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    Name:
    Nb BtsI
    Description:
    Nb BtsI 1 000 units
    Catalog Number:
    R0707L
    Price:
    70
    Category:
    Restriction Enzymes
    Size:
    1 000 units
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    New England Biolabs nb btsi
    Nb BtsI
    Nb BtsI 1 000 units
    https://www.bioz.com/result/nb btsi/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nb btsi - by Bioz Stars, 2021-09
    94/100 stars

    Images

    1) Product Images from "Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq"

    Article Title: Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq

    Journal: Nature protocols

    doi: 10.1038/nprot.2015.099

    Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p
    Figure Legend Snippet: Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p

    Techniques Used: Sonication, Agarose Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Selection

    2) Product Images from "Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq"

    Article Title: Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq

    Journal: Nature protocols

    doi: 10.1038/nprot.2015.099

    Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p
    Figure Legend Snippet: Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p

    Techniques Used: Sonication, Agarose Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Selection

    3) Product Images from "Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research"

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research

    Journal: Plants

    doi: 10.3390/plants9091090

    Schematic diagram of NEMDA strategy for plant CRISPR/Cas9 multiplex CRISPR/Cas9 vector construction and its mutation detection of transgenic plants. ( A ) Structural features of pYLCRISPR/Cas9Pubi-H with two Bsa I sites in the flanking sides of a lethal ccdB gene [ 22 ]. ( B ) The five sgRNA expression cassettes have Bsa I and Nb.BtsI recognition sites with differently designed target sequence adaptors (different colors). ( C ) One-step construction of multiple sgRNA expression cassettes into the CRISPR/Cas9 vector. Each sgRNA expression cassette is PCR amplified using primers carrying Bas I and Nb.BtsI sites and 10-bp complementary overhanging sequences. These PCR products were mixed with pYLCRISPR/Cas9Pubi-H vector, and treated with Nb.BtvCI and Bsa I, in one tube, for digestion, heating, annealing, and ligation. The reaction product was transferred into E. coli competent cells to produce pYLCRISPR/Cas9Pubi-T5s ( D ). ( E ) Results of multiplex genome editing for three rice genes (five targets) using the above plasmid. Each target site was effectively edited by randomly selecting five T-A clones for sequencing. Nucleotides variations of insertion or deletion present as red in targets. The PAM (protospacer adjacent motif) sequences of the target site are underlined. The number in brackets indicates the proportion of variants.
    Figure Legend Snippet: Schematic diagram of NEMDA strategy for plant CRISPR/Cas9 multiplex CRISPR/Cas9 vector construction and its mutation detection of transgenic plants. ( A ) Structural features of pYLCRISPR/Cas9Pubi-H with two Bsa I sites in the flanking sides of a lethal ccdB gene [ 22 ]. ( B ) The five sgRNA expression cassettes have Bsa I and Nb.BtsI recognition sites with differently designed target sequence adaptors (different colors). ( C ) One-step construction of multiple sgRNA expression cassettes into the CRISPR/Cas9 vector. Each sgRNA expression cassette is PCR amplified using primers carrying Bas I and Nb.BtsI sites and 10-bp complementary overhanging sequences. These PCR products were mixed with pYLCRISPR/Cas9Pubi-H vector, and treated with Nb.BtvCI and Bsa I, in one tube, for digestion, heating, annealing, and ligation. The reaction product was transferred into E. coli competent cells to produce pYLCRISPR/Cas9Pubi-T5s ( D ). ( E ) Results of multiplex genome editing for three rice genes (five targets) using the above plasmid. Each target site was effectively edited by randomly selecting five T-A clones for sequencing. Nucleotides variations of insertion or deletion present as red in targets. The PAM (protospacer adjacent motif) sequences of the target site are underlined. The number in brackets indicates the proportion of variants.

    Techniques Used: CRISPR, Multiplex Assay, Plasmid Preparation, Mutagenesis, Transgenic Assay, Expressing, Sequencing, Polymerase Chain Reaction, Amplification, Ligation, Clone Assay

    Schematic diagram of the nicking endonucleases-mediated DNA assembly (NEMDA) strategy for plant ihpRNA vector constructions. ( A ) The pRNAi-NE includes the 35S CaMV promoter, the Catalase intron, the ccdB gene, and four Nb.BtvCI and Xba I recognition sites with differently designed adaptors (different colors). ( B ) The sense and antisense PCR products have four Nb.BtsI recognition sites with differently designed adaptors (different colors). ( C ) One-step construction of an ihpRNA vector. The target fragments of the gene of interest are PCR amplified using gene-specific primers carrying Nb.BtsI sites and adaptors complementary to the appropriate sequences on the vector. The unpurified PCR products digested by Nb.BtsI are mixed, in one tube, with unpurified pRNAi-NE vector digested by Nb.BtvCI and Xba I, for heat-inactivation of these restriction endonucleases and melting out of the nicked end strands, annealing. The T4 DNA ligase also can be used to increase cloning efficiency. The reaction product is transferred into E. coli competent cells to produce the pRNAi plasmid ( D ).
    Figure Legend Snippet: Schematic diagram of the nicking endonucleases-mediated DNA assembly (NEMDA) strategy for plant ihpRNA vector constructions. ( A ) The pRNAi-NE includes the 35S CaMV promoter, the Catalase intron, the ccdB gene, and four Nb.BtvCI and Xba I recognition sites with differently designed adaptors (different colors). ( B ) The sense and antisense PCR products have four Nb.BtsI recognition sites with differently designed adaptors (different colors). ( C ) One-step construction of an ihpRNA vector. The target fragments of the gene of interest are PCR amplified using gene-specific primers carrying Nb.BtsI sites and adaptors complementary to the appropriate sequences on the vector. The unpurified PCR products digested by Nb.BtsI are mixed, in one tube, with unpurified pRNAi-NE vector digested by Nb.BtvCI and Xba I, for heat-inactivation of these restriction endonucleases and melting out of the nicked end strands, annealing. The T4 DNA ligase also can be used to increase cloning efficiency. The reaction product is transferred into E. coli competent cells to produce the pRNAi plasmid ( D ).

