bfuai restriction enzyme  (New England Biolabs)


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    Name:
    BfuAI
    Description:
    BfuAI 1 250 units
    Catalog Number:
    r0701l
    Price:
    269
    Size:
    1 250 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs bfuai restriction enzyme
    BfuAI
    BfuAI 1 250 units
    https://www.bioz.com/result/bfuai restriction enzyme/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bfuai restriction enzyme - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Transduction:

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme. .. Following transduction and selection with puromycin (3 μg/mL) and blasticidin (500 μg/mL) for five days, cells were cultured in the presence of 3 μg/mL doxycycline (Sigma) for at least five days to induce sgRNA expression.

    Clone Assay:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: Paragraph title: Cloning of the sgRNA and crRNA libraries ... The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C.

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: .. Following amplification in E.coli, library plasmids with the first position cloned were digested with BfuAI (NEB) to allow cloning of SaCas9 sgRNAs into the second position. .. To remove undigested orthogonal sgRNA library plasmid from the pool, the purified (Nucleospin, Macherey-Nagel) BfuAI digested plasmid was subsequently digested with AscI for which restriction sites exist in the stuffer sequences in both sgRNA positions 1 and 2.

    Article Title: Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
    Article Snippet: To optimize this plasmid for cloning the library, we first replaced the sgRNA with a 1.9kb stuffer derived from the lentiGuide-Puro plasmid (Addgene, plasmid #52963) with flanking BfuAI cut sites. .. This stuffer was excised using the BfuAI restriction enzyme (NEB, #R0701) and the linear backbone was gel purified (Zymo, #D4007).

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme. .. Following transduction and selection with puromycin (3 μg/mL) and blasticidin (500 μg/mL) for five days, cells were cultured in the presence of 3 μg/mL doxycycline (Sigma) for at least five days to induce sgRNA expression.

    Centrifugation:

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. For 19 F labeling, cells were then collected by centrifugation and re-suspended in M9 minimal growth medium containing 1 g/L (NH4 )2 SO4 (for 15 N labeling, (NH4 )2 SO4 (Cambridge Isotope Laboratories, Tewksbury, MA)), 2 g/L D-glucose, 4 mL/L of 1M MgSO4 , and 1.8 ml/L of 1 mM FeSO4 .

    Amplification:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: The sgRNA/crRNA pools were amplified in 10 parallel reactions (25 μL, 1 ng input) by PCR with the CloneAmp HiFi premix kit (Clontech) and PCR products were purified with the QIAQuick Nucleotide Removal kit (QiaGen). .. The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C.

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: .. Following amplification in E.coli, library plasmids with the first position cloned were digested with BfuAI (NEB) to allow cloning of SaCas9 sgRNAs into the second position. .. To remove undigested orthogonal sgRNA library plasmid from the pool, the purified (Nucleospin, Macherey-Nagel) BfuAI digested plasmid was subsequently digested with AscI for which restriction sites exist in the stuffer sequences in both sgRNA positions 1 and 2.

    Synthesized:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: All appended sgRNA and crRNA sequences were synthesized as pools by Customarray Inc. (Bothell, WA) on a 12K (9/10 gene libraries) or 90K (“non-FDA-target”/”FDA-target”) chip. .. The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C.

    Construct:

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: KB1P-G3 cells were transduced with the FUCCI plasmids mKO2-hCdt1(30/120) and mAG-hGem(1/110) ( ) and were sorted for red fluorescence and green fluorescence in two subsequent sorting rounds to ensure the presence of both constructs in each cell. .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme.

    Real-time Polymerase Chain Reaction:

    Article Title: Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein
    Article Snippet: Restriction enzymes AciI, AscI, BanI, BfuAI, BsrFI, BsrBI, BseYI, BfuAI, BspEI, Cac81, FspI, NciI, NruI (New England Biolabs) and HpyCH4III/Taal (Fermentas) were used. .. Restriction digestions were then analyzed by real time PCR using Universal SYBR Green (BioRad) and primers spanning the promoters regions and primers spanning the promoters regions (BRF2-forward, 5’-GGC CTC CAA AAG CGT T-3’; BRF2-reverse, 5’-AGC TGG CTC TGC GAA TAG T-3’; BRF1-forward, 5’-GGG GTT GGG TCC CAG GTC GC-3’; BRF1-reverse, 5’-GTC CTC CAG CAC TGA GCC GC-3’; U6-forward, 5’- AAG TAT TTC GAT TTC TTG GC-3’; U6-reverse, 5’- AAT ATG GAA CGC TTC ACG-3’; tRNAi Met -forward, 5’-TAG ATA GCA GAG TGG CGC A-3’; tRNAi Met -reverse, 5’-AAC TCC GAT AGC AGA GGA TG-3’).

