spei hf  (New England Biolabs)


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  • 90
    Name:
    SpeI HF
    Description:
    SpeI HF 2 500 units
    Catalog Number:
    r3133l
    Price:
    277
    Size:
    2 500 units
    Category:
    Restriction Enzymes
    Buy from Supplier


    Structured Review

    New England Biolabs spei hf
    SpeI HF
    SpeI HF 2 500 units
    https://www.bioz.com/result/spei hf/product/New England Biolabs
    Average 90 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    spei hf - by Bioz Stars, 2020-01
    90/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: .. Positive clones were confirmed by restriction digestion of the isolated plasmid with EcoRI HF and SpeI-HF (New England Biolabs, Massachusetts, USA) sequentially, at 37°C overnight and electrophoresing the digested products on 0.8% agarose gels. .. Overlapping primers were designed for hdac6 ORF using Primer 3 software to amplify and sequence the 3.56 kb gene by Sanger sequencing ( ).

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: Paragraph title: Cloning ... The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes.

    Article Title: CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters
    Article Snippet: In addition, the CMV promoter cassette was removed by digestion with NheI-HF and SpeI-HF (NEB) and replaced with the GRK1 or CAR promoter sequences. .. Guide RNA sequences targeting either promoter were cloned between the SapI sites between the U6 promoter and scaffold.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain
    Article Snippet: This was done by PCR mutagenesis with Q5 polymerase (M0491S; NEB), primers i75 and i76 , digestion with SpeI (R3133S; NEB), and subsequent religation. .. Inducible constructs were then cloned into J23100-mRFP1-pSEVA331Bb, replacing the constitutive J23100 promoter (thus controlling downstream mRFP1 expression) to create pTet01, pTet02, pLux01, and pLux02.

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: .. This was cloned into pJET cloning vector and transformed in TOP10 competent cells, and the plasmids from the positive colonies were double digested with EcoRI HF and SpeI-HF. .. Two bands of 2.9 Kb and 3.5 kb were observed for vector and hdac6 gene, respectively (data not shown).

    Amplification:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease
    Article Snippet: The PacBio No-Amp targeted sequencing procedure, currently in development, uses the CRISPR-Cas9 system to target and enrich a region of interest without PCR amplification (Fig. ) [ ]. .. Genome complexity reduction was then performed by incubating each sample with high fidelity restriction enzymes KpnI-HF and SpeI-HF (New England Biolabs, PN R3142S and R3133S, respectively) and Exonuclease III and VII (Pacific Biosciences, part of SMRTbell Template Prep Kit 1.0, PN 100–259-100).

    Article Title: CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters
    Article Snippet: The eGFP-KASH coding sequence was removed by digestion with AgeI-HF and EcoRI-HIF (NEB, Ipswich, MA, USA) and the wild-type eGFP sequence was amplified and restored between these sites. .. In addition, the CMV promoter cassette was removed by digestion with NheI-HF and SpeI-HF (NEB) and replaced with the GRK1 or CAR promoter sequences.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Synthesized:

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: Construction of Cas9 expression cassettes The DNA sequence of the Cas9 gene was synthesized from the MLM3613 plasmid (Addgene, Catalog no. 42251). .. For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions.

    Construct:

    Article Title: Structure of a human translation termination complex
    Article Snippet: In vitro transcription Plasmids were linearized with SpeI-HF (NEB) in CutSmart (NEB) buffer at 37°C overnight and purified (QIAquick PCR purification kit (Qiagen)) before in vitro transcription (tc) to ensure the right length of the mRNA product. .. The final mRNA construct encoded a CrPV IGR IRES sequence for translation initiation, an N-terminal HA- and (His)6 -tag, the well characterized DP75 dipeptidyl-aminopeptidase B (DPAPB) , the hCMV uORF2 stalling sequence and a polyA-tail.

    Article Title: Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain
    Article Snippet: Paragraph title: Engineering of Constitutive Promoters, Inducible Promoters, CBD Fusions, and sRNA Constructs. ... This was done by PCR mutagenesis with Q5 polymerase (M0491S; NEB), primers i75 and i76 , digestion with SpeI (R3133S; NEB), and subsequent religation.