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

    4) Product Images from "Disintegration promotes proto-spacer integration by the Cas1-Cas2 complex"

    Article Title: Disintegration promotes proto-spacer integration by the Cas1-Cas2 complex

    Journal: bioRxiv

    doi: 10.1101/2020.12.21.423798

    Cas1-Cas2 integrates and disintegrates proto-spacers at the CRISPR locus in a supercoiled plasmid. A . Illustration of protospacer (PS, green) integration into a minimal CRISPR locus comprised of the leader (L, red), repeat (R, yellow) and spacer (S, blue). The reaction follows the cut- and-paste DNA transposition mechanism with a duplication of the repeat sequence. The 3’-hydroxyls that perform nucleophilic attacks at the L-R junction (top strand) and the R-S junction (bottom strand) are indicated by the split arrowheads. B . In vitro reactions were performed with a supercoiled acceptor plasmid containing the minimal CRISPR locus (L = 11 bp; R = 36 bp; S = 27 bp). Each strand of the proto-spacer was 26 nt long, with four single stranded 3’-proximal nucleotides. The products of two-ended (complete) and one-ended (partial) proto-spacer integration and of protospacer integration-disintegration are diagrammed. Plasmid supercoiling will be reduced if the free DNA end undergoes rotation between integration and disintegration. C . The target plasmid and the proto-spacer were reacted with Cas1-Cas2 mixtures in the indicated molar ratios. After 2 hr incubation, reactions were analyzed by agarose gel electrophoresis and ethidium bromide staining. A split view of the top and bottom portions of the gel is presented to show the plasmid and proto-spacer bands. D . Reactions were similar to those shown in C, and utilized a Cas1 to Cas2 molar ratio of 2:1. E . The substrate plasmid used for the reactions in C and D was treated with a sub-optimal amount of E. coli topoisomerase I to follow the pattern of DNA relaxation over time. F . The T4 ligase reactions (lanes 3-7) were performed on the plasmid containing the L-R-S target site nicked with Nb BtsI (lane 2). The Cas1-Cas2 integration-disintegration reaction (lane 8) utilized the same plasmid in its supercoiled form (lane 1). Reactions were analyzed by gel electrophoresis in the presence of chloroquine (0.4 μg /ml). The DNA bands from the T4 ligase (lanes 3-7) and Cas1-Cas2 (lane 8) reactions are offset by one-half twist/writhe, as indicated by their staggered patterns. SC = supercoiled; N = nicked; PS = proto-spacer.
    Figure Legend Snippet: Cas1-Cas2 integrates and disintegrates proto-spacers at the CRISPR locus in a supercoiled plasmid. A . Illustration of protospacer (PS, green) integration into a minimal CRISPR locus comprised of the leader (L, red), repeat (R, yellow) and spacer (S, blue). The reaction follows the cut- and-paste DNA transposition mechanism with a duplication of the repeat sequence. The 3’-hydroxyls that perform nucleophilic attacks at the L-R junction (top strand) and the R-S junction (bottom strand) are indicated by the split arrowheads. B . In vitro reactions were performed with a supercoiled acceptor plasmid containing the minimal CRISPR locus (L = 11 bp; R = 36 bp; S = 27 bp). Each strand of the proto-spacer was 26 nt long, with four single stranded 3’-proximal nucleotides. The products of two-ended (complete) and one-ended (partial) proto-spacer integration and of protospacer integration-disintegration are diagrammed. Plasmid supercoiling will be reduced if the free DNA end undergoes rotation between integration and disintegration. C . The target plasmid and the proto-spacer were reacted with Cas1-Cas2 mixtures in the indicated molar ratios. After 2 hr incubation, reactions were analyzed by agarose gel electrophoresis and ethidium bromide staining. A split view of the top and bottom portions of the gel is presented to show the plasmid and proto-spacer bands. D . Reactions were similar to those shown in C, and utilized a Cas1 to Cas2 molar ratio of 2:1. E . The substrate plasmid used for the reactions in C and D was treated with a sub-optimal amount of E. coli topoisomerase I to follow the pattern of DNA relaxation over time. F . The T4 ligase reactions (lanes 3-7) were performed on the plasmid containing the L-R-S target site nicked with Nb BtsI (lane 2). The Cas1-Cas2 integration-disintegration reaction (lane 8) utilized the same plasmid in its supercoiled form (lane 1). Reactions were analyzed by gel electrophoresis in the presence of chloroquine (0.4 μg /ml). The DNA bands from the T4 ligase (lanes 3-7) and Cas1-Cas2 (lane 8) reactions are offset by one-half twist/writhe, as indicated by their staggered patterns. SC = supercoiled; N = nicked; PS = proto-spacer.

