paci  (New England Biolabs)


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    Structured Review

    New England Biolabs paci
    Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme <t>PacI</t> and the nicking enzyme <t>Nt.BbvCI</t> , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .
    Paci, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    paci - by Bioz Stars, 2022-07
    86/100 stars

    Images

    1) Product Images from "UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals"

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals

    Journal: Plant Methods

    doi: 10.1186/1746-4811-6-15

    Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .
    Figure Legend Snippet: Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction

    2) Product Images from "RNA aptamer inhibitors of a restriction endonuclease"

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv702

    In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.
    Figure Legend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Techniques Used: In Vitro, Selection

    3) Product Images from "Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments"

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl635

    Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.
    Figure Legend Snippet: Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Techniques Used: Clone Assay, Sequencing, Synthesized, Construct, Plasmid Preparation, Generated

    Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.
    Figure Legend Snippet: Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Incubation, Transformation Assay, Ligation, Generated

    4) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

    5) Product Images from "RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences"

    Article Title: RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2016.02.003

    The RNA-ID reporter and its derivatives. (A) In the RNA-ID vector, expression of two reporters GFP and RFP is driven by the bidirectional GAL1,10 promoter. The GFP reporter is a fusion protein encoding a 3C protease site, an HA epitope, His6, (marked by dark box) followed by superfolder GFP. Putative regulatory sequences are inserted into the PacI, BbrPI sites (GFP) or into the SwaI site (RFP) using LIC cloning. The GFP lacks a start codon, allowing insertion of sequences upstream or within the coding sequence. (B) The Renilla luciferase-GFP fusion protein allows for analysis of the upstream nascent polypeptide and uses the same restriction sites for cloning. (C) In the GLN4 -GFP fusion protein, varying lengths of GLN4 can be PCR amplified to allow for the insertion of sequences at different locations relative to the start codon.
    Figure Legend Snippet: The RNA-ID reporter and its derivatives. (A) In the RNA-ID vector, expression of two reporters GFP and RFP is driven by the bidirectional GAL1,10 promoter. The GFP reporter is a fusion protein encoding a 3C protease site, an HA epitope, His6, (marked by dark box) followed by superfolder GFP. Putative regulatory sequences are inserted into the PacI, BbrPI sites (GFP) or into the SwaI site (RFP) using LIC cloning. The GFP lacks a start codon, allowing insertion of sequences upstream or within the coding sequence. (B) The Renilla luciferase-GFP fusion protein allows for analysis of the upstream nascent polypeptide and uses the same restriction sites for cloning. (C) In the GLN4 -GFP fusion protein, varying lengths of GLN4 can be PCR amplified to allow for the insertion of sequences at different locations relative to the start codon.

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

    6) Product Images from "Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state"

