bsp q i  (New England Biolabs)


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    BspQI
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    BspQI 2 500 units
    Catalog Number:
    r0712l
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    290
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    New England Biolabs bsp q i
    BspQI
    BspQI 2 500 units
    https://www.bioz.com/result/bsp q i/product/New England Biolabs
    Average 96 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    bsp q i - by Bioz Stars, 2020-07
    96/100 stars

    Images

    1) Product Images from "A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells"

    Article Title: A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

    Journal: Journal of Genetics and Genomics

    doi: 10.1016/j.jgg.2015.03.011

    Cloning of sgRNA library. A:  sgRNA expression vector. sgRNAs (blue) are expressed from a  Drosophila U6:2  promoter, along with the Cas9 protein from an  Actin-5C  promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term).  B:  Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector.  C:  Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the  Bsp Q I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the  dU6:2  promoter (G) is indicated by an arrow.
    Figure Legend Snippet: Cloning of sgRNA library. A: sgRNA expression vector. sgRNAs (blue) are expressed from a Drosophila U6:2 promoter, along with the Cas9 protein from an Actin-5C promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term). B: Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector. C: Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the Bsp Q I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the dU6:2 promoter (G) is indicated by an arrow.

    Techniques Used: Clone Assay, Expressing, Plasmid Preparation, Oligo Synthesis, Amplification, Polymerase Chain Reaction, Ligation

    2) Product Images from "Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿"

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02340-10

    Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and
    Figure Legend Snippet: Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and

    Techniques Used: Functional Assay, Polymerase Chain Reaction, Clone Assay, Transfection

    3) Product Images from "Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9"

    Article Title: Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9

    Journal: Biology Open

    doi: 10.1242/bio.20137120

    CRISPR/Cas9 expression system for Drosophila cell culture. (A) The CRISPR/Cas9 system adapted from S. pyogenes for inducing double strand breaks. The synthetic guide RNA (sgRNA) contains 20 nt complementarity to the target site within the DNA, and the RNA structure necessary for incorporation into the Cas9 protein. Cas9 is indicated by a yellow circle, cleavage sites by arrowheads and protospacer adjacent motif (PAM, NGG) required for cleavage in red. (B) Schematic of the expression vector. The sgRNA is produced from a Drosophila U6 promoter by RNA polymerase III, which produces an uncapped transcript. Human codon-optimised Cas9 mRNA (orange oval) containing N- and C-terminal SV40 nuclear localisation signals (grey ovals) is produced from the strong, constitutive actin5C promoter by RNA polymerase II as the first half of a bicistronic transcript with the puromycin N-acetyltransferase gene (purple oval). The two open reading frames are separated by a viral 2A ribosome skipping site (red oval) to allow bicistronic expression, and transcription is terminated by a polyadenylation signal from the SV40 virus. (C) Strategy for cloning of target oligos. Two Bsp QI sites (yellow) cause cleavage at the end of the U6 promoter (blue) and sgRNA backbone (red), leaving 3 nt 5′ overhangs. Transcription from the dU6 promoter begins with the indicated G nucleotide (arrow). Target oligos (orange) are designed to provide complementary overhangs flanking the 20 nt target sequence. If the target sequence does not begin with a G, this is appended to its 5′ end to reconstitute the G nucleotide required by the dU6 promoter (as indicated here). See also supplementary material Table S1 .
    Figure Legend Snippet: CRISPR/Cas9 expression system for Drosophila cell culture. (A) The CRISPR/Cas9 system adapted from S. pyogenes for inducing double strand breaks. The synthetic guide RNA (sgRNA) contains 20 nt complementarity to the target site within the DNA, and the RNA structure necessary for incorporation into the Cas9 protein. Cas9 is indicated by a yellow circle, cleavage sites by arrowheads and protospacer adjacent motif (PAM, NGG) required for cleavage in red. (B) Schematic of the expression vector. The sgRNA is produced from a Drosophila U6 promoter by RNA polymerase III, which produces an uncapped transcript. Human codon-optimised Cas9 mRNA (orange oval) containing N- and C-terminal SV40 nuclear localisation signals (grey ovals) is produced from the strong, constitutive actin5C promoter by RNA polymerase II as the first half of a bicistronic transcript with the puromycin N-acetyltransferase gene (purple oval). The two open reading frames are separated by a viral 2A ribosome skipping site (red oval) to allow bicistronic expression, and transcription is terminated by a polyadenylation signal from the SV40 virus. (C) Strategy for cloning of target oligos. Two Bsp QI sites (yellow) cause cleavage at the end of the U6 promoter (blue) and sgRNA backbone (red), leaving 3 nt 5′ overhangs. Transcription from the dU6 promoter begins with the indicated G nucleotide (arrow). Target oligos (orange) are designed to provide complementary overhangs flanking the 20 nt target sequence. If the target sequence does not begin with a G, this is appended to its 5′ end to reconstitute the G nucleotide required by the dU6 promoter (as indicated here). See also supplementary material Table S1 .