    Techniques Used: CRISPR, Plasmid Preparation, Sequencing, In Vitro, Incubation, Agarose Gel Electrophoresis, Staining, Nucleic Acid Electrophoresis

    5) Product Images from "T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR"

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-18

    T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.
    Figure Legend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Techniques Used: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    6) Product Images from "Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries"

    Article Title: Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094752

    Alkaline denaturation method. (A) Experimental design. (B) lane A – emulsion PCR product (step2), lane B – Nb.BtsI nicked ssDNA (step 3), lane C – nicked DNA (step 4), lane D alkali-melting of nicked ssDNA (step 6), lane E – one of negative selection (step 7), lane F – exonuclease I hydrolysis step 7 products.
    Figure Legend Snippet: Alkaline denaturation method. (A) Experimental design. (B) lane A – emulsion PCR product (step2), lane B – Nb.BtsI nicked ssDNA (step 3), lane C – nicked DNA (step 4), lane D alkali-melting of nicked ssDNA (step 6), lane E – one of negative selection (step 7), lane F – exonuclease I hydrolysis step 7 products.

    Techniques Used: Polymerase Chain Reaction, Selection

    7) Product Images from "Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research"

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research

    Journal: Plants

    doi: 10.3390/plants9091090

    Schematic diagram of NEMDA strategy for plant CRISPR/Cas9 multiplex CRISPR/Cas9 vector construction and its mutation detection of transgenic plants. ( A ) Structural features of pYLCRISPR/Cas9Pubi-H with two Bsa I sites in the flanking sides of a lethal ccdB gene [ 22 ]. ( B ) The five sgRNA expression cassettes have Bsa I and Nb.BtsI recognition sites with differently designed target sequence adaptors (different colors). ( C ) One-step construction of multiple sgRNA expression cassettes into the CRISPR/Cas9 vector. Each sgRNA expression cassette is PCR amplified using primers carrying Bas I and Nb.BtsI sites and 10-bp complementary overhanging sequences. These PCR products were mixed with pYLCRISPR/Cas9Pubi-H vector, and treated with Nb.BtvCI and Bsa I, in one tube, for digestion, heating, annealing, and ligation. The reaction product was transferred into E. coli competent cells to produce pYLCRISPR/Cas9Pubi-T5s ( D ). ( E ) Results of multiplex genome editing for three rice genes (five targets) using the above plasmid. Each target site was effectively edited by randomly selecting five T-A clones for sequencing. Nucleotides variations of insertion or deletion present as red in targets. The PAM (protospacer adjacent motif) sequences of the target site are underlined. The number in brackets indicates the proportion of variants.
    Figure Legend Snippet: Schematic diagram of NEMDA strategy for plant CRISPR/Cas9 multiplex CRISPR/Cas9 vector construction and its mutation detection of transgenic plants. ( A ) Structural features of pYLCRISPR/Cas9Pubi-H with two Bsa I sites in the flanking sides of a lethal ccdB gene [ 22 ]. ( B ) The five sgRNA expression cassettes have Bsa I and Nb.BtsI recognition sites with differently designed target sequence adaptors (different colors). ( C ) One-step construction of multiple sgRNA expression cassettes into the CRISPR/Cas9 vector. Each sgRNA expression cassette is PCR amplified using primers carrying Bas I and Nb.BtsI sites and 10-bp complementary overhanging sequences. These PCR products were mixed with pYLCRISPR/Cas9Pubi-H vector, and treated with Nb.BtvCI and Bsa I, in one tube, for digestion, heating, annealing, and ligation. The reaction product was transferred into E. coli competent cells to produce pYLCRISPR/Cas9Pubi-T5s ( D ). ( E ) Results of multiplex genome editing for three rice genes (five targets) using the above plasmid. Each target site was effectively edited by randomly selecting five T-A clones for sequencing. Nucleotides variations of insertion or deletion present as red in targets. The PAM (protospacer adjacent motif) sequences of the target site are underlined. The number in brackets indicates the proportion of variants.

    Techniques Used: CRISPR, Multiplex Assay, Plasmid Preparation, Mutagenesis, Transgenic Assay, Expressing, Sequencing, Polymerase Chain Reaction, Amplification, Ligation, Clone Assay

    Related Articles

    Plasmid Preparation:

    Article Title: A phage parasite deploys a nicking nuclease effector to inhibit replication of its viral host
    Article Snippet: .. To generate cleaved controls, 100ng of vector was digested for 1 hour at 37°C with 10 U of BamHI-HF (NEB) to create a single double-stranded cut or 10 U Nb.BtsI (NEB) to create a single nick, then the enzymes heat inactivated at 80°C for 20 minutes. ..

    Article Title: Disintegration promotes proto-spacer integration by the Cas1-Cas2 complex
    Article Snippet: .. Ligation of nicked plasmid DNAPlasmid DNA, nicked at a single site using Nb.BtsI (New England Biolabs), was ligated using T4 DNA ligase and the ligation buffer (obtained from New England Biolabs). ..

    Article Title: Disintegration promotes protospacer integration by the Cas1-Cas2 complex
    Article Snippet: .. Plasmid DNA, nicked at a single site using Nb.BtsI (New England Biolabs), was ligated using T4 DNA ligase and the ligation buffer (obtained from the supplier). ..

    Purification:

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research
    Article Snippet: .. Purified PCR fragments (each ~500 ng) were digested by five units Nb.BtsI (NEB) at 37 °C for 1 h in a total volume 20 μL. pRNAi-NE was digested with NbBtvCI (NEB) and Xba I (NEB) at 37 °C for 1 h following 20 min of heat inactivation at 80 °C. ..

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.
    Article Snippet: .. Purified PCR fragments (each ~500 ng) were digested by five units Nb.BtsI (NEB) at 37 ◦C for 1 h in a total volume 20 µL. pRNAi-NE was digested with NbBtvCI (NEB) and XbaI (NEB) at 37 ◦C for 1 h following 20 min of heat inactivation at 80 ◦C. ..