    Activity Assay:

    Article Title: Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
    Article Snippet: This stuffer was excised using the BfuAI restriction enzyme (NEB, #R0701) and the linear backbone was gel purified (Zymo, #D4007). .. We designed a targeted library to include all genes matching Gene Ontology for “Cell Surface”, “‘T cell receptor signaling pathway”, or “cytokine receptor activity”.

    Expressing:

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: KB1P-G3 BRCA1 reconstituted cells were generated by transfecting KB1P-G3 cells with a human BRCA1 cDNA expression construct ( ) using Lipofectamine 2000 (Thermo Fisher Scientific). .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme.

    Article Title: Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein
    Article Snippet: Restriction enzymes AciI, AscI, BanI, BfuAI, BsrFI, BsrBI, BseYI, BfuAI, BspEI, Cac81, FspI, NciI, NruI (New England Biolabs) and HpyCH4III/Taal (Fermentas) were used. .. Results were quantified using the ΔΔCt method and normalized to RPS13 expression levels.

    Modification:

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    Transformation Assay:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C. .. Transformed cells were then plated in 15-cm-diameter petridishes containing prewarmed LB agar with 100 µg/mL ampicilin and grown overnight at 32 °C.

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    Derivative Assay:

    Article Title: Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
    Article Snippet: To optimize this plasmid for cloning the library, we first replaced the sgRNA with a 1.9kb stuffer derived from the lentiGuide-Puro plasmid (Addgene, plasmid #52963) with flanking BfuAI cut sites. .. This stuffer was excised using the BfuAI restriction enzyme (NEB, #R0701) and the linear backbone was gel purified (Zymo, #D4007).

    Transfection:

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: One day after transfection, cells were passaged and cultured with 300 μg/ml G418 (Thermo Fisher Scientific) to select for human BRCA1 complemented colonies. .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme.

    Inverse PCR:

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: In brief, individual tryptophan mutations were introduced on a previously descried pDW1 plasmid (pDrive vector containing the W1 gene) through inverse PCR with mutagenic primers ( ). .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes.

    Ligation:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C. .. After digestion, six ligation reactions containing 33 ng of digested sgRNA/crRNAs and 500 ng of the BfuAI and NsiI predigested pLCKO vector were performed overnight at 16 °C with T4 DNA ligase (NEB).

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: .. After digestion, six ligation reactions containing 33 ng of digested sgRNA/crRNAs and 500 ng of the BfuAI and NsiI predigested pLCKO vector were performed overnight at 16 °C with T4 DNA ligase (NEB). .. The pooled mixture of ligated pLCKO vectors was then purified with the QIAquick nucleotide removal kit (Qiagen) and electroporated into Endura competent cells (Lucigen) with a Gene Pulser system (Biorad) according to the manufacturer’s instructions.

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    Cell Culture:

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: One day after transfection, cells were passaged and cultured with 300 μg/ml G418 (Thermo Fisher Scientific) to select for human BRCA1 complemented colonies. .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme.

    Generated:

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme. .. Following transduction and selection with puromycin (3 μg/mL) and blasticidin (500 μg/mL) for five days, cells were cultured in the presence of 3 μg/mL doxycycline (Sigma) for at least five days to induce sgRNA expression.

    other:

    Article Title: O6-Methylguanine induces altered proteins at the level of transcription in human cells
    Article Snippet: Chemicals and biochemicals PspOMI, DpnI, BfuAI, NotI, SalI, T4 PNK, T4 DNA ligase and helper phage M13K07 were purchased from New England Biolabs (Waltham, MA, USA).

    Sequencing:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: Duplicate sgRNA sequences were removed on a gene-per-gene basis, sgRNAs containing a TTTTT sequence were removed and sgRNAs were then appended with additional sequences to facilitate PCR and the generation of subpools. .. The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C.

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: Paragraph title: Plasmids, genome editing and sequence analysis ... Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme.

    Cellular Antioxidant Activity Assay:

    Article Title: Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein
    Article Snippet: Restriction enzymes AciI, AscI, BanI, BfuAI, BsrFI, BsrBI, BseYI, BfuAI, BspEI, Cac81, FspI, NciI, NruI (New England Biolabs) and HpyCH4III/Taal (Fermentas) were used. .. Restriction digestions were then analyzed by real time PCR using Universal SYBR Green (BioRad) and primers spanning the promoters regions and primers spanning the promoters regions (BRF2-forward, 5’-GGC CTC CAA AAG CGT T-3’; BRF2-reverse, 5’-AGC TGG CTC TGC GAA TAG T-3’; BRF1-forward, 5’-GGG GTT GGG TCC CAG GTC GC-3’; BRF1-reverse, 5’-GTC CTC CAG CAC TGA GCC GC-3’; U6-forward, 5’- AAG TAT TTC GAT TTC TTG GC-3’; U6-reverse, 5’- AAT ATG GAA CGC TTC ACG-3’; tRNAi Met -forward, 5’-TAG ATA GCA GAG TGG CGC A-3’; tRNAi Met -reverse, 5’-AAC TCC GAT AGC AGA GGA TG-3’).