    Techniques Used: CRISPR, Expressing, Cell Culture, Plasmid Preparation, Produced, Clone Assay, Sequencing

    4) Product Images from "Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization"

    Article Title: Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-4939-6700-1_18

    Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal BspQI sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with SphI and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct
    Figure Legend Snippet: Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal BspQI sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with SphI and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct

    Techniques Used: Flow Cytometry, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, In Vitro, Hybridization, Produced, Construct

    5) Product Images from "Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease"

    Article Title: Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease

    Journal: Plasmid

    doi: 10.1016/j.plasmid.2011.07.007

    Strategy for construction of lacZ gene fusions using the nicking endonuclease Nt Bsp QI Top. The site of insertion into pLacCOs1 is shown, including the Stu I restriction enzyme recognition site (shown in bold) for linearizing the vector and the two Nt. Bsp QI recognition sites (shown in bold italics). The arrows indicate the positions where the DNA strands are cut by each endonuclease. Bottom. The compatible single-stranded overhangs, generated as described in Materials and Methods , of both vector and PCR product are shown. The location of a promoter (P) is shown within the PCR product.
    Figure Legend Snippet: Strategy for construction of lacZ gene fusions using the nicking endonuclease Nt Bsp QI Top. The site of insertion into pLacCOs1 is shown, including the Stu I restriction enzyme recognition site (shown in bold) for linearizing the vector and the two Nt. Bsp QI recognition sites (shown in bold italics). The arrows indicate the positions where the DNA strands are cut by each endonuclease. Bottom. The compatible single-stranded overhangs, generated as described in Materials and Methods , of both vector and PCR product are shown. The location of a promoter (P) is shown within the PCR product.

