plasmid puc19  (New England Biolabs)


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    SmaI
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    SmaI 10 000 units
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    R0141L
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    Restriction Enzymes
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    New England Biolabs plasmid puc19
    SmaI
    SmaI 10 000 units
    https://www.bioz.com/result/plasmid puc19/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid puc19 - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky219

    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.
    Figure Legend 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.

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

    2) Product Images from "Protective Effect of Qnr on Agents Other than Quinolones That Target DNA Gyrase"

    Article Title: Protective Effect of Qnr on Agents Other than Quinolones That Target DNA Gyrase

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01292-15

    Protective effect of QnrB1 against gyrase inhibition by PAT. Lane 1, control reaction of relaxed pUC19 substrate and DNA gyrase. Lanes 2 to 12, DNA substrate, gyrase, and 8 μM PAT with no QnrB1 (lane 2) or with QnrB1 at concentrations of 0.11
    Figure Legend Snippet: Protective effect of QnrB1 against gyrase inhibition by PAT. Lane 1, control reaction of relaxed pUC19 substrate and DNA gyrase. Lanes 2 to 12, DNA substrate, gyrase, and 8 μM PAT with no QnrB1 (lane 2) or with QnrB1 at concentrations of 0.11

    Techniques Used: Inhibition

    Lack of protection by QnrB1 against gyrase inhibition by simocyclinone D8. Lane 1, control reaction of pUC19 substrate and DNA gyrase. Lanes 2 to 11, DNA substrate, gyrase, and 0.5 μM simocyclinone D8 with no QnrB1 (lane 2) or with QnrB1 at concentrations
    Figure Legend Snippet: Lack of protection by QnrB1 against gyrase inhibition by simocyclinone D8. Lane 1, control reaction of pUC19 substrate and DNA gyrase. Lanes 2 to 11, DNA substrate, gyrase, and 0.5 μM simocyclinone D8 with no QnrB1 (lane 2) or with QnrB1 at concentrations

    Techniques Used: Inhibition

    3) Product Images from "The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ?H contains DNA helicase activity"

    Article Title: The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ?H contains DNA helicase activity

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

    doi:

    The mthMCM protein contains DNA-dependent ATPase activity. ( A ) The influence of various DNA preparations on the ATPase activity of the mthMCM protein. The ATPase activity of the multimeric glycerol gradient peak was determined in the absence or presence of the various indicated DNAs (30 ng/μl). The numbers at the bottom of the autoradiogram indicate the amount of 32 P i released (quantitated by phosphorimager analysis). ( B ) Influence of DNA concentration on ATPase activity. ATPase activity assay was measured in the presence of the indicated amount of φX174 ssDNA (○) or pUC19 dsDNA (●) and the mthMCM multimeric glycerol gradient peak fraction (80 ng). The activity observed with no DNA added (5 pmol) was set at a value of 1; ATP hydrolysis in the presence of DNA at various concentrations was calculated relative to this value.
    Figure Legend Snippet: The mthMCM protein contains DNA-dependent ATPase activity. ( A ) The influence of various DNA preparations on the ATPase activity of the mthMCM protein. The ATPase activity of the multimeric glycerol gradient peak was determined in the absence or presence of the various indicated DNAs (30 ng/μl). The numbers at the bottom of the autoradiogram indicate the amount of 32 P i released (quantitated by phosphorimager analysis). ( B ) Influence of DNA concentration on ATPase activity. ATPase activity assay was measured in the presence of the indicated amount of φX174 ssDNA (○) or pUC19 dsDNA (●) and the mthMCM multimeric glycerol gradient peak fraction (80 ng). The activity observed with no DNA added (5 pmol) was set at a value of 1; ATP hydrolysis in the presence of DNA at various concentrations was calculated relative to this value.

    Techniques Used: Activity Assay, Concentration Assay

    4) Product Images from "Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky219

    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 ) Mapping of spacers acquired from the G-1T variant target protospacer plasmid to the pUC19 backbone (see Supplementary Figure S7 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 ) Position-dependent acquisition frequency for targets with seed mutation plotted over the acquisition frequency for the G-1T PAM mutation target. A high correlation between spacer acquisition patterns of all tested target variants (see Supplementary Figure S7B 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.
    Figure Legend 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 ) Mapping of spacers acquired from the G-1T variant target protospacer plasmid to the pUC19 backbone (see Supplementary Figure S7 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 ) Position-dependent acquisition frequency for targets with seed mutation plotted over the acquisition frequency for the G-1T PAM mutation target. A high correlation between spacer acquisition patterns of all tested target variants (see Supplementary Figure S7B 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.