    Incubation:

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: Positive colonies were inoculated in 2 ml L.B broth containing 100 ug/ml ampicillin and incubated at 37°C, 230 rpm O/N. .. Positive clones were confirmed by restriction digestion of the isolated plasmid with EcoRI HF and SpeI-HF (New England Biolabs, Massachusetts, USA) sequentially, at 37°C overnight and electrophoresing the digested products on 0.8% agarose gels.

    Article Title: Origin plasticity during budding yeast DNA replication in vitro
    Article Snippet: pARS1 was digested with AfeI, NdeI, SpeI-HF, and XmnI (all DNA modifying enzymes were from NEB); pARS305 was digested with BglI, Bsu36I, and PstI. .. To identify ORC binding sites, restriction fragment mixtures containing 11 nM of each fragment were incubated with ORC as indicated in 40 μl of 25 mM Hepes-KOH pH 7.6/0.12 M KOAc/10 mM Mg(OAc)2 /0.02% NP-40/5% glycerol/1 mM DTT/5 mM ATP for 30 min at 30°C.

    Expressing:

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: .. For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions. .. The expression cassette consists of the following elements: Ef1α -Cas9-NLS-bGH poly (A).

    Article Title: Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain
    Article Snippet: This was done by PCR mutagenesis with Q5 polymerase (M0491S; NEB), primers i75 and i76 , digestion with SpeI (R3133S; NEB), and subsequent religation. .. Inducible constructs were then cloned into J23100-mRFP1-pSEVA331Bb, replacing the constitutive J23100 promoter (thus controlling downstream mRFP1 expression) to create pTet01, pTet02, pLux01, and pLux02.

    Modification:

    Article Title: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease
    Article Snippet: Genome complexity reduction was then performed by incubating each sample with high fidelity restriction enzymes KpnI-HF and SpeI-HF (New England Biolabs, PN R3142S and R3133S, respectively) and Exonuclease III and VII (Pacific Biosciences, part of SMRTbell Template Prep Kit 1.0, PN 100–259-100). .. Oligos comprising the guide RNA (crRNA and tracrRNA) were obtained from Integrated DNA Technologies containing an Alt-R modification to prevent RNase degradation.

    Transformation Assay:

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: Briefly, the 3.5 Kb band excised and extracted from the gel was cloned into pJET cloning vector in 1 : 3 ratio and the mixture incubated at 22°C for 1 h. Ligation mixture was ethanol precipitated and then transformed in Top10 competent cells and the cells plated on LB agar containing 100 ug/ml ampicillin and incubated at 37°C overnight. .. Positive clones were confirmed by restriction digestion of the isolated plasmid with EcoRI HF and SpeI-HF (New England Biolabs, Massachusetts, USA) sequentially, at 37°C overnight and electrophoresing the digested products on 0.8% agarose gels.

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: The PCR conditions are set out below: Denature 98 °C 30 s 1X Denature 98 °C 8 s Anneal 57–60 °C 20 s 35X Extension 72 °C 15 s Final Extension 72 °C 5 min 1X The second PCR was performed using 120 ng DNA generated in the first PCR reaction and 70 ng of the pEF1α-V5/His vector as the destination vector using the following PCR conditions: Denature 98 °C 30 s 1X Denature 98 °C 8 s Extension 72°2 min 15 s 15X Final extension 72 °C 5 min 1X Competent cells were transformed with 1 µl of the final PCR product using One Shot® MAX Efficiency® DH5α™-T1R chemically-competent cells (Invitrogen) and single colonies grown for plasmid DNA extraction. .. The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes.

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: .. This was cloned into pJET cloning vector and transformed in TOP10 competent cells, and the plasmids from the positive colonies were double digested with EcoRI HF and SpeI-HF. .. Two bands of 2.9 Kb and 3.5 kb were observed for vector and hdac6 gene, respectively (data not shown).

    Gel Purification:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)). .. The rx1 gfp plasmid was cloned with homology flanks (5’HF 430 bp, primers rx1 5’HF f/rx1 5’HF r; 3’ HF 508 bp, rx1 3’HF f/rx1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)). .. The rx1 gfp plasmid was cloned with homology flanks (5’HF 430 bp, primers rx1 5’HF f/rx1 5’HF r; 3’ HF 508 bp, rx1 3’HF f/rx1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA.

    Transfection:

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes. .. The vector was transfected into cells seeded into a 6-well plate using Lipofectamine 2000 (Thermo Fisher, Cat. No 11668027) using 2.5 µg plasmid DNA and 9 µl Lipofectamine 2000 reagent per well.