    Polymerase Chain Reaction:

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research
    Article Snippet: .. Purified PCR fragments (each ~500 ng) were digested by five units Nb.BtsI (NEB) at 37 °C for 1 h in a total volume 20 μL. pRNAi-NE was digested with NbBtvCI (NEB) and Xba I (NEB) at 37 °C for 1 h following 20 min of heat inactivation at 80 °C. ..

    Article Title: Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-yeast Rad53 Susan Gasser; Hauer et al., 2017 N/A Rat monoclonal anti-tubulin, clone YL1/2 Sigma-Aldrich Cat# 92092402-1VL, RRID: CVCL_J781 Rabbit polyclonal anti-DNA Ligase 1 Elabscience Cat# E-AB-31210 Mouse monoclonal anti-Chk1, clone 2G1D5 Cell Signaling Technology Cat# 2360, RRID: AB_2080320 Rabbit polyclonal anti-phospho-Chk1 (Ser345) Cell Signaling Technology Cat# 2341, RRID: AB_330023 Goat anti-mouse immunoglobulins, HRP Dako Cat# P0447, RRID: AB_2617137 Goat anti-rabbit immunoglobulins, HRP Dako Cat# P0448, RRID: AB_2617138 Goat anti-rat immunoglobulins, HRP Dako Cat# P0450, RRID: AB_2630354 Goat anti-rat IgG Secondary Antibody, IRDye 800CW LI-COR Cat# 926-32219, RRID: AB_1850025 Chemicals, Peptides, and Recombinant Proteins Zymolyase 20T AMS Biotechnology Cat# 120491-1 T4 DNA ligase, 20,000,000 U/mL New England Biolabs Cat# B0202 Q5 High-Fidelity DNA polymerase New England Biolabs Cat# M0491 RNase A from bovine pancreas Sigma-Aldrich Cat# 10109169001 Proteinase K Roche Cat# 3115801001 Phenylmethanesulfonyl fluoride Sigma-Aldrich Cat# P7626 b-Agarase I, 1000 U/mL New England Biolabs Cat# M0392 Alpha-Factor peptide (WHWLQLKPGQPMY), > 95% ProteoGenix SAS Cat# GM-PT001 BsrDI New England Biolabs Cat# R0574 Nb.BsrDI New England Biolabs Cat# R0648 NotI New England Biolabs Cat# R0189 Nb.BtsI New England Biolabs Cat# R0707 Antarctic Phosphatase New England Biolabs Cat# M0289 T4 Endonuclease V (T4 PDG) New England Biolabs Cat# M0308 APE1 New England Biolabs Cat# M0282 hAAG New England Biolabs Cat# M0313 Lipofectamine RNAiMAX Transfection Reagent Thermo Fisher Scientific Cat# 13778150 Benzonase Sigma-Aldrich Cat# E1014-25KU PhosSTOP Phosphatase Inhibitor Sigma-Aldrich Cat# 04906837001 Opti-MEM Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 Critical Commercial Assays AMPure XP beads Beckman Coulter Cat# A63881 NEBNext Ultra II DNA Library Prep Kit for Illumina New England Biolabs Cat# E7645 Phusion Flash high-fidelity PCR master mix Thermo Fisher Scientific Cat# F-548 Dynabeads MyOne Streptavidin C1 Life Technologies Cat# 65001 High Sensitivity D1000 ScreenTape Agilent Technologies Cat# 5067-5584 High Sensitivity D1000 ScreenTape reagents Agilent Technologies Cat# 5067-5585 RNA ScreenTape Agilent Technologies Cat# 5067-5576 RNA ScreenTape sample buffer Agilent Technologies Cat# 5067-5577 RNA ScreenTape ladder Agilent Technologies Cat# 5067-5578 Qubit dsDNA HS Assay Kit Invitrogen Cat# Q32854 Agilent Bioanalyzer High Sensitivity DNA Kit Agilent Technologies Cat# 5067-4646 (Continued on next page) e1 Molecular Cell 78, 1–11.e1–e7, June 4, 2020 ..

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.
    Article Snippet: .. Purified PCR fragments (each ~500 ng) were digested by five units Nb.BtsI (NEB) at 37 ◦C for 1 h in a total volume 20 µL. pRNAi-NE was digested with NbBtvCI (NEB) and XbaI (NEB) at 37 ◦C for 1 h following 20 min of heat inactivation at 80 ◦C. ..

    Ligation:

    Article Title: Disintegration promotes proto-spacer integration by the Cas1-Cas2 complex
    Article Snippet: .. Ligation of nicked plasmid DNAPlasmid DNA, nicked at a single site using Nb.BtsI (New England Biolabs), was ligated using T4 DNA ligase and the ligation buffer (obtained from New England Biolabs). ..

    Article Title: Disintegration promotes protospacer integration by the Cas1-Cas2 complex
    Article Snippet: .. Plasmid DNA, nicked at a single site using Nb.BtsI (New England Biolabs), was ligated using T4 DNA ligase and the ligation buffer (obtained from the supplier). ..