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

    5) Product Images from "Transposition-Based Method for the Rapid Generation of Gene-Targeting Vectors to Produce Cre/Flp-Modifiable Conditional Knock-Out Mice"

    Article Title: Transposition-Based Method for the Rapid Generation of Gene-Targeting Vectors to Produce Cre/Flp-Modifiable Conditional Knock-Out Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004341

    Details of the genetic tools used. (A) Structure and characteristic elements of the multicopy cloning vector pHTH22 with unique restriction sites indicated. The gene region encoding HSV thymidine kinase (TK) under the control of mouse phosphoglycerate kinase promoter and terminator is shown with a black curved arrow. The vector portion of the plasmid including the gene for ampicillin resistance (Amp R ) and pUC19 origin of replication (ori) are shown with gray symbols (curved arrow and rectangle). (B) The nucleotide sequence of the polylinker and its flanking regions in pHTH22. The polylinker is shown in boldface with enzyme recognition sites underlined. The arrows indicate the binding sites of primers that were used for the confirmatory sequencing of genomic inserts' ends. (C) Structures of the Kan/Neo-loxP-Mu and Kan/Neo-loxP-FRT-Mu transposons. The transposons contain bacterial (p bact ) and eukaryotic (p SV40 ) promoters (short arrows), a marker gene (Kan/Neo) conferring resistance to kanamycin in bacteria and G418 in eukaryotes (black arrow), and the HSV thymidine kinase polyadenylation (HSV TK polyA) signal (small rectangle). The loxP sites are indicated by triangles and FRT sites by pentagons. The rectangles in the transposon ends indicate 50 bp of Mu R-end DNA sequences in inverted orientation relative to each other. For the sake of clarity, the features are not in scale. The BglII sites in the ends are used to excise the transposons from their carrier plasmids. The sequences of the transposon-containing plasmids pHTH19 (Kan/Neo-loxP-Mu) and pHTH24 (Kan/Neo-loxP-FRT-Mu) are available upon request.
    Figure Legend Snippet: Details of the genetic tools used. (A) Structure and characteristic elements of the multicopy cloning vector pHTH22 with unique restriction sites indicated. The gene region encoding HSV thymidine kinase (TK) under the control of mouse phosphoglycerate kinase promoter and terminator is shown with a black curved arrow. The vector portion of the plasmid including the gene for ampicillin resistance (Amp R ) and pUC19 origin of replication (ori) are shown with gray symbols (curved arrow and rectangle). (B) The nucleotide sequence of the polylinker and its flanking regions in pHTH22. The polylinker is shown in boldface with enzyme recognition sites underlined. The arrows indicate the binding sites of primers that were used for the confirmatory sequencing of genomic inserts' ends. (C) Structures of the Kan/Neo-loxP-Mu and Kan/Neo-loxP-FRT-Mu transposons. The transposons contain bacterial (p bact ) and eukaryotic (p SV40 ) promoters (short arrows), a marker gene (Kan/Neo) conferring resistance to kanamycin in bacteria and G418 in eukaryotes (black arrow), and the HSV thymidine kinase polyadenylation (HSV TK polyA) signal (small rectangle). The loxP sites are indicated by triangles and FRT sites by pentagons. The rectangles in the transposon ends indicate 50 bp of Mu R-end DNA sequences in inverted orientation relative to each other. For the sake of clarity, the features are not in scale. The BglII sites in the ends are used to excise the transposons from their carrier plasmids. The sequences of the transposon-containing plasmids pHTH19 (Kan/Neo-loxP-Mu) and pHTH24 (Kan/Neo-loxP-FRT-Mu) are available upon request.

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Binding Assay, Marker

    6) Product Images from "Mycofumigation by the Volatile Organic Compound-Producing Fungus Muscodor albus Induces Bacterial Cell Death through DNA Damage"

    Article Title: Mycofumigation by the Volatile Organic Compound-Producing Fungus Muscodor albus Induces Bacterial Cell Death through DNA Damage

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03294-14

    Growth curve of E. coli DH10B and E. coli DH10B expressing recA during treatment with VOCs. (a) DH10B growth curve (black) as measured spectrophotometrically by OD 600 and upon treatment with M. albus VOCs (red). (b) Growth curve of DH10B expressing pUC19
    Figure Legend Snippet: Growth curve of E. coli DH10B and E. coli DH10B expressing recA during treatment with VOCs. (a) DH10B growth curve (black) as measured spectrophotometrically by OD 600 and upon treatment with M. albus VOCs (red). (b) Growth curve of DH10B expressing pUC19