    Article Title: Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3383

    Symmetric barcoding and amplification of chromatin fragments A) Barcode adapters (top) are 64 bp double-stranded oligonucleotides with universal primers, barcode sequences and restriction sites, whose symmetric design allows ligation on either side. Schematic (bottom left) depicts possible outcomes of ligation in drops, including symmetrically labeled nucleosomes, asymmetrically labeled nucleosomes, and adapter concatemers. Concatemers are removed by digestion of PacI sites formed by adapter juxtaposition (bottom center), allowing selective PCR amplification of symmetrically adapted chromatin fragments (bottom right). See also Supplementary Figure 2 . B) Gel electrophoresis for DNA products at successive assay stages: left : DNA ladder; MNase : DNA fragments purified after capture, lysis and MNase digestion of single cells in drops confirm efficient digestion to mononucleosomes (∼1 million drops collected); Concat : Illumina library prepared from adaptor-ligated chromatin fragments without PacI digestion reveals overwhelming concatemer bias. Library : Illumina library prepared from adaptor-ligated chromatin fragments digested with PacI, reveals appropriate MNase digestion pattern, shifted by the size of barcode and Illumina adapters. C) Pie charts depict numbers of uniquely aligned sequencing read that satisfy successive filtering criteria (values reflect data from 100 single cells, averaged over 82 trials). We select reads that have barcode sequences on both ends (top) with matching sequence (middle). We then apply a Poisson model to identify barcodes that represent single cells (bottom). D) Heatmap depicts homogeneity of barcode selection. Barcodes (rows) are colored according to their relative prevalence (rank order) across 37 experiments (columns). The absence of bias towards particular barcodes (light or dark horizontal stripes) indicates the homogeneity of the barcode library. The mean normalized rank over all barcodes (right) is close to 0.5, consistent with balanced representation. E) Stability of the barcode library emulsion over time. The fraction of reads with matching barcodes on both ends is plotted as a function of time from encapsulation of the barcode library. F) The microfluidics system was applied to barcode a mixed suspension of human and mouse cells. For each barcode, plot depicts the number of reads aligning to the mouse genome (y-axis) versus the number of reads aligning to the human genome (x-axis). The data suggest that a vast majority of barcodes is unique to a single cell.
    Figure Legend Snippet: Symmetric barcoding and amplification of chromatin fragments A) Barcode adapters (top) are 64 bp double-stranded oligonucleotides with universal primers, barcode sequences and restriction sites, whose symmetric design allows ligation on either side. Schematic (bottom left) depicts possible outcomes of ligation in drops, including symmetrically labeled nucleosomes, asymmetrically labeled nucleosomes, and adapter concatemers. Concatemers are removed by digestion of PacI sites formed by adapter juxtaposition (bottom center), allowing selective PCR amplification of symmetrically adapted chromatin fragments (bottom right). See also Supplementary Figure 2 . B) Gel electrophoresis for DNA products at successive assay stages: left : DNA ladder; MNase : DNA fragments purified after capture, lysis and MNase digestion of single cells in drops confirm efficient digestion to mononucleosomes (∼1 million drops collected); Concat : Illumina library prepared from adaptor-ligated chromatin fragments without PacI digestion reveals overwhelming concatemer bias. Library : Illumina library prepared from adaptor-ligated chromatin fragments digested with PacI, reveals appropriate MNase digestion pattern, shifted by the size of barcode and Illumina adapters. C) Pie charts depict numbers of uniquely aligned sequencing read that satisfy successive filtering criteria (values reflect data from 100 single cells, averaged over 82 trials). We select reads that have barcode sequences on both ends (top) with matching sequence (middle). We then apply a Poisson model to identify barcodes that represent single cells (bottom). D) Heatmap depicts homogeneity of barcode selection. Barcodes (rows) are colored according to their relative prevalence (rank order) across 37 experiments (columns). The absence of bias towards particular barcodes (light or dark horizontal stripes) indicates the homogeneity of the barcode library. The mean normalized rank over all barcodes (right) is close to 0.5, consistent with balanced representation. E) Stability of the barcode library emulsion over time. The fraction of reads with matching barcodes on both ends is plotted as a function of time from encapsulation of the barcode library. F) The microfluidics system was applied to barcode a mixed suspension of human and mouse cells. For each barcode, plot depicts the number of reads aligning to the mouse genome (y-axis) versus the number of reads aligning to the human genome (x-axis). The data suggest that a vast majority of barcodes is unique to a single cell.

    Techniques Used: Amplification, Ligation, Labeling, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Purification, Lysis, Sequencing, Selection

    7) Product Images from "Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91"

    Article Title: Identification and Characterization of Putative Virulence Genes and Gene Clusters in Aeromonas hydrophila PPD134/91

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.71.8.4469-4477.2005

    Physical map of A. hydrophila PPD134/91. The locations of virulence genes and clusters were determined with respect to PacI fragments.
    Figure Legend Snippet: Physical map of A. hydrophila PPD134/91. The locations of virulence genes and clusters were determined with respect to PacI fragments.

    Techniques Used:

    8) Product Images from "Identification and Characterization of a Phase-Variable Element That Regulates the Autotransporter UpaE in Uropathogenic Escherichia coli"

    Article Title: Identification and Characterization of a Phase-Variable Element That Regulates the Autotransporter UpaE in Uropathogenic Escherichia coli