    Recombinant:

    Article Title: Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-yeast Rad53 Susan Gasser; Hauer et al., 2017 N/A Rat monoclonal anti-tubulin, clone YL1/2 Sigma-Aldrich Cat# 92092402-1VL, RRID: CVCL_J781 Rabbit polyclonal anti-DNA Ligase 1 Elabscience Cat# E-AB-31210 Mouse monoclonal anti-Chk1, clone 2G1D5 Cell Signaling Technology Cat# 2360, RRID: AB_2080320 Rabbit polyclonal anti-phospho-Chk1 (Ser345) Cell Signaling Technology Cat# 2341, RRID: AB_330023 Goat anti-mouse immunoglobulins, HRP Dako Cat# P0447, RRID: AB_2617137 Goat anti-rabbit immunoglobulins, HRP Dako Cat# P0448, RRID: AB_2617138 Goat anti-rat immunoglobulins, HRP Dako Cat# P0450, RRID: AB_2630354 Goat anti-rat IgG Secondary Antibody, IRDye 800CW LI-COR Cat# 926-32219, RRID: AB_1850025 Chemicals, Peptides, and Recombinant Proteins Zymolyase 20T AMS Biotechnology Cat# 120491-1 T4 DNA ligase, 20,000,000 U/mL New England Biolabs Cat# B0202 Q5 High-Fidelity DNA polymerase New England Biolabs Cat# M0491 RNase A from bovine pancreas Sigma-Aldrich Cat# 10109169001 Proteinase K Roche Cat# 3115801001 Phenylmethanesulfonyl fluoride Sigma-Aldrich Cat# P7626 b-Agarase I, 1000 U/mL New England Biolabs Cat# M0392 Alpha-Factor peptide (WHWLQLKPGQPMY), > 95% ProteoGenix SAS Cat# GM-PT001 BsrDI New England Biolabs Cat# R0574 Nb.BsrDI New England Biolabs Cat# R0648 NotI New England Biolabs Cat# R0189 Nb.BtsI New England Biolabs Cat# R0707 Antarctic Phosphatase New England Biolabs Cat# M0289 T4 Endonuclease V (T4 PDG) New England Biolabs Cat# M0308 APE1 New England Biolabs Cat# M0282 hAAG New England Biolabs Cat# M0313 Lipofectamine RNAiMAX Transfection Reagent Thermo Fisher Scientific Cat# 13778150 Benzonase Sigma-Aldrich Cat# E1014-25KU PhosSTOP Phosphatase Inhibitor Sigma-Aldrich Cat# 04906837001 Opti-MEM Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 Critical Commercial Assays AMPure XP beads Beckman Coulter Cat# A63881 NEBNext Ultra II DNA Library Prep Kit for Illumina New England Biolabs Cat# E7645 Phusion Flash high-fidelity PCR master mix Thermo Fisher Scientific Cat# F-548 Dynabeads MyOne Streptavidin C1 Life Technologies Cat# 65001 High Sensitivity D1000 ScreenTape Agilent Technologies Cat# 5067-5584 High Sensitivity D1000 ScreenTape reagents Agilent Technologies Cat# 5067-5585 RNA ScreenTape Agilent Technologies Cat# 5067-5576 RNA ScreenTape sample buffer Agilent Technologies Cat# 5067-5577 RNA ScreenTape ladder Agilent Technologies Cat# 5067-5578 Qubit dsDNA HS Assay Kit Invitrogen Cat# Q32854 Agilent Bioanalyzer High Sensitivity DNA Kit Agilent Technologies Cat# 5067-4646 (Continued on next page) e1 Molecular Cell 78, 1–11.e1–e7, June 4, 2020 ..

    Transfection:

    Article Title: Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-yeast Rad53 Susan Gasser; Hauer et al., 2017 N/A Rat monoclonal anti-tubulin, clone YL1/2 Sigma-Aldrich Cat# 92092402-1VL, RRID: CVCL_J781 Rabbit polyclonal anti-DNA Ligase 1 Elabscience Cat# E-AB-31210 Mouse monoclonal anti-Chk1, clone 2G1D5 Cell Signaling Technology Cat# 2360, RRID: AB_2080320 Rabbit polyclonal anti-phospho-Chk1 (Ser345) Cell Signaling Technology Cat# 2341, RRID: AB_330023 Goat anti-mouse immunoglobulins, HRP Dako Cat# P0447, RRID: AB_2617137 Goat anti-rabbit immunoglobulins, HRP Dako Cat# P0448, RRID: AB_2617138 Goat anti-rat immunoglobulins, HRP Dako Cat# P0450, RRID: AB_2630354 Goat anti-rat IgG Secondary Antibody, IRDye 800CW LI-COR Cat# 926-32219, RRID: AB_1850025 Chemicals, Peptides, and Recombinant Proteins Zymolyase 20T AMS Biotechnology Cat# 120491-1 T4 DNA ligase, 20,000,000 U/mL New England Biolabs Cat# B0202 Q5 High-Fidelity DNA polymerase New England Biolabs Cat# M0491 RNase A from bovine pancreas Sigma-Aldrich Cat# 10109169001 Proteinase K Roche Cat# 3115801001 Phenylmethanesulfonyl fluoride Sigma-Aldrich Cat# P7626 b-Agarase I, 1000 U/mL New England Biolabs Cat# M0392 Alpha-Factor peptide (WHWLQLKPGQPMY), > 95% ProteoGenix SAS Cat# GM-PT001 BsrDI New England Biolabs Cat# R0574 Nb.BsrDI New England Biolabs Cat# R0648 NotI New England Biolabs Cat# R0189 Nb.BtsI New England Biolabs Cat# R0707 Antarctic Phosphatase New England Biolabs Cat# M0289 T4 Endonuclease V (T4 PDG) New England Biolabs Cat# M0308 APE1 New England Biolabs Cat# M0282 hAAG New England Biolabs Cat# M0313 Lipofectamine RNAiMAX Transfection Reagent Thermo Fisher Scientific Cat# 13778150 Benzonase Sigma-Aldrich Cat# E1014-25KU PhosSTOP Phosphatase Inhibitor Sigma-Aldrich Cat# 04906837001 Opti-MEM Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 Critical Commercial Assays AMPure XP beads Beckman Coulter Cat# A63881 NEBNext Ultra II DNA Library Prep Kit for Illumina New England Biolabs Cat# E7645 Phusion Flash high-fidelity PCR master mix Thermo Fisher Scientific Cat# F-548 Dynabeads MyOne Streptavidin C1 Life Technologies Cat# 65001 High Sensitivity D1000 ScreenTape Agilent Technologies Cat# 5067-5584 High Sensitivity D1000 ScreenTape reagents Agilent Technologies Cat# 5067-5585 RNA ScreenTape Agilent Technologies Cat# 5067-5576 RNA ScreenTape sample buffer Agilent Technologies Cat# 5067-5577 RNA ScreenTape ladder Agilent Technologies Cat# 5067-5578 Qubit dsDNA HS Assay Kit Invitrogen Cat# Q32854 Agilent Bioanalyzer High Sensitivity DNA Kit Agilent Technologies Cat# 5067-4646 (Continued on next page) e1 Molecular Cell 78, 1–11.e1–e7, June 4, 2020 ..