    Techniques Used: Expressing

    7) Product Images from "Cooperative cluster formation, DNA bending and base-flipping by O6-alkylguanine-DNA alkyltransferase"

    Article Title: Cooperative cluster formation, DNA bending and base-flipping by O6-alkylguanine-DNA alkyltransferase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks574

    Analysis of AGT–DNA interactions by analytical ultracentrifugation. ( A ) Sedimentation equilibrium data for an AGT–DNA mixture obtained at 20 ± 0.1°C. This sample contained the 1000-bp DNA fragment (0.015 µM) and AGT protein (3.5 µM) in 10 mM Tris (pH 7.6 at 20°C), 1 mM EDTA, 100 mM NaCl and 1 mM DTT. Radial scans taken at 3000 rpm (red), 4500 rpm (blue) and 6000 rpm (green) are shown with vertical offsets for clarity. The smooth curves correspond to fits of equation 1 to these data. Small, symmetrically distributed residuals (upper panel) indicate that the two-species model represented by equation 1 is consistent with the mass distributions of DNA in these samples. ( B ) Dependence of binding stoichiometry on free AGT concentration for the 1000-bp fragment (upper panel) and linear pUC19 DNA (lower panel). Stoichiometries were inferred from weight-average molecular weights measured at sedimentation equilibrium. Error bars are 95% confidence limits for the individual parameters. The smooth curve is an isotherm calculated with equation 4 using parameters determined from the Scatchard plots shown in the insets. Insets: Scatchard plots for the data ensembles shown in the main panels. The solid curves are fits of equation 4 , returning K = 9667 ± 1499, ω = 35.9 ± 6.8 and s = 6.32 ± 0.12 for binding the 1000-bp fragment and K = 7960 ± 916, ω = 44.2 ± 3.8 and s = 6.81 ± 0.14 for binding linear pUC19 DNA.
    Figure Legend Snippet: Analysis of AGT–DNA interactions by analytical ultracentrifugation. ( A ) Sedimentation equilibrium data for an AGT–DNA mixture obtained at 20 ± 0.1°C. This sample contained the 1000-bp DNA fragment (0.015 µM) and AGT protein (3.5 µM) in 10 mM Tris (pH 7.6 at 20°C), 1 mM EDTA, 100 mM NaCl and 1 mM DTT. Radial scans taken at 3000 rpm (red), 4500 rpm (blue) and 6000 rpm (green) are shown with vertical offsets for clarity. The smooth curves correspond to fits of equation 1 to these data. Small, symmetrically distributed residuals (upper panel) indicate that the two-species model represented by equation 1 is consistent with the mass distributions of DNA in these samples. ( B ) Dependence of binding stoichiometry on free AGT concentration for the 1000-bp fragment (upper panel) and linear pUC19 DNA (lower panel). Stoichiometries were inferred from weight-average molecular weights measured at sedimentation equilibrium. Error bars are 95% confidence limits for the individual parameters. The smooth curve is an isotherm calculated with equation 4 using parameters determined from the Scatchard plots shown in the insets. Insets: Scatchard plots for the data ensembles shown in the main panels. The solid curves are fits of equation 4 , returning K = 9667 ± 1499, ω = 35.9 ± 6.8 and s = 6.32 ± 0.12 for binding the 1000-bp fragment and K = 7960 ± 916, ω = 44.2 ± 3.8 and s = 6.81 ± 0.14 for binding linear pUC19 DNA.

    Techniques Used: Sedimentation, Binding Assay, Concentration Assay

    Comparison of predicted torsional free energies (Δ G (twist)) and cooperative free energies (Δ G (cooperative)) for clusters of 4–12 AGT proteins. Δ G is given as its absolute value. The quadratic model ( equation 6 ) was used to calculate the cumulative Δ G (twist) as functions of proteins/cluster ( N ) for displacements of 6–10°/protein. The red and blue lines give |Δ G (cooperative)| for the addition of a single protein molecule to complexes formed on linear pUC19 DNA and the 1000-bp fragment, respectively. The intersections of these functions indicate where Δ G (twist) = −Δ G (cooperative). A vertical gray line is plotted for a cluster length of 6.7 proteins, the mean of all length estimates for [AGT] ≥ 6 µM, for the 1000-bp DNA. The lighter gray zone spans between means of minimum and maximum estimates of cluster length.
    Figure Legend Snippet: Comparison of predicted torsional free energies (Δ G (twist)) and cooperative free energies (Δ G (cooperative)) for clusters of 4–12 AGT proteins. Δ G is given as its absolute value. The quadratic model ( equation 6 ) was used to calculate the cumulative Δ G (twist) as functions of proteins/cluster ( N ) for displacements of 6–10°/protein. The red and blue lines give |Δ G (cooperative)| for the addition of a single protein molecule to complexes formed on linear pUC19 DNA and the 1000-bp fragment, respectively. The intersections of these functions indicate where Δ G (twist) = −Δ G (cooperative). A vertical gray line is plotted for a cluster length of 6.7 proteins, the mean of all length estimates for [AGT] ≥ 6 µM, for the 1000-bp DNA. The lighter gray zone spans between means of minimum and maximum estimates of cluster length.