    Journal: mBio

    doi: 10.1128/mBio.01360-18

    Assessment of each recombinase’s ability to catalyze inversion of ipuS in vitro . Ethidium bromide-stained electrophoretic gels of PacI-digested PCR products are shown. Lanes 1 and 2 contain digested PCR products from CFT073 ipuS phase locked-ON (WAM5064) and -OFF (WAM5065) strains generated by 5-way recombinase deletion. Lanes 3 and 4 contain digested PCR products from vector-only controls (WAM5070, WAM5079, WAM5074, and WAM5083). Lanes 5 to 10 contain PCR products from the locked-OFF strain WAM5065 (top panel) or locked-ON strain WAM5064 (bottom panel) after transforming each with a recombinant plasmid containing the indicated recombinase. Both the full-length and truncated forms of ipuA were tested for activity (lanes 9 and 10).
    Figure Legend Snippet: Assessment of each recombinase’s ability to catalyze inversion of ipuS in vitro . Ethidium bromide-stained electrophoretic gels of PacI-digested PCR products are shown. Lanes 1 and 2 contain digested PCR products from CFT073 ipuS phase locked-ON (WAM5064) and -OFF (WAM5065) strains generated by 5-way recombinase deletion. Lanes 3 and 4 contain digested PCR products from vector-only controls (WAM5070, WAM5079, WAM5074, and WAM5083). Lanes 5 to 10 contain PCR products from the locked-OFF strain WAM5065 (top panel) or locked-ON strain WAM5064 (bottom panel) after transforming each with a recombinant plasmid containing the indicated recombinase. Both the full-length and truncated forms of ipuA were tested for activity (lanes 9 and 10).

    Techniques Used: In Vitro, Staining, Polymerase Chain Reaction, Generated, Plasmid Preparation, Recombinant, Activity Assay

    9) Product Images from "Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains ▿ PAO1 Laboratory Strains ▿ †"

    Article Title: Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains ▿ PAO1 Laboratory Strains ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01515-09

    Genomic differences in PAO1 sublines. (A) PFGE of SwaI- and PacI-digested DNA of PAO1 sublines PAO1-DSM (lanes a) and PAO1-UW (lanes b). Restriction fragments that are altered due to the chromosomal inversion are labeled. (B) Circular maps of PAO1 sublines
    Figure Legend Snippet: Genomic differences in PAO1 sublines. (A) PFGE of SwaI- and PacI-digested DNA of PAO1 sublines PAO1-DSM (lanes a) and PAO1-UW (lanes b). Restriction fragments that are altered due to the chromosomal inversion are labeled. (B) Circular maps of PAO1 sublines

    Techniques Used: Labeling

    10) Product Images from "Identification and Characterization of a Phase-Variable Element That Regulates the Autotransporter UpaE in Uropathogenic Escherichia coli"

    Article Title: Identification and Characterization of a Phase-Variable Element That Regulates the Autotransporter UpaE in Uropathogenic Escherichia coli

    Journal: mBio

    doi: 10.1128/mBio.01360-18

    Assessment of each recombinase’s ability to catalyze inversion of ipuS in vitro . Ethidium bromide-stained electrophoretic gels of PacI-digested PCR products are shown. Lanes 1 and 2 contain digested PCR products from CFT073 ipuS phase locked-ON (WAM5064) and -OFF (WAM5065) strains generated by 5-way recombinase deletion. Lanes 3 and 4 contain digested PCR products from vector-only controls (WAM5070, WAM5079, WAM5074, and WAM5083). Lanes 5 to 10 contain PCR products from the locked-OFF strain WAM5065 (top panel) or locked-ON strain WAM5064 (bottom panel) after transforming each with a recombinant plasmid containing the indicated recombinase. Both the full-length and truncated forms of ipuA were tested for activity (lanes 9 and 10).
    Figure Legend Snippet: Assessment of each recombinase’s ability to catalyze inversion of ipuS in vitro . Ethidium bromide-stained electrophoretic gels of PacI-digested PCR products are shown. Lanes 1 and 2 contain digested PCR products from CFT073 ipuS phase locked-ON (WAM5064) and -OFF (WAM5065) strains generated by 5-way recombinase deletion. Lanes 3 and 4 contain digested PCR products from vector-only controls (WAM5070, WAM5079, WAM5074, and WAM5083). Lanes 5 to 10 contain PCR products from the locked-OFF strain WAM5065 (top panel) or locked-ON strain WAM5064 (bottom panel) after transforming each with a recombinant plasmid containing the indicated recombinase. Both the full-length and truncated forms of ipuA were tested for activity (lanes 9 and 10).

    Techniques Used: In Vitro, Staining, Polymerase Chain Reaction, Generated, Plasmid Preparation, Recombinant, Activity Assay

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    New England Biolabs paci
    Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme <t>PacI</t> and the nicking enzyme <t>Nt.BbvCI</t> , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .
    Paci, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/paci/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    paci - by Bioz Stars, 2022-07
    86/100 stars
      Buy from Supplier

    95
    New England Biolabs restriction enzyme paci
    Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of <t>nucleosome</t> and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d <t>PacI</t> digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.
    Restriction Enzyme Paci, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction enzyme paci/product/New England Biolabs
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    Price from $9.99 to $1999.99
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    Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .

