puc19  (New England Biolabs)


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    Name:
    pUC19 Vector
    Description:
    pUC19 Vector 250 ug
    Catalog Number:
    N3041L
    Price:
    300
    Category:
    Vectors Plasmids
    Size:
    250 ug
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    Structured Review

    New England Biolabs puc19
    pUC19 Vector
    pUC19 Vector 250 ug
    https://www.bioz.com/result/puc19/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    puc19 - by Bioz Stars, 2021-05
    86/100 stars

    Images

    1) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    2) Product Images from "EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA"

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.884692

    EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.
    Figure Legend Snippet: EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.

    Techniques Used: Methylation, CpG Methylation Assay

    3) Product Images from "A Conserved Sequence Extending Motif III of the Motor Domain in the Snf2-Family DNA Translocase Rad54 Is Critical for ATPase Activity"

    Article Title: A Conserved Sequence Extending Motif III of the Motor Domain in the Snf2-Family DNA Translocase Rad54 Is Critical for ATPase Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0082184

    Rad54-2A exhibits a dsDNA binding defect. A . Rad54 (left panel) and Rad54-2A (right panel) were incubated with pUC19 supercoiled dsDNA at the indicated stoichiometries (bp DNA:Rad54 monomer) in the absence of nucleotide cofactor (upper panels), in the presence of 5 mM ATP and an ATP-regeneraton system (middle panels) or in the presence of 5 mM ATP-γ-S (lower panels) at 30°C for 15 minutes. The DNA-protein complexes were fixed by glutaraldehyde (GA) and visualized on a 1% agarose gel. B . Quantification of the DNA protein complexes from A .
    Figure Legend Snippet: Rad54-2A exhibits a dsDNA binding defect. A . Rad54 (left panel) and Rad54-2A (right panel) were incubated with pUC19 supercoiled dsDNA at the indicated stoichiometries (bp DNA:Rad54 monomer) in the absence of nucleotide cofactor (upper panels), in the presence of 5 mM ATP and an ATP-regeneraton system (middle panels) or in the presence of 5 mM ATP-γ-S (lower panels) at 30°C for 15 minutes. The DNA-protein complexes were fixed by glutaraldehyde (GA) and visualized on a 1% agarose gel. B . Quantification of the DNA protein complexes from A .

    Techniques Used: Binding Assay, Incubation, Agarose Gel Electrophoresis

    4) Product Images from "Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake"

    Article Title: Ehrlichia chaffeensis Proliferation Begins with NtrY/NtrX and PutA/GlnA Upregulation and CtrA Degradation Induced by Proline and Glutamine Uptake

    Journal: mBio

    doi: 10.1128/mBio.02141-14

    E. chaffeensis PutA and GlnA are functional enzymes, and E. chaffeensis GlnA is sensitive to MSX. (A) E. chaffeensis PutA complements an E. coli putA mutant (JT31). wt (CSH4) and JT31 transformed with the E. chaffeensis putA construct or pUC19 (negative control) were cultured on redox indicator TTC-proline plates. (B) E. chaffeensis GlnA complements an E. coli glnA mutant (YMC11). wt (XL1-Blue) and YMC11 transformed with the E. chaffeensis glnA construct or pUC19 (negative control) were cultured on M9 plates containing 20 mM (NH4) 2 SO 4 as the sole nitrogen source. (C) MSX inhibits E. chaffeensis GlnA activity. YMC11 transformed with the E. chaffeensis glnA construct was cultured on M9 plates containing 2 mM glutamine (Gln) or 20 mM (NH4) 2 SO 4 with or without 200 µM MSX.
    Figure Legend Snippet: E. chaffeensis PutA and GlnA are functional enzymes, and E. chaffeensis GlnA is sensitive to MSX. (A) E. chaffeensis PutA complements an E. coli putA mutant (JT31). wt (CSH4) and JT31 transformed with the E. chaffeensis putA construct or pUC19 (negative control) were cultured on redox indicator TTC-proline plates. (B) E. chaffeensis GlnA complements an E. coli glnA mutant (YMC11). wt (XL1-Blue) and YMC11 transformed with the E. chaffeensis glnA construct or pUC19 (negative control) were cultured on M9 plates containing 20 mM (NH4) 2 SO 4 as the sole nitrogen source. (C) MSX inhibits E. chaffeensis GlnA activity. YMC11 transformed with the E. chaffeensis glnA construct was cultured on M9 plates containing 2 mM glutamine (Gln) or 20 mM (NH4) 2 SO 4 with or without 200 µM MSX.