    Techniques Used:

    Visualization of AGT cooperative units on DNA. AFM images of the 1000-bp DNA fragment in the absence of protein ( A ) and after incubation with AGT at 6 μM at a protein:DNA ratio of 100:1 ( B ) or 12 μM AGT and protein:DNA ratio of 200:1 ( C ) show increasing numbers of AGT clusters on the DNA with increasing protein concentration. A three-dimensional projection of the data from (C) is shown in ( D ). Results are not dependent on the DNA substrate used: the image in ( E ) shows similar results for a linearized pUC19 plasmid substrate after incubation with 12 μM AGT. To test whether protein distributions were affected by the deposition process, samples containing linear pUC19 (60 nM) and AGT (12 µM) were crosslinked with glutaraldehyde (0.1% glutaraldehyde, 10 min at 37°C) and then applied to the mica substrate ( F ). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis ( Supplementary Figure S4 ) showed that > 50% of AGT molecules were crosslinked to a neighbor by this treatment. Arrows in (B) indicate AGT clusters on the DNA fragments. Images are 1 μm × 1 μm (A, B, E, F) and 170 nm × 170 nm (C, D).
    Figure Legend Snippet: Visualization of AGT cooperative units on DNA. AFM images of the 1000-bp DNA fragment in the absence of protein ( A ) and after incubation with AGT at 6 μM at a protein:DNA ratio of 100:1 ( B ) or 12 μM AGT and protein:DNA ratio of 200:1 ( C ) show increasing numbers of AGT clusters on the DNA with increasing protein concentration. A three-dimensional projection of the data from (C) is shown in ( D ). Results are not dependent on the DNA substrate used: the image in ( E ) shows similar results for a linearized pUC19 plasmid substrate after incubation with 12 μM AGT. To test whether protein distributions were affected by the deposition process, samples containing linear pUC19 (60 nM) and AGT (12 µM) were crosslinked with glutaraldehyde (0.1% glutaraldehyde, 10 min at 37°C) and then applied to the mica substrate ( F ). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis ( Supplementary Figure S4 ) showed that > 50% of AGT molecules were crosslinked to a neighbor by this treatment. Arrows in (B) indicate AGT clusters on the DNA fragments. Images are 1 μm × 1 μm (A, B, E, F) and 170 nm × 170 nm (C, D).

    Techniques Used: Incubation, Protein Concentration, Plasmid Preparation, Polyacrylamide Gel Electrophoresis

    8) Product Images from "Use of DNA Microarrays for Rapid Genotyping of TEM Beta-Lactamases That Confer Resistance"

    Article Title: Use of DNA Microarrays for Rapid Genotyping of TEM Beta-Lactamases That Confer Resistance

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.42.8.3766-3774.2004

    (a) Mean signal intensities for each SNP position and corresponding probe (A, G, C, and T refer to the base at the central position) from a hybridization experiment with 100 ng of bla TEM from pUC19 (NT/F = 57) ( n = 3). (b) Number of probe MMs depending on the MM/PM ratio from the experiment for which the results are shown in panel a.
    Figure Legend Snippet: (a) Mean signal intensities for each SNP position and corresponding probe (A, G, C, and T refer to the base at the central position) from a hybridization experiment with 100 ng of bla TEM from pUC19 (NT/F = 57) ( n = 3). (b) Number of probe MMs depending on the MM/PM ratio from the experiment for which the results are shown in panel a.

    Techniques Used: Hybridization, Transmission Electron Microscopy

    Related Articles

    Sequencing:

    Article Title: High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042 ▿
    Article Snippet: The mixture was vortexed for 30 s, incubated at 42°C for 40 min, and spread on uracil dropout plates. .. S. cerevisiae centromere/autonomously replicating sequence (ARS) plasmid pRS316 ( ) containing Sc URA3 as a marker was used for K. marxianus transformations. pRS316 (4.8 kb) was linearized by digestion with SmaI (New England Biolabs, MA). .. Chromosomal DNA from BY4704, K. marxianus DMKU3-1042, a K. marxianus ura3 mutant (RAK3605), and Sc URA3 transformants of the ura3 mutant was isolated and digested with BamHI (Roche Diagnostics GmbH, Mannheim, Germany).