    Techniques Used: Plasmid Preparation, Generated, Polymerase Chain Reaction

    6) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001164

    Preexisting nicks act as preferential nicking (but not loading) sites for Mlh1-Mlh3. (A) Mlh1-Mlh3 creates approximately the same amount of linear product regardless of how many preexisting nicks are in the circular substrate. 300 nM yeast Mlh1-Mlh3 on either closed circular 2.7 kb plasmid substrate (0 preexisting nicks), Nt . Bsp QI-treated substrate (one preexisting nick), Nt . Bst NBI-treated substrate (four preexisting nicks), or Nt . Alw I-treated substrate (ten preexisting nicks). Nicked (n) and linear (black triangle) products are shown. (B) Wild-type Mlh1-Mlh3 (300 nM) creates linear product on either closed circular 2.7 kb plasmid substrate or Nt . Bst NBI-treated substrate, while Mlh1-mlh3D523N (300 nM) is inactive on both. (C) Mapping the formation of Mlh1-Mlh3–induced nick opposite a preexisting nick. Top: experimental setup. Plasmid was either linearized with Sap I, which creates a 3-bp overhang, or substrate with a preexisting nick generated by Nt . Bsp QI was linearized by Mlh1-Mlh3. For each, linear product was gel extracted and annealed to a primer either complementary to the strand with the preexisting nick (primer A) or to the opposite strand (primer B). Primers were extended by T4 polymerase where indicated. Primer extension products were resolved by denaturing PAGE. For the Sap I linear control, extension of primer A gives a 60-mer and extension of primer B gives a 63-mer product. Bottom: lanes 1, 6, and 11 are radiolabeled 60-mer used as a primer extension marker. Lanes 2–5 show primer extension for the linear control, while lanes 7–10 show primer extension for Mlh1-Mlh3 linear product. Lane 12 is a duplicate of the material in lane 5.
    Figure Legend Snippet: Preexisting nicks act as preferential nicking (but not loading) sites for Mlh1-Mlh3. (A) Mlh1-Mlh3 creates approximately the same amount of linear product regardless of how many preexisting nicks are in the circular substrate. 300 nM yeast Mlh1-Mlh3 on either closed circular 2.7 kb plasmid substrate (0 preexisting nicks), Nt . Bsp QI-treated substrate (one preexisting nick), Nt . Bst NBI-treated substrate (four preexisting nicks), or Nt . Alw I-treated substrate (ten preexisting nicks). Nicked (n) and linear (black triangle) products are shown. (B) Wild-type Mlh1-Mlh3 (300 nM) creates linear product on either closed circular 2.7 kb plasmid substrate or Nt . Bst NBI-treated substrate, while Mlh1-mlh3D523N (300 nM) is inactive on both. (C) Mapping the formation of Mlh1-Mlh3–induced nick opposite a preexisting nick. Top: experimental setup. Plasmid was either linearized with Sap I, which creates a 3-bp overhang, or substrate with a preexisting nick generated by Nt . Bsp QI was linearized by Mlh1-Mlh3. For each, linear product was gel extracted and annealed to a primer either complementary to the strand with the preexisting nick (primer A) or to the opposite strand (primer B). Primers were extended by T4 polymerase where indicated. Primer extension products were resolved by denaturing PAGE. For the Sap I linear control, extension of primer A gives a 60-mer and extension of primer B gives a 63-mer product. Bottom: lanes 1, 6, and 11 are radiolabeled 60-mer used as a primer extension marker. Lanes 2–5 show primer extension for the linear control, while lanes 7–10 show primer extension for Mlh1-Mlh3 linear product. Lane 12 is a duplicate of the material in lane 5.

    Techniques Used: Activated Clotting Time Assay, Plasmid Preparation, Generated, Polyacrylamide Gel Electrophoresis, Marker

    7) Product Images from "The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans"

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001164

    Preexisting nicks act as preferential nicking (but not loading) sites for Mlh1-Mlh3. (A) Mlh1-Mlh3 creates approximately the same amount of linear product regardless of how many preexisting nicks are in the circular substrate. 300 nM yeast Mlh1-Mlh3 on either closed circular 2.7 kb plasmid substrate (0 preexisting nicks), Nt . Bsp QI-treated substrate (one preexisting nick), Nt . Bst NBI-treated substrate (four preexisting nicks), or Nt . Alw I-treated substrate (ten preexisting nicks). Nicked (n) and linear (black triangle) products are shown. (B) Wild-type Mlh1-Mlh3 (300 nM) creates linear product on either closed circular 2.7 kb plasmid substrate or Nt . Bst NBI-treated substrate, while Mlh1-mlh3D523N (300 nM) is inactive on both. (C) Mapping the formation of Mlh1-Mlh3–induced nick opposite a preexisting nick. Top: experimental setup. Plasmid was either linearized with Sap I, which creates a 3-bp overhang, or substrate with a preexisting nick generated by Nt . Bsp QI was linearized by Mlh1-Mlh3. For each, linear product was gel extracted and annealed to a primer either complementary to the strand with the preexisting nick (primer A) or to the opposite strand (primer B). Primers were extended by T4 polymerase where indicated. Primer extension products were resolved by denaturing PAGE. For the Sap I linear control, extension of primer A gives a 60-mer and extension of primer B gives a 63-mer product. Bottom: lanes 1, 6, and 11 are radiolabeled 60-mer used as a primer extension marker. Lanes 2–5 show primer extension for the linear control, while lanes 7–10 show primer extension for Mlh1-Mlh3 linear product. Lane 12 is a duplicate of the material in lane 5.
    Figure Legend Snippet: Preexisting nicks act as preferential nicking (but not loading) sites for Mlh1-Mlh3. (A) Mlh1-Mlh3 creates approximately the same amount of linear product regardless of how many preexisting nicks are in the circular substrate. 300 nM yeast Mlh1-Mlh3 on either closed circular 2.7 kb plasmid substrate (0 preexisting nicks), Nt . Bsp QI-treated substrate (one preexisting nick), Nt . Bst NBI-treated substrate (four preexisting nicks), or Nt . Alw I-treated substrate (ten preexisting nicks). Nicked (n) and linear (black triangle) products are shown. (B) Wild-type Mlh1-Mlh3 (300 nM) creates linear product on either closed circular 2.7 kb plasmid substrate or Nt . Bst NBI-treated substrate, while Mlh1-mlh3D523N (300 nM) is inactive on both. (C) Mapping the formation of Mlh1-Mlh3–induced nick opposite a preexisting nick. Top: experimental setup. Plasmid was either linearized with Sap I, which creates a 3-bp overhang, or substrate with a preexisting nick generated by Nt . Bsp QI was linearized by Mlh1-Mlh3. For each, linear product was gel extracted and annealed to a primer either complementary to the strand with the preexisting nick (primer A) or to the opposite strand (primer B). Primers were extended by T4 polymerase where indicated. Primer extension products were resolved by denaturing PAGE. For the Sap I linear control, extension of primer A gives a 60-mer and extension of primer B gives a 63-mer product. Bottom: lanes 1, 6, and 11 are radiolabeled 60-mer used as a primer extension marker. Lanes 2–5 show primer extension for the linear control, while lanes 7–10 show primer extension for Mlh1-Mlh3 linear product. Lane 12 is a duplicate of the material in lane 5.