    Techniques Used: Functional Assay, Mutagenesis, Transformation Assay, Construct, Negative Control, Cell Culture, Activity Assay

    5) Product Images from "A bacterial DNA repair pathway specific to a natural antibiotic"

    Article Title: A bacterial DNA repair pathway specific to a natural antibiotic

    Journal: Molecular microbiology

    doi: 10.1111/mmi.14158

    MrfB is a metal-dependent exonuclease. (A) A schematic of MrfB depicting putative catalytic residues and C-terminal tetratrichopeptide repeat (TPR) domain. (B) Spot titer assay using strains with the indicated genotypes spotted on the indicated media. (C) 1 μg of purified MrfB stained with Coomassie brilliant blue. (D) Exonuclease assay using pUC19 linearized with BamHI (lanes 3–7). Reactions were incubated at 37°C for 15 minutes with or without MrfB, MgCl 2 , or EDTA as indicated, and separated on an agarose gel stained with ethidium bromide. Lane 1 is a 1 kb plus molecular weight marker (M) and lane 2 is undigested pUC19 plasmid. (E) Exonuclease assay testing substrate preference. The indicated exonucleases were incubated with a closed covalent circular plasmid (CCC), a nicked plasmid (Nicked) or a linear plasmid (Linear) in the presence of Mg 2+ at 37°C for 10 minutes. Reaction products were separated on an agarose gel stained with ethidium bromide. Lane 1 is a 1 kb plus molecular weight marker (M).
    Figure Legend Snippet: MrfB is a metal-dependent exonuclease. (A) A schematic of MrfB depicting putative catalytic residues and C-terminal tetratrichopeptide repeat (TPR) domain. (B) Spot titer assay using strains with the indicated genotypes spotted on the indicated media. (C) 1 μg of purified MrfB stained with Coomassie brilliant blue. (D) Exonuclease assay using pUC19 linearized with BamHI (lanes 3–7). Reactions were incubated at 37°C for 15 minutes with or without MrfB, MgCl 2 , or EDTA as indicated, and separated on an agarose gel stained with ethidium bromide. Lane 1 is a 1 kb plus molecular weight marker (M) and lane 2 is undigested pUC19 plasmid. (E) Exonuclease assay testing substrate preference. The indicated exonucleases were incubated with a closed covalent circular plasmid (CCC), a nicked plasmid (Nicked) or a linear plasmid (Linear) in the presence of Mg 2+ at 37°C for 10 minutes. Reaction products were separated on an agarose gel stained with ethidium bromide. Lane 1 is a 1 kb plus molecular weight marker (M).

    Techniques Used: Titer Assay, Purification, Staining, Incubation, Agarose Gel Electrophoresis, Molecular Weight, Marker, Plasmid Preparation, Countercurrent Chromatography

    6) Product Images from "Extrachromosomal circular DNA is common in yeast"

    Article Title: Extrachromosomal circular DNA is common in yeast

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

    doi: 10.1073/pnas.1508825112

    Detection of known circular DNA elements. ( A ) Scatter plot shows fraction of uniquely mapped reads for plasmids spiked into samples after cell lysis and before column purification. Plasmids added in ratios per cell: pBR322 (crosses) 1:1, pUC19 (circles)
    Figure Legend Snippet: Detection of known circular DNA elements. ( A ) Scatter plot shows fraction of uniquely mapped reads for plasmids spiked into samples after cell lysis and before column purification. Plasmids added in ratios per cell: pBR322 (crosses) 1:1, pUC19 (circles)

    Techniques Used: Lysis, Purification

    7) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.
    Figure Legend Snippet: ( A ) Influence of ionic strength on pUC19 condensation (400 ng) by OC3 and MC3 (25 μM) opioid compounds. Condensation reactions on pUC19 (400 ng) by opioid compounds in ( B ) acidic NaOAc buffer (80 mM, pH = 4.0), and ( C ) basic Tris buffer (80 mM, pH = 9.0) in the presence of 25 mM NaCl.