    Polyacrylamide Gel Electrophoresis:

    Article Title: Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-yeast Rad53 Susan Gasser; Hauer et al., 2017 N/A Rat monoclonal anti-tubulin, clone YL1/2 Sigma-Aldrich Cat# 92092402-1VL, RRID: CVCL_J781 Rabbit polyclonal anti-DNA Ligase 1 Elabscience Cat# E-AB-31210 Mouse monoclonal anti-Chk1, clone 2G1D5 Cell Signaling Technology Cat# 2360, RRID: AB_2080320 Rabbit polyclonal anti-phospho-Chk1 (Ser345) Cell Signaling Technology Cat# 2341, RRID: AB_330023 Goat anti-mouse immunoglobulins, HRP Dako Cat# P0447, RRID: AB_2617137 Goat anti-rabbit immunoglobulins, HRP Dako Cat# P0448, RRID: AB_2617138 Goat anti-rat immunoglobulins, HRP Dako Cat# P0450, RRID: AB_2630354 Goat anti-rat IgG Secondary Antibody, IRDye 800CW LI-COR Cat# 926-32219, RRID: AB_1850025 Chemicals, Peptides, and Recombinant Proteins Zymolyase 20T AMS Biotechnology Cat# 120491-1 T4 DNA ligase, 20,000,000 U/mL New England Biolabs Cat# B0202 Q5 High-Fidelity DNA polymerase New England Biolabs Cat# M0491 RNase A from bovine pancreas Sigma-Aldrich Cat# 10109169001 Proteinase K Roche Cat# 3115801001 Phenylmethanesulfonyl fluoride Sigma-Aldrich Cat# P7626 b-Agarase I, 1000 U/mL New England Biolabs Cat# M0392 Alpha-Factor peptide (WHWLQLKPGQPMY), > 95% ProteoGenix SAS Cat# GM-PT001 BsrDI New England Biolabs Cat# R0574 Nb.BsrDI New England Biolabs Cat# R0648 NotI New England Biolabs Cat# R0189 Nb.BtsI New England Biolabs Cat# R0707 Antarctic Phosphatase New England Biolabs Cat# M0289 T4 Endonuclease V (T4 PDG) New England Biolabs Cat# M0308 APE1 New England Biolabs Cat# M0282 hAAG New England Biolabs Cat# M0313 Lipofectamine RNAiMAX Transfection Reagent Thermo Fisher Scientific Cat# 13778150 Benzonase Sigma-Aldrich Cat# E1014-25KU PhosSTOP Phosphatase Inhibitor Sigma-Aldrich Cat# 04906837001 Opti-MEM Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 Critical Commercial Assays AMPure XP beads Beckman Coulter Cat# A63881 NEBNext Ultra II DNA Library Prep Kit for Illumina New England Biolabs Cat# E7645 Phusion Flash high-fidelity PCR master mix Thermo Fisher Scientific Cat# F-548 Dynabeads MyOne Streptavidin C1 Life Technologies Cat# 65001 High Sensitivity D1000 ScreenTape Agilent Technologies Cat# 5067-5584 High Sensitivity D1000 ScreenTape reagents Agilent Technologies Cat# 5067-5585 RNA ScreenTape Agilent Technologies Cat# 5067-5576 RNA ScreenTape sample buffer Agilent Technologies Cat# 5067-5577 RNA ScreenTape ladder Agilent Technologies Cat# 5067-5578 Qubit dsDNA HS Assay Kit Invitrogen Cat# Q32854 Agilent Bioanalyzer High Sensitivity DNA Kit Agilent Technologies Cat# 5067-4646 (Continued on next page) e1 Molecular Cell 78, 1–11.e1–e7, June 4, 2020 ..

    Detection Assay:

    Article Title: Pancreatitis-Associated Extracellular Vesicle Identification through an Allosteric Probe-Initiated Cascade Amplification System
    Article Snippet: .. For EV detection assay, 0.25 μL of linear probes (10 μM) was added into the polymerization solution (25 μL) containing different concentrations of target EVs, 5 U of DNA polymerase I, 2.5 μL of 10× CutSmart buffer, 8 U of Nb.BtsI, 2.5 μL of 10× NEBuffer 2, 1 U of lambda exonuclease, 2.5 μL of 10× lambda exonuclease reaction buffer, 300 nM hairpin probes, 150 μM dNTPs, and 20 U of RNase inhibitor and then incubated for 45 min at 37 °C to perform the quencher-free cascade amplification reaction. ..

