bfuai  (New England Biolabs)


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  • 95
    Name:
    BfuAI
    Description:
    BfuAI 1 250 units
    Catalog Number:
    R0701L
    Price:
    269
    Category:
    Restriction Enzymes
    Size:
    1 250 units
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    Structured Review

    New England Biolabs bfuai
    BfuAI
    BfuAI 1 250 units
    https://www.bioz.com/result/bfuai/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bfuai - by Bioz Stars, 2021-06
    95/100 stars

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    Related Articles

    Agarose Gel Electrophoresis:

    Article Title: High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli
    Article Snippet: Refer to for the appropriate restriction enzyme to be used for any given vector. .. 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.

    Gel Extraction:

    Article Title: High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli
    Article Snippet: Refer to for the appropriate restriction enzyme to be used for any given vector. .. 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.

    Plasmid Preparation:

    Article Title: High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli
    Article Snippet: Refer to for the appropriate restriction enzyme to be used for any given vector. .. 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-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
    Article Snippet: The resulting PCR product was purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey & Nagel), and the correct fragment size was confirmed using a High Sensitivity Bioanalyzer DNA Kit (Agilent). .. For cloning, the HDCRISPRv1 vector was digested with BfuAI (NEB) and BsrGI-HF (NEB) overnight and dephosphorylated using CIP (NEB). .. The resulting linearized ~ 7000 bp vector missing the eGFP stuffer was gel purified again using the NucleoSpin Gel and PCR Clean-up kit (Macherey & Nagel) and used for library cloning in a one-step digestion-ligation-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). .. 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: Inhibition of mitochondrial translation suppresses glioblastoma stem cell growth
    Article Snippet: sgRNA cloning pLKO.1-puro U6 sgRNA BfuAI large stuffer plasmid where the puromycin gene was removed and replaced with a puro-GFP fusion protein as previously described by Phelan et al., 2018 was used (gift from Dr. Louis Staudt, National Cancer Institute, USA). .. The resulting plasmid was digested with BfuAI (NEB cat. R0701S). .. Complementary sgRNA sequences flanked by ACCG on the 5′ end, and CTTT on the 3′ of the reverse strand were annealed, diluted and ligated into the cut vector with T4 ligase (NEB cat. M0202M) according to the manufacturer’s instructions.

    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).

    Purification:

    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. .. 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: 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). .. We designed a targeted library to include all genes matching Gene Ontology for “Cell Surface”, “‘T cell receptor signaling pathway”, or “cytokine receptor activity”.

    Polymerase Chain Reaction:

    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. .. 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).

    Clone Assay:

    Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
    Article Snippet: The resulting PCR product was purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey & Nagel), and the correct fragment size was confirmed using a High Sensitivity Bioanalyzer DNA Kit (Agilent). .. For cloning, the HDCRISPRv1 vector was digested with BfuAI (NEB) and BsrGI-HF (NEB) overnight and dephosphorylated using CIP (NEB). .. The resulting linearized ~ 7000 bp vector missing the eGFP stuffer was gel purified again using the NucleoSpin Gel and PCR Clean-up kit (Macherey & Nagel) and used for library cloning in a one-step digestion-ligation-reaction [ ].

    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). .. 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.

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  • 95
    New England Biolabs bfuai
    Bfuai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bfuai/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bfuai - by Bioz Stars, 2021-06
    95/100 stars
      Buy from Supplier

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