    Techniques Used:

    ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.
    Figure Legend Snippet: ( A ) Agarose gel electrophoresis of supercoiled (400 ng) and ( B ) a 742 bp dsDNA fragment of pUC19 (400 ng) exposed to increasing concentrations of MC3, OC3 and HC3 . Reactions were carried out in the presence of 25 mM NaCl for 5 h at 37°C prior to electrophoretic analysis.

    Techniques Used: Agarose Gel Electrophoresis

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    Related Articles

    Mutagenesis:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Clone Assay:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Article Title: Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density
    Article Snippet: .. Plasmids were isolated from Escherichia coli using the PureYield Maxiprep system (Promega). pUC19-PHO8 contains ∼3.5 kb of the Saccharomyces cerevisiae PHO8 locus cloned into pUC19 via BamHI and PstI, is 6,168 bp long, and is described as “pUC19-PHO8-long” in reference . pUC19-GCY1 contains ∼3.5 kb of the S. cerevisiae GCY1 locus (PCR product using primers 5′-CAGTCGGATGGAGCTCACTTCTATTGGCTTAGGAGC-3′ and 5′-CACTGTGCATTTCTAGAACGACGAAGACGAGGATTAG-3′ and genomic DNA of strain BY4741 [EUROSCARF] as the template) cloned into pUC19 via SacI and XbaI. pUC19-PHO8 and pUC19-GCY1 were linearized with BamHI (NEB), which cleaves right at or 400 bp downstream of, respectively, the upstream border between prokaryotic and eukaryotic DNA. .. The complete plasmids were used as the template for salt gradient dialysis (SGD) reconstitution, and complete linearization was confirmed by agarose gel electrophoresis prior to chromatin assembly.

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: The E. coli serS gene, including its native promoter and terminator, was amplified from E. coli JM109 genomic DNA using primers EcserS-F and EcserS-R. .. The product was cloned into the XbaI and EcoRI sites of pUC19 to produce pUC-EcSerRS. ..

    Plasmid Preparation:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: Genomic DNA concentrations were determined using the Qubit® 2.0 fluorometer (Invitrogen) and the quality of DNA was assessed by agarose gel electrophoresis. .. Preparation of 304 bp model DNA with 4mC modifications For N 4 -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl N 4 -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents
    Article Snippet: Since condensation by C 3 opioids had been established by viscosity and turbidity measurements, visualization of DNA compaction was then followed by electrophoresis. .. Samples were titrated against both supercoiled pUC19 plasmid DNA and a 742 bp dsDNA fragment amplified from pUC19 encompassing the lacZα gene, before incubation for 5 h prior to analysis by agarose gel electrophoresis (Figure ). ..

    Expressing:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Transformation Assay:

    Article Title: Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974 ▿
    Article Snippet: Genetic complementation was used to test the in vivo activities of SerRS1 and SerRS2. .. The host strain was an E. coli mutant (K28) that is ineffective at making its own serS gene product at high temperature ( , ). serS1 and serS2 were separately cloned into a high-copy vector, pUC19, under the control of a constitutive glnS ′ promoter, which is a glnS mutant promoter used for aaRS expression in E. coli , and the resulting plasmids were transformed into E. coli K28. ..

    Article Title: Secretory expression of biologically active human Herpes virus interleukin-10 analogues in Escherichia coli via a modified Sec-dependent transporter construct
    Article Snippet: After 4 h of incubation at 37°C, the amount of TNF-α released in the medium was measured in duplicate by ELISA (R & D Systems) according to the manufacturer’s instructions. .. Periplasmic fraction of pUC19 transformed E. coli BL21 (DE3) was used as TNF-α induction control. .. Commercial EBV IL-10 (R & D Systems) served as positive control.