    Incubation:

    Article Title: Pancreatitis-Associated Extracellular Vesicle Identification through an Allosteric Probe-Initiated Cascade Amplification System
    Article Snippet: .. For EV detection assay, 0.25 μL of linear probes (10 μM) was added into the polymerization solution (25 μL) containing different concentrations of target EVs, 5 U of DNA polymerase I, 2.5 μL of 10× CutSmart buffer, 8 U of Nb.BtsI, 2.5 μL of 10× NEBuffer 2, 1 U of lambda exonuclease, 2.5 μL of 10× lambda exonuclease reaction buffer, 300 nM hairpin probes, 150 μM dNTPs, and 20 U of RNase inhibitor and then incubated for 45 min at 37 °C to perform the quencher-free cascade amplification reaction. ..

    Amplification:

    Article Title: Pancreatitis-Associated Extracellular Vesicle Identification through an Allosteric Probe-Initiated Cascade Amplification System
    Article Snippet: .. For EV detection assay, 0.25 μL of linear probes (10 μM) was added into the polymerization solution (25 μL) containing different concentrations of target EVs, 5 U of DNA polymerase I, 2.5 μL of 10× CutSmart buffer, 8 U of Nb.BtsI, 2.5 μL of 10× NEBuffer 2, 1 U of lambda exonuclease, 2.5 μL of 10× lambda exonuclease reaction buffer, 300 nM hairpin probes, 150 μM dNTPs, and 20 U of RNase inhibitor and then incubated for 45 min at 37 °C to perform the quencher-free cascade amplification reaction. ..

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    New England Biolabs nb btsi
    Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using <t>Nb.BtsI</t> are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p
    Nb Btsi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nb btsi/product/New England Biolabs
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    94
    New England Biolabs nb btsi nicking endonuclease
    h-PARP1 domains and nicked-DNA ligand. (a) Scheme of h-PARP1 domain organization. NLS is the nuclear localization signal of h-PARP1 at amino acid residues 207–209 and 221–226. (b) Sequence and the nick position (indicated by arrowhead) of the designed <t>dsDNA.</t> The recognition sequence of <t>Nb.BtsI</t> nicking endonuclease is underlined.
    Nb Btsi Nicking Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nb btsi nicking endonuclease/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nb btsi nicking endonuclease - by Bioz Stars, 2021-09
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    Image Search Results


    Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p

    Journal: Nature protocols

    Article Title: Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq

    doi: 10.1038/nprot.2015.099

    Figure Lengend Snippet: Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p

    Article Snippet: CRITICAL: alternative sources of recombinant type 2 RNase H enzymes may be used 10% (wt/vol) Bovine Serum Albumin Fraction V (BSA, Roche, cat. no. 10 735 086 001) Magnesium chloride (MgCl2 , Sigma, cat. no. M2670) Nb.BtsI, Supplied with 10x CutSmart® buffer (New England Biolabs, cat. no. R0707) BciVI, Supplied with 10x CutSmart® buffer (New England Biolabs, cat. no. R0596) Shrimp Alkaline Phosphatase (SAP), supplied with 10x reaction buffer (Affymetrix USB® , cat. no. 70092Z) Dynabeads® M-280 Streptavidin (Life Technologies, cat. no. 11205D) Tri-sodium citrate (Sigma, cat. no. C8532) Glycogen (Roche, cat. no. 10 901 393 001) Sodium acetate (NaOAc, Sigma, cat. no. S2889) Sodium hydroxide (NaOH, Sigma, cat. no. 38215) CAUTION Sodium hydroxide is corrosive.

    Techniques: Sonication, Agarose Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Selection

    Schematic diagram of NEMDA strategy for plant CRISPR/Cas9 multiplex CRISPR/Cas9 vector construction and its mutation detection of transgenic plants. ( A ) Structural features of pYLCRISPR/Cas9Pubi-H with two Bsa I sites in the flanking sides of a lethal ccdB gene [ 22 ]. ( B ) The five sgRNA expression cassettes have Bsa I and Nb.BtsI recognition sites with differently designed target sequence adaptors (different colors). ( C ) One-step construction of multiple sgRNA expression cassettes into the CRISPR/Cas9 vector. Each sgRNA expression cassette is PCR amplified using primers carrying Bas I and Nb.BtsI sites and 10-bp complementary overhanging sequences. These PCR products were mixed with pYLCRISPR/Cas9Pubi-H vector, and treated with Nb.BtvCI and Bsa I, in one tube, for digestion, heating, annealing, and ligation. The reaction product was transferred into E. coli competent cells to produce pYLCRISPR/Cas9Pubi-T5s ( D ). ( E ) Results of multiplex genome editing for three rice genes (five targets) using the above plasmid. Each target site was effectively edited by randomly selecting five T-A clones for sequencing. Nucleotides variations of insertion or deletion present as red in targets. The PAM (protospacer adjacent motif) sequences of the target site are underlined. The number in brackets indicates the proportion of variants.

