroporator (Pulse 2.5 kV, number of impulse 1) in a 0.2 cm electroporation cuvette.

    Expressing:

    Article Title: Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
    Article Snippet: .. After confirmation of the inserts´ identities and correct orientation by sequencing (Seqlab, Göttingen, Germany), plasmid DNA isolated from E . coli DH5α (Zippy Plasmid Miniprep kit, Epigenetics) was electroporated into E . coli BL21(DE3) expression host (New England Biolabs) as above. .. The cdt V-B deletion mutant (cdt V-ACΔB ) was constructed by inverse PCR using primer pair F-del-cdtB and R-del-cdtB ( ) and plasmid DNA from strain BL21(DE3)/pET23b(+)cdt V-ABC ( ) as a template.

    Sequencing:

    Article Title: Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes
    Article Snippet: .. Cells were cultivated overnight at 37 °C and plasmid DNA, carrying LmChdC M149A, Q187A, and M149A/Q187A variants, was extracted using the Monarch Plasmid Mini Prep Kit (New England BioLabs Inc.) and sent for sequencing. .. Expression and Purification of LmChdC Wild-Type and Variants WT and variants were subcloned into a modified version of the pET21(+) expression vector with an N-terminal StrepII-tag, cleavable TEV protease, or into a pETM11 expression vector with an N-terminal, TEV cleavable 6×His-tag, expressed in E. coli Tuner (DE3) cells (Merck/Novagen) and purified via a StrepTrap HP or a HisTrap HP 5 mL column (GE Healthcare).

    Article Title: Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
    Article Snippet: .. After confirmation of the inserts´ identities and correct orientation by sequencing (Seqlab, Göttingen, Germany), plasmid DNA isolated from E . coli DH5α (Zippy Plasmid Miniprep kit, Epigenetics) was electroporated into E . coli BL21(DE3) expression host (New England Biolabs) as above. .. The cdt V-B deletion mutant (cdt V-ACΔB ) was constructed by inverse PCR using primer pair F-del-cdtB and R-del-cdtB ( ) and plasmid DNA from strain BL21(DE3)/pET23b(+)cdt V-ABC ( ) as a template.

    Article Title: Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors
    Article Snippet: .. Cosmid DNA was obtained from each bioactive clone using a Monarch Plasmid Miniprep kit (NEB, Ipswich, MA) and sequenced by Sanger sequencing using primers targeting the flanking regions of the ScaI site on pJWC1 vector ( ). ..

    Transformation Assay:

    Article Title: High Nuclease Activity of Long Persisting Staphylococcus aureus Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing
    Article Snippet: .. For transformation, electro-competent cells of the CF isolate were mixed with 5 μl of the purified pCM28nuc plasmid (NEB Monarch Plasmid Miniprep Kit). .. Electroporation was executed by the Ec2 program of the BIORad MicroPulser Electroporator (Pulse 2.5 kV, number of impulse 1) in a 0.2 cm electroporation cuvette.

    Cosmid DNA:

    Article Title: Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors
    Article Snippet: .. Cosmid DNA was obtained from each bioactive clone using a Monarch Plasmid Miniprep kit (NEB, Ipswich, MA) and sequenced by Sanger sequencing using primers targeting the flanking regions of the ScaI site on pJWC1 vector ( ). ..

    Plasmid Preparation:

    Article Title: Insights into the Active Site of Coproheme Decarboxylase from Listeria monocytogenes
    Article Snippet: .. Cells were cultivated overnight at 37 °C and plasmid DNA, carrying LmChdC M149A, Q187A, and M149A/Q187A variants, was extracted using the Monarch Plasmid Mini Prep Kit (New England BioLabs Inc.) and sent for sequencing. .. Expression and Purification of LmChdC Wild-Type and Variants WT and variants were subcloned into a modified version of the pET21(+) expression vector with an N-terminal StrepII-tag, cleavable TEV protease, or into a pETM11 expression vector with an N-terminal, TEV cleavable 6×His-tag, expressed in E. coli Tuner (DE3) cells (Merck/Novagen) and purified via a StrepTrap HP or a HisTrap HP 5 mL column (GE Healthcare).

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

    Article Title: Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury
    Article Snippet: .. After confirmation of the inserts´ identities and correct orientation by sequencing (Seqlab, Göttingen, Germany), plasmid DNA isolated from E . coli DH5α (Zippy Plasmid Miniprep kit, Epigenetics) was electroporated into E . coli BL21(DE3) expression host (New England Biolabs) as above. .. The cdt V-B deletion mutant (cdt V-ACΔB ) was constructed by inverse PCR using primer pair F-del-cdtB and R-del-cdtB ( ) and plasmid DNA from strain BL21(DE3)/pET23b(+)cdt V-ABC ( ) as a template.

    Article Title: TET enzymes control antibody production and shape the mutational landscape in germinal centre B cells
    Article Snippet: .. Plasmid DNA was extracted using the Monarch Plasmid Miniprep Kit (New England BioLabs, T1010L) as per manufacturer's instructions. .. Subsequently, Sanger sequencing was performed using the pJet1.2 F primer detailed above.

    Article Title: Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors
    Article Snippet: .. Cosmid DNA was obtained from each bioactive clone using a Monarch Plasmid Miniprep kit (NEB, Ipswich, MA) and sequenced by Sanger sequencing using primers targeting the flanking regions of the ScaI site on pJWC1 vector ( ). ..

    Article Title: High Nuclease Activity of Long Persisting Staphylococcus aureus Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing
    Article Snippet: .. For transformation, electro-competent cells of the CF isolate were mixed with 5 μl of the purified pCM28nuc plasmid (NEB Monarch Plasmid Miniprep Kit). .. Electroporation was executed by the Ec2 program of the BIORad MicroPulser Electroporator (Pulse 2.5 kV, number of impulse 1) in a 0.2 cm electroporation cuvette.

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  • 96
    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.
    Monarch Plasmid Miniprep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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).
    Monarch Pcr, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs monarch dna cleanup kit
    RcGTA gp1 in vitro <t>DNA</t> 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 <t>PCR</t> 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.
    Monarch Dna Cleanup Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    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.

    Journal: Journal of Virology

    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

    doi: 10.1128/JVI.01328-19

    Figure Lengend 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.

    Article Snippet: DNA substrates were prepared by PCR amplification with oligonucleotides indicated in and cleaned with a Monarch DNA cleanup kit (NEB).

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