    Isolation:

    Article Title: Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density
    Article Snippet: .. Plasmids were isolated from Escherichia coli using the PureYield Maxiprep system (Promega). pUC19-PHO8 contains ∼3.5 kb of the Saccharomyces cerevisiae PHO8 locus cloned into pUC19 via BamHI and PstI, is 6,168 bp long, and is described as “pUC19-PHO8-long” in reference . pUC19-GCY1 contains ∼3.5 kb of the S. cerevisiae GCY1 locus (PCR product using primers 5′-CAGTCGGATGGAGCTCACTTCTATTGGCTTAGGAGC-3′ and 5′-CACTGTGCATTTCTAGAACGACGAAGACGAGGATTAG-3′ and genomic DNA of strain BY4741 [EUROSCARF] as the template) cloned into pUC19 via SacI and XbaI. pUC19-PHO8 and pUC19-GCY1 were linearized with BamHI (NEB), which cleaves right at or 400 bp downstream of, respectively, the upstream border between prokaryotic and eukaryotic DNA. .. The complete plasmids were used as the template for salt gradient dialysis (SGD) reconstitution, and complete linearization was confirmed by agarose gel electrophoresis prior to chromatin assembly.

    Polymerase Chain Reaction:

    Article Title: Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density
    Article Snippet: .. Plasmids were isolated from Escherichia coli using the PureYield Maxiprep system (Promega). pUC19-PHO8 contains ∼3.5 kb of the Saccharomyces cerevisiae PHO8 locus cloned into pUC19 via BamHI and PstI, is 6,168 bp long, and is described as “pUC19-PHO8-long” in reference . pUC19-GCY1 contains ∼3.5 kb of the S. cerevisiae GCY1 locus (PCR product using primers 5′-CAGTCGGATGGAGCTCACTTCTATTGGCTTAGGAGC-3′ and 5′-CACTGTGCATTTCTAGAACGACGAAGACGAGGATTAG-3′ and genomic DNA of strain BY4741 [EUROSCARF] as the template) cloned into pUC19 via SacI and XbaI. pUC19-PHO8 and pUC19-GCY1 were linearized with BamHI (NEB), which cleaves right at or 400 bp downstream of, respectively, the upstream border between prokaryotic and eukaryotic DNA. .. The complete plasmids were used as the template for salt gradient dialysis (SGD) reconstitution, and complete linearization was confirmed by agarose gel electrophoresis prior to chromatin assembly.

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: Genomic DNA concentrations were determined using the Qubit® 2.0 fluorometer (Invitrogen) and the quality of DNA was assessed by agarose gel electrophoresis. .. Preparation of 304 bp model DNA with 4mC modifications For N 4 -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl N 4 -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Amplification:

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: Genomic DNA concentrations were determined using the Qubit® 2.0 fluorometer (Invitrogen) and the quality of DNA was assessed by agarose gel electrophoresis. .. Preparation of 304 bp model DNA with 4mC modifications For N 4 -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl N 4 -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents
    Article Snippet: Since condensation by C 3 opioids had been established by viscosity and turbidity measurements, visualization of DNA compaction was then followed by electrophoresis. .. Samples were titrated against both supercoiled pUC19 plasmid DNA and a 742 bp dsDNA fragment amplified from pUC19 encompassing the lacZα gene, before incubation for 5 h prior to analysis by agarose gel electrophoresis (Figure ). ..

    Incubation:

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents
    Article Snippet: Since condensation by C 3 opioids had been established by viscosity and turbidity measurements, visualization of DNA compaction was then followed by electrophoresis. .. Samples were titrated against both supercoiled pUC19 plasmid DNA and a 742 bp dsDNA fragment amplified from pUC19 encompassing the lacZα gene, before incubation for 5 h prior to analysis by agarose gel electrophoresis (Figure ). ..

    Agarose Gel Electrophoresis:

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents
    Article Snippet: Since condensation by C 3 opioids had been established by viscosity and turbidity measurements, visualization of DNA compaction was then followed by electrophoresis. .. Samples were titrated against both supercoiled pUC19 plasmid DNA and a 742 bp dsDNA fragment amplified from pUC19 encompassing the lacZα gene, before incubation for 5 h prior to analysis by agarose gel electrophoresis (Figure ). ..