    Techniques Used: Activated Clotting Time Assay, Plasmid Preparation, Generated, Polyacrylamide Gel Electrophoresis, Marker

    Related Articles

    Clone Assay:

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿
    Article Snippet: .. A single copy of the full-length HBV genome was released by the EcoRI digestion of the EcoRI monomer, ApaI or SphI digestion of the EcoRI dimer, and digestion of the monomeric PCR clones or clone pools at 50°C with BspQI (New England BioLabs). .. The digested DNA was purified through QIAquick PCR purification columns (Qiagen) and resuspended in endotoxin-free Tris-EDTA buffer.

    Isolation:

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: .. Mapping Mlh1-Mlh3–generated nicks opposite preexisting nicks pUC18 plasmid was either nicked with Nt .Bsp QI or linearized using Sap I (NEB) according to the manufacturer’s instructions with heat inactivation and gel isolation of the nicked or linear product. .. Nicked plasmid was then used as an endonuclease substrate in a reaction performed with 24 replicates containing 300 nM Mlh1-Mlh3.

    Purification:

    Article Title: Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9
    Article Snippet: .. 2 µg pAc-sgRNA-Cas9 vector was digested with 20 U Bsp QI (NEB) for 2 h at 50°C, and treated with 10 U calf intestinal alkaline phosphatase for 10 min at 37°C (NEB) before PCR purification (Qiagen). .. Ligations were performed with approximately 50 ng vector and 2 µl annealed diluted oligonucleotides with T4 DNA ligase (NEB) in a 10 µl reaction volume for 2 h at 18°C, and transformed into chemically competent E. coli DH5α cells (Life Technologies).

    Article Title: A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells
    Article Snippet: .. PCR products were purified using a PCR purification kit (Qiagen, UK), digested with Bsp Q I (NEB), and 20 nt fragments were extracted from a 20% acrylamide-TBE gel (Life Technologies, UK). .. Gel extraction was performed by homogenising gel pieces and overnight incubated in 600 μL 0.3 mol/L NaCl.

    Generated:

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: .. Prenicked circular substrates used in were generated as previously described using Nt .Bsp QI, Nt .Bst NBI, or Nt .Alw I purchased from New England Biolabs [ ]. .. Large linear substrates The 2.7, 7 kb, and 12 kb linear DNA substrates were generated by digesting pUC18, pEAE399, or pEAE99 with Hin dIII (NEB) according to the manufacturer’s instructions.