    Fluorescence:

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: KB1P-G3 cells were transduced with the FUCCI plasmids mKO2-hCdt1(30/120) and mAG-hGem(1/110) ( ) and were sorted for red fluorescence and green fluorescence in two subsequent sorting rounds to ensure the presence of both constructs in each cell. .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme.

    Methylation:

    Article Title: Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein
    Article Snippet: Paragraph title: Methylation analysis ... Restriction enzymes AciI, AscI, BanI, BfuAI, BsrFI, BsrBI, BseYI, BfuAI, BspEI, Cac81, FspI, NciI, NruI (New England Biolabs) and HpyCH4III/Taal (Fermentas) were used.

    Mutagenesis:

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    Labeling:

    Article Title: Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase II? *Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase II? * ♦
    Article Snippet: For multiple labeling with biotin or digoxigenin, 10–20% of appropriately labeled dUTP (Roche Applied Science) was added to the PCR reaction. .. After PCR, the products were purified (Qiagen PCR purification kit) and cut with restriction endonucleases BsaI (New England Biolabs) for the digoxigenin-labeled DNA handle, BfuAI (New England Biolabs) for the biotin-labeled DNA handle, and both enzymes for the 5-kb DNA fragment.

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. For 19 F labeling, cells were then collected by centrifugation and re-suspended in M9 minimal growth medium containing 1 g/L (NH4 )2 SO4 (for 15 N labeling, (NH4 )2 SO4 (Cambridge Isotope Laboratories, Tewksbury, MA)), 2 g/L D-glucose, 4 mL/L of 1M MgSO4 , and 1.8 ml/L of 1 mM FeSO4 .

    Purification:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: .. The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C. .. After digestion, six ligation reactions containing 33 ng of digested sgRNA/crRNAs and 500 ng of the BfuAI and NsiI predigested pLCKO vector were performed overnight at 16 °C with T4 DNA ligase (NEB).

    Article Title: Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase II? *Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase II? * ♦
    Article Snippet: .. After PCR, the products were purified (Qiagen PCR purification kit) and cut with restriction endonucleases BsaI (New England Biolabs) for the digoxigenin-labeled DNA handle, BfuAI (New England Biolabs) for the biotin-labeled DNA handle, and both enzymes for the 5-kb DNA fragment. ..

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C. .. After digestion, six ligation reactions containing 33 ng of digested sgRNA/crRNAs and 500 ng of the BfuAI and NsiI predigested pLCKO vector were performed overnight at 16 °C with T4 DNA ligase (NEB).

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: Following amplification in E.coli, library plasmids with the first position cloned were digested with BfuAI (NEB) to allow cloning of SaCas9 sgRNAs into the second position. .. To remove undigested orthogonal sgRNA library plasmid from the pool, the purified (Nucleospin, Macherey-Nagel) BfuAI digested plasmid was subsequently digested with AscI for which restriction sites exist in the stuffer sequences in both sgRNA positions 1 and 2.

    Article Title: Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
    Article Snippet: .. This stuffer was excised using the BfuAI restriction enzyme (NEB, #R0701) and the linear backbone was gel purified (Zymo, #D4007). .. We designed a targeted library to include all genes matching Gene Ontology for “Cell Surface”, “‘T cell receptor signaling pathway”, or “cytokine receptor activity”.

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: Paragraph title: W1 and W2 mutagenesis, expression, 5F-Trp-labeling, and purification ... W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes.

    Polymerase Chain Reaction:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: .. The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C. .. After digestion, six ligation reactions containing 33 ng of digested sgRNA/crRNAs and 500 ng of the BfuAI and NsiI predigested pLCKO vector were performed overnight at 16 °C with T4 DNA ligase (NEB).

    Article Title: Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase II? *Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase II? * ♦
    Article Snippet: .. After PCR, the products were purified (Qiagen PCR purification kit) and cut with restriction endonucleases BsaI (New England Biolabs) for the digoxigenin-labeled DNA handle, BfuAI (New England Biolabs) for the biotin-labeled DNA handle, and both enzymes for the 5-kb DNA fragment. ..