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    New England Biolabs puc19 vector dna
    Data analysis of spike-in controls from MethylC-seq and 4mC-TAB-seq in the context of C. kristjanssonii genomic <t>DNA.</t> ( A ) Composition of <t>pUC19</t> DNA, lambda DNA, and C. kristjanssonii genomic DNA. ( B ) The percentage of detected as cytosine reads on 4mC sites in untreated and Tet-treated samples. ( C ) The detected as cytosine reads percentage on unmodified cytosine sites (non-CpG context) and 5mC sites (CpG context) in untreated and Tet-treated samples. ( D ) Quantification of 4mC and 5mC in C. kristjanssonii genomic DNA, determined by LC-MS/MS and deep-sequencing respectively. Error bars indicate mean ± SD, n = 4.
    Puc19 Vector Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs plasmid puc19
    Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with <t>pUC19</t> plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.
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    Data analysis of spike-in controls from MethylC-seq and 4mC-TAB-seq in the context of C. kristjanssonii genomic DNA. ( A ) Composition of pUC19 DNA, lambda DNA, and C. kristjanssonii genomic DNA. ( B ) The percentage of detected as cytosine reads on 4mC sites in untreated and Tet-treated samples. ( C ) The detected as cytosine reads percentage on unmodified cytosine sites (non-CpG context) and 5mC sites (CpG context) in untreated and Tet-treated samples. ( D ) Quantification of 4mC and 5mC in C. kristjanssonii genomic DNA, determined by LC-MS/MS and deep-sequencing respectively. Error bars indicate mean ± SD, n = 4.

    Journal: Nucleic Acids Research

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing

    doi: 10.1093/nar/gkv738

    Figure Lengend Snippet: Data analysis of spike-in controls from MethylC-seq and 4mC-TAB-seq in the context of C. kristjanssonii genomic DNA. ( A ) Composition of pUC19 DNA, lambda DNA, and C. kristjanssonii genomic DNA. ( B ) The percentage of detected as cytosine reads on 4mC sites in untreated and Tet-treated samples. ( C ) The detected as cytosine reads percentage on unmodified cytosine sites (non-CpG context) and 5mC sites (CpG context) in untreated and Tet-treated samples. ( D ) Quantification of 4mC and 5mC in C. kristjanssonii genomic DNA, determined by LC-MS/MS and deep-sequencing respectively. Error bars indicate mean ± SD, n = 4.

    Article Snippet: Preparation of 304 bp model DNA with 4mC modifications For N 4 -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl N 4 -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG).

    Techniques: Lambda DNA Preparation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

    EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.

    Journal: bioRxiv

    Article Title: EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

    doi: 10.1101/2019.12.20.884692

    Figure Lengend Snippet: EM-seq accurately represents methylation EM-seq and bisulfite libraries were made using 10, 50 and 200 ng of NA12878 DNA with control DNA (2 ng unmethylated lambda and 0.1 ng CpG methylated pUC19). Libraries were sequenced on an Illumina NovaSeq 6000 (2 x 100 bases). 324 million paired reads for each library were aligned to a human + control reference genome (see supplemental materials) using bwa-meth 0.2.2. and methylation information was extracted from the alignments using MethylDackel. The top and bottom strand CpGs were counted independently, yielding a maximum of 56 million possible CpG sites. (A) NA12878 EM-seq and whole genome bisulfite library (WGBS) methylation in CpG, CHH and CHG contexts are similarly represented. Methylation state for unmethylated lambda control and CpG methylated pUC19 control DNAs are shown in Supplemental Figure 5. (B) The number of CpGs covered for EM-seq and bisulfite libraries were calculated and graphed at minimum coverage depths of 1x through 21x. (C) The number of CpGs detected were compared between EM-seq and bisulfite libraries at 1x and 8x coverage depths. CpGs unique to EM-seq libraries, bisulfite libraries or those that were common to both are represented in the Venn diagrams. (D, E) Methylkit analysis at minimum 1x coverage shows good CpG methylation correlation between 10 ng and 200 ng NA12878 EM-seq libraries (D) and WGBS libraries (E). Methylation level correlations between inputs and replicates of EM-seq libraries are better than for WGBS libraries. The reduction in observations of disagreement (upper left and lower right corners) is particularly striking. Correlation between EM-seq and WGBS libraries at 10 ng, 50 ng, and 200 ng NA12878 DNA input are shown in Supplemental Figure 7.