    Journal: Plants

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research

    doi: 10.3390/plants9091090

    Figure Lengend Snippet: Schematic diagram of NEMDA strategy for plant CRISPR/Cas9 multiplex CRISPR/Cas9 vector construction and its mutation detection of transgenic plants. ( A ) Structural features of pYLCRISPR/Cas9Pubi-H with two Bsa I sites in the flanking sides of a lethal ccdB gene [ 22 ]. ( B ) The five sgRNA expression cassettes have Bsa I and Nb.BtsI recognition sites with differently designed target sequence adaptors (different colors). ( C ) One-step construction of multiple sgRNA expression cassettes into the CRISPR/Cas9 vector. Each sgRNA expression cassette is PCR amplified using primers carrying Bas I and Nb.BtsI sites and 10-bp complementary overhanging sequences. These PCR products were mixed with pYLCRISPR/Cas9Pubi-H vector, and treated with Nb.BtvCI and Bsa I, in one tube, for digestion, heating, annealing, and ligation. The reaction product was transferred into E. coli competent cells to produce pYLCRISPR/Cas9Pubi-T5s ( D ). ( E ) Results of multiplex genome editing for three rice genes (five targets) using the above plasmid. Each target site was effectively edited by randomly selecting five T-A clones for sequencing. Nucleotides variations of insertion or deletion present as red in targets. The PAM (protospacer adjacent motif) sequences of the target site are underlined. The number in brackets indicates the proportion of variants.

    Article Snippet: Purified PCR fragments (each ~500 ng) were digested by five units Nb.BtsI (NEB) at 37 °C for 1 h in a total volume 20 μL. pRNAi-NE was digested with NbBtvCI (NEB) and Xba I (NEB) at 37 °C for 1 h following 20 min of heat inactivation at 80 °C.

    Techniques: CRISPR, Multiplex Assay, Plasmid Preparation, Mutagenesis, Transgenic Assay, Expressing, Sequencing, Polymerase Chain Reaction, Amplification, Ligation, Clone Assay

    Schematic diagram of the nicking endonucleases-mediated DNA assembly (NEMDA) strategy for plant ihpRNA vector constructions. ( A ) The pRNAi-NE includes the 35S CaMV promoter, the Catalase intron, the ccdB gene, and four Nb.BtvCI and Xba I recognition sites with differently designed adaptors (different colors). ( B ) The sense and antisense PCR products have four Nb.BtsI recognition sites with differently designed adaptors (different colors). ( C ) One-step construction of an ihpRNA vector. The target fragments of the gene of interest are PCR amplified using gene-specific primers carrying Nb.BtsI sites and adaptors complementary to the appropriate sequences on the vector. The unpurified PCR products digested by Nb.BtsI are mixed, in one tube, with unpurified pRNAi-NE vector digested by Nb.BtvCI and Xba I, for heat-inactivation of these restriction endonucleases and melting out of the nicked end strands, annealing. The T4 DNA ligase also can be used to increase cloning efficiency. The reaction product is transferred into E. coli competent cells to produce the pRNAi plasmid ( D ).

    Journal: Plants

    Article Title: Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research

    doi: 10.3390/plants9091090

    Figure Lengend Snippet: Schematic diagram of the nicking endonucleases-mediated DNA assembly (NEMDA) strategy for plant ihpRNA vector constructions. ( A ) The pRNAi-NE includes the 35S CaMV promoter, the Catalase intron, the ccdB gene, and four Nb.BtvCI and Xba I recognition sites with differently designed adaptors (different colors). ( B ) The sense and antisense PCR products have four Nb.BtsI recognition sites with differently designed adaptors (different colors). ( C ) One-step construction of an ihpRNA vector. The target fragments of the gene of interest are PCR amplified using gene-specific primers carrying Nb.BtsI sites and adaptors complementary to the appropriate sequences on the vector. The unpurified PCR products digested by Nb.BtsI are mixed, in one tube, with unpurified pRNAi-NE vector digested by Nb.BtvCI and Xba I, for heat-inactivation of these restriction endonucleases and melting out of the nicked end strands, annealing. The T4 DNA ligase also can be used to increase cloning efficiency. The reaction product is transferred into E. coli competent cells to produce the pRNAi plasmid ( D ).

    Article Snippet: Purified PCR fragments (each ~500 ng) were digested by five units Nb.BtsI (NEB) at 37 °C for 1 h in a total volume 20 μL. pRNAi-NE was digested with NbBtvCI (NEB) and Xba I (NEB) at 37 °C for 1 h following 20 min of heat inactivation at 80 °C.

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

    h-PARP1 domains and nicked-DNA ligand. (a) Scheme of h-PARP1 domain organization. NLS is the nuclear localization signal of h-PARP1 at amino acid residues 207–209 and 221–226. (b) Sequence and the nick position (indicated by arrowhead) of the designed dsDNA. The recognition sequence of Nb.BtsI nicking endonuclease is underlined.

    Journal: Biophysics and Physicobiology

    Article Title: Single-particle analysis of full-length human poly(ADP-ribose) polymerase 1

    doi: 10.2142/biophysico.16.0_59

    Figure Lengend Snippet: h-PARP1 domains and nicked-DNA ligand. (a) Scheme of h-PARP1 domain organization. NLS is the nuclear localization signal of h-PARP1 at amino acid residues 207–209 and 221–226. (b) Sequence and the nick position (indicated by arrowhead) of the designed dsDNA. The recognition sequence of Nb.BtsI nicking endonuclease is underlined.

    Article Snippet: A part of the dsDNA was digested for 4 h at 37°C with Nb.BtsI nicking endonuclease (New England Biolabs), which recognized 5′-GCAGTG(nick)-3′ sequence and introduced single-strand break, to produce a 105 bp nicked-dsDNA in the middle of the amplified sequence ( ).

    Techniques: Sequencing