    Journal: Plant Methods

    Article Title: UCE: A uracil excision (USER(TM))-based toolbox for transformation of cereals

    doi: 10.1186/1746-4811-6-15

    Figure Lengend Snippet: Overview of the USER™ reaction . A . The vector carrying the USER™ cassette is digested simultaneously with the restriction enzyme PacI and the nicking enzyme Nt.BbvCI , which creates 9 nt long 3' overhangs. The insert, which is to be fused with the vector, is amplified by PCR using standard PCR primers for the insert, but with the addition of 9 nt complementary to the USER™ site, including a uracil base. In the USER™ reaction USER ™ enzymes remove the uracil creating 9 nt long 5' overhangs complementary to the overhangs of the digested vector. Spontaneous annealing will then result in the desired fusion of the PCR product and vector. Bases in bold indicates the recognition sites of the relevant enzymes, and the arrowheads indicates the site of cleavage. B . The two USER™ cassettes designed and used in this study, the USER TC-TG and the USER TC-CC .

    Article Snippet: Generation of single strand DNA overhangs in vectors containing a USER™ site Vectors containing a USER™ site were made ready for cloning by digesting 5 μg of the vector with 6 μl of the enzyme PacI (New England Biolabs) for 15 hours at 37°C in a total volume of 200 μl, followed by 2 hours of digest with freshly added 2 μl PacI and 3 μl Nt.BbvCI (New England Biolabs) for 2 hours at 37°C.

    Techniques: Plasmid Preparation, Amplification, Polymerase Chain Reaction

    In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Article Snippet: Protein expression and purification Commercial REases and purified preparations of BamHI (E111A mutant), KpnI and PacI for in vitro selection and binding assays were obtained from New England Biolabs.

    Techniques: In Vitro, Selection

    Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Journal: Nucleic Acids Research

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    doi: 10.1093/nar/gkl635

    Figure Lengend Snippet: Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.

    Article Snippet: A total of 5 μg plasmid DNA of the constructed vectors were digested with 40 U PacI (New England Biolabs) overnight at 37°C in a total volume of 200 μl.

    Techniques: Clone Assay, Sequencing, Synthesized, Construct, Plasmid Preparation, Generated

    Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Journal: Nucleic Acids Research

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    doi: 10.1093/nar/gkl635

    Figure Lengend Snippet: Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo ® C x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

    Article Snippet: A total of 5 μg plasmid DNA of the constructed vectors were digested with 40 U PacI (New England Biolabs) overnight at 37°C in a total volume of 200 μl.

    Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Incubation, Transformation Assay, Ligation, Generated

    Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of nucleosome and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d PacI digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.

    Journal: Nature Communications

    Article Title: Site-specific ubiquitylation acts as a regulator of linker histone H1

    doi: 10.1038/s41467-021-23636-5

    Figure Lengend Snippet: Impact of H1.2 KxUb variants on chromatosome assembly. a Schematic overview of 12-mer chromatosome array formation containing H1.2 or H1.2 KxUbs. b EMSA of different arrays analyzed; crDNA indicates competitor DNA. c Overview of MNase digestion assay which probes DNA accessibility of nucleosome and chromatosome arrays (left). The arrays were treated with MNase for indicated times, digested by proteinase K, and analyzed by agarose gel electrophoresis. Undigested arrays were quantified and plotted over time (middle). Shown are mean values +/− standard deviation, n = 3. For statistical analysis, the area under the curve for the digestion of the different arrays was determined and analyzed (right); shown are mean values +/− standard deviation, n = 3, one-way ANOVA with Tukey’s multiple comparisons test with α = 0.05, 95% confidence interval, exact p values with p ≤ 0.05 are shown, ns (not significant) p > 0.05. d PacI digestion assay. PacI cleaves the array site-specifically within the linker DNA between the repetitive nucleosome positioning sequences resulting in the formation of mono-nucleosomes and mono-chromatosomes.

    Article Snippet: Analysis of nucleosome and chromatosome arrays PacI digest : To analyze saturation of the nucleosome array and evaluate H1.2-binding to the nucleosome, 79 fmol arrays were digested with 5 U of the restriction enzyme PacI (NEB) in 10 mM HEPES/KOH (pH 7.6), 50 mM KCl, 1.5 mM MgCl2, 0.5 mM EGTA for 1 h at 37 °C.

    Techniques: Agarose Gel Electrophoresis, Standard Deviation