    Plasmid Preparation:

    Article Title: High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042 ▿
    Article Snippet: The mixture was vortexed for 30 s, incubated at 42°C for 40 min, and spread on uracil dropout plates. .. S. cerevisiae centromere/autonomously replicating sequence (ARS) plasmid pRS316 ( ) containing Sc URA3 as a marker was used for K. marxianus transformations. pRS316 (4.8 kb) was linearized by digestion with SmaI (New England Biolabs, MA). .. Chromosomal DNA from BY4704, K. marxianus DMKU3-1042, a K. marxianus ura3 mutant (RAK3605), and Sc URA3 transformants of the ura3 mutant was isolated and digested with BamHI (Roche Diagnostics GmbH, Mannheim, Germany).

    Marker:

    Article Title: High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042 ▿
    Article Snippet: The mixture was vortexed for 30 s, incubated at 42°C for 40 min, and spread on uracil dropout plates. .. S. cerevisiae centromere/autonomously replicating sequence (ARS) plasmid pRS316 ( ) containing Sc URA3 as a marker was used for K. marxianus transformations. pRS316 (4.8 kb) was linearized by digestion with SmaI (New England Biolabs, MA). .. Chromosomal DNA from BY4704, K. marxianus DMKU3-1042, a K. marxianus ura3 mutant (RAK3605), and Sc URA3 transformants of the ura3 mutant was isolated and digested with BamHI (Roche Diagnostics GmbH, Mannheim, Germany).

    other:

    Article Title: An efficient method for the construction of artificial, concatemeric DNA, RNA and proteins with genetically programmed functions, using a novel, vector-enzymatic DNA fragment amplification-expression technology
    Article Snippet: Media and reagentsREases SapI, BspQI, SmaI were from New England Biolabs (Ipswich, MA, USA).

    Methylation:

    Article Title: Maternal protein restriction induces renal AT2R promoter hypomethylation in salt‐sensitive, hypertensive rats, et al. Maternal protein restriction induces renal AT2R promoter hypomethylation in salt‐sensitive, hypertensive rats
    Article Snippet: Partial methylation was performed as according to Tamura et al. ( ). .. Methylated DNA levels were determined via digestion with SmaI and XbaI (New England Biolabs), followed by electrophoresis. .. 2.2 Cell culture and transfection Human embryonic kidney 293t cells were cultured in Dulbecco's modified eagle medium (DMEM; Sigma‐Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin‐streptomycin (Thermo Fisher Scientific).

    Electrophoresis:

    Article Title: Maternal protein restriction induces renal AT2R promoter hypomethylation in salt‐sensitive, hypertensive rats, et al. Maternal protein restriction induces renal AT2R promoter hypomethylation in salt‐sensitive, hypertensive rats
    Article Snippet: Partial methylation was performed as according to Tamura et al. ( ). .. Methylated DNA levels were determined via digestion with SmaI and XbaI (New England Biolabs), followed by electrophoresis. .. 2.2 Cell culture and transfection Human embryonic kidney 293t cells were cultured in Dulbecco's modified eagle medium (DMEM; Sigma‐Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin‐streptomycin (Thermo Fisher Scientific).

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  • 99
    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.
    Plasmid Puc19, 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
    https://www.bioz.com/result/plasmid puc19/product/New England Biolabs
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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
    https://www.bioz.com/result/puc19 vector dna/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    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

    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

    Calcium Competent C. novyi Transformations. (A) Schematic representation of experimental flow. (B) Resulting colony forming units after Clostridium novyi cells underwent calcium competent transformation with the E. coli plasmid pUC19, which contains a gene encoding ampicillin resistance. Statistical significance was determined to be p

    Journal: Frontiers in Microbiology

    Article Title: Methods and Techniques to Facilitate the Development of Clostridium novyi NT as an Effective, Therapeutic Oncolytic Bacteria

    doi: 10.3389/fmicb.2021.624618

    Figure Lengend Snippet: Calcium Competent C. novyi Transformations. (A) Schematic representation of experimental flow. (B) Resulting colony forming units after Clostridium novyi cells underwent calcium competent transformation with the E. coli plasmid pUC19, which contains a gene encoding ampicillin resistance. Statistical significance was determined to be p

    Article Snippet: Subsequently, 5 μg of either purified pUC19 control plasmid (New England BioLabs Inc.) or the purified pKMD002 plasmid was added to an empty prechilled 15 mL tube (4°C).

    Techniques: Transformation Assay, Plasmid Preparation