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C. .. After digestion, six ligation reactions containing 33 ng of digested sgRNA/crRNAs and 500 ng of the BfuAI and NsiI predigested pLCKO vector were performed overnight at 16 °C with T4 DNA ligase (NEB).

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: G418 resistant colonies were tested for human BRCA1 integration by PCR with human BRCA1 exon 11 specific primers: Fwd-5’-TCCAGGAAATGCAGAAGAGG-3’, Rv-5’-ACTGGAGCCCACTTCATTAG-3’. .. Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme.

    Positron Emission Tomography:

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    CRISPR:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: For orthogonal CRISPR libraries, CRISPRa sgRNA pools of 174 sgRNA against 87 selected target genes (2 sgRNAs/gene) plus 18 non-target control sgRNAs were cloned into position 1 of the AarI-digested plasmid sgLenti-orthogonal exactly as described for the CRISPRa library. .. Following amplification in E.coli, library plasmids with the first position cloned were digested with BfuAI (NEB) to allow cloning of SaCas9 sgRNAs into the second position.

    Chromatin Immunoprecipitation:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: All appended sgRNA and crRNA sequences were synthesized as pools by Customarray Inc. (Bothell, WA) on a 12K (9/10 gene libraries) or 90K (“non-FDA-target”/”FDA-target”) chip. .. The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C.

    Plasmid Preparation:

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: The purified PCR products were then subjected to restriction digestion (six parallel reactions) with BfuAI (NEB) overnight at 50 °C. .. After digestion, six ligation reactions containing 33 ng of digested sgRNA/crRNAs and 500 ng of the BfuAI and NsiI predigested pLCKO vector were performed overnight at 16 °C with T4 DNA ligase (NEB).

    Article Title: Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase II? *Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase II? * ♦
    Article Snippet: Briefly, the 5-kb DNA was made by PCR from pET28 plasmid (EMD4 Biosciences) with primers (forward, 5′-GGACCTGCTTTCCAACGCCATATTCAACGGGAAACG-3′ at position 3999; and reverse, 5′GGGTCTCGACCAAACAGCTGATTGCCCTTCAC-3′ at position 1728, Invitrogen) that were both extended to include nonpalindromic restriction sites at their 5′ ends to generate ssDNA regions complementary to the DNA handles after digestion. .. After PCR, the products were purified (Qiagen PCR purification kit) and cut with restriction endonucleases BsaI (New England Biolabs) for the digoxigenin-labeled DNA handle, BfuAI (New England Biolabs) for the biotin-labeled DNA handle, and both enzymes for the 5-kb DNA fragment.

    Article Title: Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes
    Article Snippet: .. After digestion, six ligation reactions containing 33 ng of digested sgRNA/crRNAs and 500 ng of the BfuAI and NsiI predigested pLCKO vector were performed overnight at 16 °C with T4 DNA ligase (NEB). .. The pooled mixture of ligated pLCKO vectors was then purified with the QIAquick nucleotide removal kit (Qiagen) and electroporated into Endura competent cells (Lucigen) with a Gene Pulser system (Biorad) according to the manufacturer’s instructions.

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: For orthogonal CRISPR libraries, CRISPRa sgRNA pools of 174 sgRNA against 87 selected target genes (2 sgRNAs/gene) plus 18 non-target control sgRNAs were cloned into position 1 of the AarI-digested plasmid sgLenti-orthogonal exactly as described for the CRISPRa library. .. Following amplification in E.coli, library plasmids with the first position cloned were digested with BfuAI (NEB) to allow cloning of SaCas9 sgRNAs into the second position.

    Article Title: High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli
    Article Snippet: .. 10× NEB Buffers 100× BSA (10 mg/ml) Restriction enzymes Sna BI, Bfu AI, Ssp I and Sma I (NEB) Incubators set at 25°C, 37°C, 50°C and 65°C Agarose gel electrophoresis system Gel extraction kit (Qiagen 28706) or similar Digest 50-100 μg of plasmid DNA in a total volume of 100-150 μl, using the conditions recommended by the manufacturer (New England Biolabs). .. In order to achieve complete digestion of large quantities of plasmid DNA, we find that overnight digests are acceptable, as long as one avoids using a large excess of enzyme.

    Article Title: Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
    Article Snippet: To optimize this plasmid for cloning the library, we first replaced the sgRNA with a 1.9kb stuffer derived from the lentiGuide-Puro plasmid (Addgene, plasmid #52963) with flanking BfuAI cut sites. .. This stuffer was excised using the BfuAI restriction enzyme (NEB, #R0701) and the linear backbone was gel purified (Zymo, #D4007).