    Activity Assay:

    Article Title: The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases
    Article Snippet: .. We also thank Derek Robinson for the initiation of star activity measurement; Mathieu-Sheltry Vanessa for providing pXba and pBC4 plasmid; Richard Morgan for providing T7 DNA; Richard Roberts, Elisabeth Raleigh, Bill Jack, Shuangyong Xu, Richard Grandoni, Peter Nathan, Geoffery Wilson for critical reviewing; Penghua Zhang for initial FI measurement on BspQI and; Dapeng Sun for the FI measurement of BclI, SexAI and BtsI; and New England Biolabs for financial support. .. Funding to pay the Open Access publication charges for this article was provided by New England Biolabs, Inc.

    Polymerase Chain Reaction:

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿
    Article Snippet: .. A single copy of the full-length HBV genome was released by the EcoRI digestion of the EcoRI monomer, ApaI or SphI digestion of the EcoRI dimer, and digestion of the monomeric PCR clones or clone pools at 50°C with BspQI (New England BioLabs). .. The digested DNA was purified through QIAquick PCR purification columns (Qiagen) and resuspended in endotoxin-free Tris-EDTA buffer.

    Article Title: Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9
    Article Snippet: .. 2 µg pAc-sgRNA-Cas9 vector was digested with 20 U Bsp QI (NEB) for 2 h at 50°C, and treated with 10 U calf intestinal alkaline phosphatase for 10 min at 37°C (NEB) before PCR purification (Qiagen). .. Ligations were performed with approximately 50 ng vector and 2 µl annealed diluted oligonucleotides with T4 DNA ligase (NEB) in a 10 µl reaction volume for 2 h at 18°C, and transformed into chemically competent E. coli DH5α cells (Life Technologies).

    Article Title: A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells
    Article Snippet: .. PCR products were purified using a PCR purification kit (Qiagen, UK), digested with Bsp Q I (NEB), and 20 nt fragments were extracted from a 20% acrylamide-TBE gel (Life Technologies, UK). .. Gel extraction was performed by homogenising gel pieces and overnight incubated in 600 μL 0.3 mol/L NaCl.

    Plasmid Preparation:

    Article Title: The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases
    Article Snippet: .. We also thank Derek Robinson for the initiation of star activity measurement; Mathieu-Sheltry Vanessa for providing pXba and pBC4 plasmid; Richard Morgan for providing T7 DNA; Richard Roberts, Elisabeth Raleigh, Bill Jack, Shuangyong Xu, Richard Grandoni, Peter Nathan, Geoffery Wilson for critical reviewing; Penghua Zhang for initial FI measurement on BspQI and; Dapeng Sun for the FI measurement of BclI, SexAI and BtsI; and New England Biolabs for financial support. .. Funding to pay the Open Access publication charges for this article was provided by New England Biolabs, Inc.

    Article Title: Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9
    Article Snippet: .. 2 µg pAc-sgRNA-Cas9 vector was digested with 20 U Bsp QI (NEB) for 2 h at 50°C, and treated with 10 U calf intestinal alkaline phosphatase for 10 min at 37°C (NEB) before PCR purification (Qiagen). .. Ligations were performed with approximately 50 ng vector and 2 µl annealed diluted oligonucleotides with T4 DNA ligase (NEB) in a 10 µl reaction volume for 2 h at 18°C, and transformed into chemically competent E. coli DH5α cells (Life Technologies).

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans
    Article Snippet: .. Mapping Mlh1-Mlh3–generated nicks opposite preexisting nicks pUC18 plasmid was either nicked with Nt .Bsp QI or linearized using Sap I (NEB) according to the manufacturer’s instructions with heat inactivation and gel isolation of the nicked or linear product. .. Nicked plasmid was then used as an endonuclease substrate in a reaction performed with 24 replicates containing 300 nM Mlh1-Mlh3.