    Article Snippet: CpG methylated pUC19, was made by transforming pUC19 plasmid (NEB, Ipswich, MA) into dam-/dcm-competent E.coli cells (NEB, Ipswich, MA).

    Techniques: Methylation, CpG Methylation Assay

    ISWI, ACF, and Chd1 have clamping activity, i.e., they generate very similar and constant nucleosomal repeat lengths regardless of nucleosome density. (A) Limited digests with the indicated MNase concentrations of SGD chromatin (plasmid pUC19-PHO8) at the indicated assembly degrees after incubation with (+ISWI) or without (−ISWI) ISWI remodeler. Asterisks denote the trinucleosomal fragment band. MNase digestion fragments were visualized by Southern blotting and probing against the yeast PHO8 gene. M, DNA marker (2-log; NEB). (B and C) As for panel A but for the ACF or Chd1 remodeler, respectively.

    Journal: Molecular and Cellular Biology

    Article Title: Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

    doi: 10.1128/MCB.01070-14

    Figure Lengend Snippet: ISWI, ACF, and Chd1 have clamping activity, i.e., they generate very similar and constant nucleosomal repeat lengths regardless of nucleosome density. (A) Limited digests with the indicated MNase concentrations of SGD chromatin (plasmid pUC19-PHO8) at the indicated assembly degrees after incubation with (+ISWI) or without (−ISWI) ISWI remodeler. Asterisks denote the trinucleosomal fragment band. MNase digestion fragments were visualized by Southern blotting and probing against the yeast PHO8 gene. M, DNA marker (2-log; NEB). (B and C) As for panel A but for the ACF or Chd1 remodeler, respectively.

    Article Snippet: Plasmids were isolated from Escherichia coli using the PureYield Maxiprep system (Promega). pUC19-PHO8 contains ∼3.5 kb of the Saccharomyces cerevisiae PHO8 locus cloned into pUC19 via BamHI and PstI, is 6,168 bp long, and is described as “pUC19-PHO8-long” in reference . pUC19-GCY1 contains ∼3.5 kb of the S. cerevisiae GCY1 locus (PCR product using primers 5′-CAGTCGGATGGAGCTCACTTCTATTGGCTTAGGAGC-3′ and 5′-CACTGTGCATTTCTAGAACGACGAAGACGAGGATTAG-3′ and genomic DNA of strain BY4741 [EUROSCARF] as the template) cloned into pUC19 via SacI and XbaI. pUC19-PHO8 and pUC19-GCY1 were linearized with BamHI (NEB), which cleaves right at or 400 bp downstream of, respectively, the upstream border between prokaryotic and eukaryotic DNA.

    Techniques: Activity Assay, Plasmid Preparation, Incubation, Southern Blot, Marker

    Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

    Journal: Nucleic Acids Research

    Article Title: Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro

    doi: 10.1093/nar/gky219

    Figure Lengend Snippet: Priming by g8 target variants. ( A ) Scheme of the E. coli KD263 CRISPR locus. The cas gene expression is controlled by inducible promoters. The CRISPR array consists of a single g8 spacer (blue boxes) surrounded by two repeats (black boxes). Priming is induced by transforming the cells with pUC19 plasmids carrying the protospacer variants. Incorporation of new spacers (green box) is revealed using PCR amplification of the CRISPR array and agarose gel electrophoresis. ( B ) Incorporation of new spacers probed at different times after induction for the indicated g8 protospacer variants. ( C for other target variant plasmids). The height of the histogram bars corresponds to the number of HTS reads found for a particular position. The location of the priming protospacer and the PAM is shown as a blue-red box. The histogram entry in orange marks the hotspot HS1, which was used for semi-quantitative measurements of the primed adaptation efficiency (see E). ( D for correlation coefficients) is apparent. ( E ) Relative frequency of priming (i.e. CRISPR array extension) probed by qPCR with a primer specific for the frequently incorporated protospacer HS1 (see C) for the different target variants. Error bars represent the standard deviation of three repeat measurements.

    Article Snippet: Constructs containing the WT g8 protospacer and its variants were cloned into plasmid pUC19 (NEB) at the single SmaI (NEB) site by blunt end ligation.

    Techniques: CRISPR, Expressing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Variant Assay, Real-time Polymerase Chain Reaction, Standard Deviation