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    SYBR Green Assay:

    Article Title: Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein
    Article Snippet: Restriction enzymes AciI, AscI, BanI, BfuAI, BsrFI, BsrBI, BseYI, BfuAI, BspEI, Cac81, FspI, NciI, NruI (New England Biolabs) and HpyCH4III/Taal (Fermentas) were used. .. Restriction digestions were then analyzed by real time PCR using Universal SYBR Green (BioRad) and primers spanning the promoters regions and primers spanning the promoters regions (BRF2-forward, 5’-GGC CTC CAA AAG CGT T-3’; BRF2-reverse, 5’-AGC TGG CTC TGC GAA TAG T-3’; BRF1-forward, 5’-GGG GTT GGG TCC CAG GTC GC-3’; BRF1-reverse, 5’-GTC CTC CAG CAC TGA GCC GC-3’; U6-forward, 5’- AAG TAT TTC GAT TTC TTG GC-3’; U6-reverse, 5’- AAT ATG GAA CGC TTC ACG-3’; tRNAi Met -forward, 5’-TAG ATA GCA GAG TGG CGC A-3’; tRNAi Met -reverse, 5’-AAC TCC GAT AGC AGA GGA TG-3’).

    Selection:

    Article Title: Radiosensitivity is an acquired vulnerability of PARPi-resistant BRCA1-deficient tumors
    Article Snippet: Cell lines targeted with the pGSC_Cas9_Neo and pLenti-sgRNA-tetR-T2A-Puro system ( ) were generated by lentiviral transduction. sgRNA sequences were cloned in the pLenti-sgRNA-tetR-T2A-Puro backbone using the BfuAI (NEB) restriction enzyme. .. Following transduction and selection with puromycin (3 μg/mL) and blasticidin (500 μg/mL) for five days, cells were cultured in the presence of 3 μg/mL doxycycline (Sigma) for at least five days to induce sgRNA expression.

    Agarose Gel Electrophoresis:

    Article Title: Dual gene activation and knockout screen reveals directional dependencies in genetic networks
    Article Snippet: Following amplification in E.coli, library plasmids with the first position cloned were digested with BfuAI (NEB) to allow cloning of SaCas9 sgRNAs into the second position. .. BfuAI/AscI digested plasmid was extracted from 1% Agarose gel (Nucleospin, Macherey-Nagel).

    Article Title: High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli
    Article Snippet: .. 10× NEB Buffers 100× BSA (10 mg/ml) Restriction enzymes Sna BI, Bfu AI, Ssp I and Sma I (NEB) Incubators set at 25°C, 37°C, 50°C and 65°C Agarose gel electrophoresis system Gel extraction kit (Qiagen 28706) or similar Digest 50-100 μg of plasmid DNA in a total volume of 100-150 μl, using the conditions recommended by the manufacturer (New England Biolabs). .. In order to achieve complete digestion of large quantities of plasmid DNA, we find that overnight digests are acceptable, as long as one avoids using a large excess of enzyme.

    Produced:

    Article Title: Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy
    Article Snippet: .. W2 Trp mutant plasmids (pDW2 ) were produced using the same cloning strategy as previously described , by ligation (using T4 DNA ligase) of two DNA fragments prepared from each of the above pDW1 Trp mutant plasmids through restriction endonuclease digestions ( Bse RI and Bam HI; Bsg I and Bam HI). pDW1 (or pDW2 ) Trp mutant plasmids were then digested by Bsa I and Bfu AI (New England Biolabs, Ipswich, MA), and the resulting W1 (or W2 ) Trp mutant genes were inserted downstream of the His6 -SUMO coding sequence in a modified pET-32 expression plasmid (EMD Millipore, Billerica, MA, USA) that was digested by the same enzymes. .. Each expression plasmid encoding a His6 -SUMO-W1 (or W2 ) Trp mutant was introduced into E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) using standard transformation protocols.

    Gel Extraction:

    Article Title: High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli
    Article Snippet: .. 10× NEB Buffers 100× BSA (10 mg/ml) Restriction enzymes Sna BI, Bfu AI, Ssp I and Sma I (NEB) Incubators set at 25°C, 37°C, 50°C and 65°C Agarose gel electrophoresis system Gel extraction kit (Qiagen 28706) or similar Digest 50-100 μg of plasmid DNA in a total volume of 100-150 μl, using the conditions recommended by the manufacturer (New England Biolabs). .. In order to achieve complete digestion of large quantities of plasmid DNA, we find that overnight digests are acceptable, as long as one avoids using a large excess of enzyme.

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    New England Biolabs bfuai restriction enzyme
    Bfuai Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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