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    New England Biolabs bsp q i
    Cloning of sgRNA library. A:  sgRNA expression vector. sgRNAs (blue) are expressed from a  Drosophila U6:2  promoter, along with the Cas9 protein from an  Actin-5C  promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term).  B:  Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector.  C:  Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the  Bsp Q I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the  dU6:2  promoter (G) is indicated by an arrow.
    Bsp Q I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsp q i/product/New England Biolabs
    Average 96 stars, based on 14 article reviews
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    Cloning of sgRNA library. A:  sgRNA expression vector. sgRNAs (blue) are expressed from a  Drosophila U6:2  promoter, along with the Cas9 protein from an  Actin-5C  promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term).  B:  Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector.  C:  Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the  Bsp Q I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the  dU6:2  promoter (G) is indicated by an arrow.

    Journal: Journal of Genetics and Genomics

    Article Title: A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

    doi: 10.1016/j.jgg.2015.03.011

    Figure Lengend Snippet: Cloning of sgRNA library. A: sgRNA expression vector. sgRNAs (blue) are expressed from a Drosophila U6:2 promoter, along with the Cas9 protein from an Actin-5C promoter. Cas9 (red box) contains N- and C-terminal nuclear localisation sequences (NLS, grey oval), and is expressed as a bicistronic transcript with a puromycin N-acetyltransferase gene (purple oval) separated by a viral 2A peptide (black oval). An SV40 transcriptional terminator is also included (SV40 term). B: Oligo synthesis. sgRNA sequences were synthesised with common 5′ and 3′adaptors, and amplified by PCR followed by digestion with restriction enzymes and cloned into the expression vector. C: Cloning strategy for sgRNAs. The synthesised oligonucleotides were amplified by PCR using common adaptor sequences, and digested with the Bsp Q I restriction enzyme, followed by ligation into a similarly digested expression vector. The first base transcribed by the dU6:2 promoter (G) is indicated by an arrow.

    Article Snippet: PCR products were purified using a PCR purification kit (Qiagen, UK), digested with Bsp Q I (NEB), and 20 nt fragments were extracted from a 20% acrylamide-TBE gel (Life Technologies, UK).

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Oligo Synthesis, Amplification, Polymerase Chain Reaction, Ligation

    Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and

    Journal: Journal of Clinical Microbiology

    Article Title: Improved Method for Rapid and Efficient Determination of Genome Replication and Protein Expression of Clinical Hepatitis B Virus Isolates ▿

    doi: 10.1128/JCM.02340-10

    Figure Lengend Snippet: Functional characterization of two High Fidelity plus PCR clones of the 4B genome. The two clones were transfected directly (lanes 1 and 4) following digestion with BspQI (lanes 2 and 5) or BspQI digestion plus treatment with T4 DNA ligase (lanes 3 and

    Article Snippet: A single copy of the full-length HBV genome was released by the EcoRI digestion of the EcoRI monomer, ApaI or SphI digestion of the EcoRI dimer, and digestion of the monomeric PCR clones or clone pools at 50°C with BspQI (New England BioLabs).

    Techniques: Functional Assay, Polymerase Chain Reaction, Clone Assay, Transfection

    CRISPR/Cas9 expression system for Drosophila cell culture. (A) The CRISPR/Cas9 system adapted from S. pyogenes for inducing double strand breaks. The synthetic guide RNA (sgRNA) contains 20 nt complementarity to the target site within the DNA, and the RNA structure necessary for incorporation into the Cas9 protein. Cas9 is indicated by a yellow circle, cleavage sites by arrowheads and protospacer adjacent motif (PAM, NGG) required for cleavage in red. (B) Schematic of the expression vector. The sgRNA is produced from a Drosophila U6 promoter by RNA polymerase III, which produces an uncapped transcript. Human codon-optimised Cas9 mRNA (orange oval) containing N- and C-terminal SV40 nuclear localisation signals (grey ovals) is produced from the strong, constitutive actin5C promoter by RNA polymerase II as the first half of a bicistronic transcript with the puromycin N-acetyltransferase gene (purple oval). The two open reading frames are separated by a viral 2A ribosome skipping site (red oval) to allow bicistronic expression, and transcription is terminated by a polyadenylation signal from the SV40 virus. (C) Strategy for cloning of target oligos. Two Bsp QI sites (yellow) cause cleavage at the end of the U6 promoter (blue) and sgRNA backbone (red), leaving 3 nt 5′ overhangs. Transcription from the dU6 promoter begins with the indicated G nucleotide (arrow). Target oligos (orange) are designed to provide complementary overhangs flanking the 20 nt target sequence. If the target sequence does not begin with a G, this is appended to its 5′ end to reconstitute the G nucleotide required by the dU6 promoter (as indicated here). See also supplementary material Table S1 .

