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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: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

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

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 "A short report on optimization of nucleic acid probes by DNA microarray synthesis and next generation sequencing"

Article Title: A short report on optimization of nucleic acid probes by DNA microarray synthesis and next generation sequencing

Journal: bioRxiv

doi: 10.1101/2021.05.04.442640

Investigation of stem-structure sequence (denoted as carrier) composition in a 2fold probe was presented by boxplot of results from experiments with Nb.BtsI (left) and Nb.BbvCI (right) in parallel following a one-step solid phase protocol.

Figure Legend Snippet: Investigation of stem-structure sequence (denoted as carrier) composition in a 2fold probe was presented by boxplot of results from experiments with Nb.BtsI (left) and Nb.BbvCI (right) in parallel following a one-step solid phase protocol.

Techniques Used: Sequencing

6) 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

7) 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