    Journal: Biology Open

    Article Title: Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9

    doi: 10.1242/bio.20137120

    Figure Lengend Snippet: CRISPR/Cas9 expression system for Drosophila cell culture. (A) The CRISPR/Cas9 system adapted from S. pyogenes for inducing double strand breaks. The synthetic guide RNA (sgRNA) contains 20 nt complementarity to the target site within the DNA, and the RNA structure necessary for incorporation into the Cas9 protein. Cas9 is indicated by a yellow circle, cleavage sites by arrowheads and protospacer adjacent motif (PAM, NGG) required for cleavage in red. (B) Schematic of the expression vector. The sgRNA is produced from a Drosophila U6 promoter by RNA polymerase III, which produces an uncapped transcript. Human codon-optimised Cas9 mRNA (orange oval) containing N- and C-terminal SV40 nuclear localisation signals (grey ovals) is produced from the strong, constitutive actin5C promoter by RNA polymerase II as the first half of a bicistronic transcript with the puromycin N-acetyltransferase gene (purple oval). The two open reading frames are separated by a viral 2A ribosome skipping site (red oval) to allow bicistronic expression, and transcription is terminated by a polyadenylation signal from the SV40 virus. (C) Strategy for cloning of target oligos. Two Bsp QI sites (yellow) cause cleavage at the end of the U6 promoter (blue) and sgRNA backbone (red), leaving 3 nt 5′ overhangs. Transcription from the dU6 promoter begins with the indicated G nucleotide (arrow). Target oligos (orange) are designed to provide complementary overhangs flanking the 20 nt target sequence. If the target sequence does not begin with a G, this is appended to its 5′ end to reconstitute the G nucleotide required by the dU6 promoter (as indicated here). See also supplementary material Table S1 .

    Article Snippet: 2 µg pAc-sgRNA-Cas9 vector was digested with 20 U Bsp QI (NEB) for 2 h at 50°C, and treated with 10 U calf intestinal alkaline phosphatase for 10 min at 37°C (NEB) before PCR purification (Qiagen).

    Techniques: CRISPR, Expressing, Cell Culture, Plasmid Preparation, Produced, Clone Assay, Sequencing

    Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal BspQI sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with SphI and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Generation of Replication-Competent Hepatitis B Virus Genome from Blood Samples for Functional Characterization

    doi: 10.1007/978-1-4939-6700-1_18

    Figure Lengend Snippet: Flow chart for the generation of replication-competent HBV genomes from blood samples. Virion-associated HBV DNA has the minus strand DNA ( thick line ) complete. The sense (S) primer anneals to its 3’ end to generate full-length plus strand, which will serve as the template for the antisense (AS) primer to generate more minus strand DNA. The HindIII and SacI sites introduced to the sense and antisense primers, respectively, will allow efficient cloning of the PCR product to pUC18 vector, whereas the internal BspQI sites allow subsequent precise release of the HBV genome. Such a linear HBV genome can be ligated in vitro to make it replication competent (capable of producing the terminally redundant pg RNA), or the ligated DNA is further digested with SphI and ligated with SphI cut, dephosphorylated pUC18 DNA. Bacterial colonies harboring tandem SphI dimer can be screened by hybridization with an oligoprobe spanning the SphI site. The 3.5-kb pg RNA can be produced from such a tandem dimer construct

    Article Snippet: Enzymes: BspQI, HindIII, SacI, ScaI, SphI, alkaline phosphatase, and Q5 DNA polymerase (New England Biolabs).

    Techniques: Flow Cytometry, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, In Vitro, Hybridization, Produced, Construct