    Ligation:

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: Briefly, the 3.5 Kb band excised and extracted from the gel was cloned into pJET cloning vector in 1 : 3 ratio and the mixture incubated at 22°C for 1 h. Ligation mixture was ethanol precipitated and then transformed in Top10 competent cells and the cells plated on LB agar containing 100 ug/ml ampicillin and incubated at 37°C overnight. .. Positive clones were confirmed by restriction digestion of the isolated plasmid with EcoRI HF and SpeI-HF (New England Biolabs, Massachusetts, USA) sequentially, at 37°C overnight and electrophoresing the digested products on 0.8% agarose gels.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)). .. The rx1 gfp plasmid was cloned with homology flanks (5’HF 430 bp, primers rx1 5’HF f/rx1 5’HF r; 3’ HF 508 bp, rx1 3’HF f/rx1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA.

    Article Title: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease
    Article Snippet: A SMRTbell library was prepared from the EcoRI-HF digested products by ligation with a hairpin adapter containing an overhang sequence complementary to the EcoRI-HF cut site using E. coli DNA ligase (New England Biolabs, PN M0205S). .. Genome complexity reduction was then performed by incubating each sample with high fidelity restriction enzymes KpnI-HF and SpeI-HF (New England Biolabs, PN R3142S and R3133S, respectively) and Exonuclease III and VII (Pacific Biosciences, part of SMRTbell Template Prep Kit 1.0, PN 100–259-100).

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: .. For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions. .. The expression cassette consists of the following elements: Ef1α -Cas9-NLS-bGH poly (A).

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)). .. The rx1 gfp plasmid was cloned with homology flanks (5’HF 430 bp, primers rx1 5’HF f/rx1 5’HF r; 3’ HF 508 bp, rx1 3’HF f/rx1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA.

    Generated:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: Donor plasmids Rx2 and actb template plasmids for gfp donor cassette amplification are described in and were generated by GoldenGATE assembly into the pGGDestSC-ATG destination vector (addgene #49322) according to . .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: .. The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes. .. The pEF1α-GFP backbone and FH insert were purified, ligated and used to transform bacterial competent cells as described above.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: Rx2 and actb template plasmids for gfp donor cassette amplification are described in and were generated by GoldenGATE assembly into the pGGDestSC-ATG destination vector (addgene #49322) according to . .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Sequencing:

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: Paragraph title: 2.5. Cloning into pJET Cloning Vector & Sequencing ... Positive clones were confirmed by restriction digestion of the isolated plasmid with EcoRI HF and SpeI-HF (New England Biolabs, Massachusetts, USA) sequentially, at 37°C overnight and electrophoresing the digested products on 0.8% agarose gels.

    Article Title: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease
    Article Snippet: Paragraph title: Sequencing ... Genome complexity reduction was then performed by incubating each sample with high fidelity restriction enzymes KpnI-HF and SpeI-HF (New England Biolabs, PN R3142S and R3133S, respectively) and Exonuclease III and VII (Pacific Biosciences, part of SMRTbell Template Prep Kit 1.0, PN 100–259-100).

    Article Title: Structure of a human translation termination complex
    Article Snippet: In vitro transcription Plasmids were linearized with SpeI-HF (NEB) in CutSmart (NEB) buffer at 37°C overnight and purified (QIAquick PCR purification kit (Qiagen)) before in vitro transcription (tc) to ensure the right length of the mRNA product. .. The final mRNA construct encoded a CrPV IGR IRES sequence for translation initiation, an N-terminal HA- and (His)6 -tag, the well characterized DP75 dipeptidyl-aminopeptidase B (DPAPB) , the hCMV uORF2 stalling sequence and a polyA-tail.

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: Primers were then designed to recognise FH cDNA that does not contain the endogenous mitochondrial targeting sequence (MTS) and included the Spe I and Eco RI restriction sites alongside a buffer sequence of GATC. .. The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes.

    Article Title: CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters
    Article Snippet: The eGFP-KASH coding sequence was removed by digestion with AgeI-HF and EcoRI-HIF (NEB, Ipswich, MA, USA) and the wild-type eGFP sequence was amplified and restored between these sites. .. In addition, the CMV promoter cassette was removed by digestion with NheI-HF and SpeI-HF (NEB) and replaced with the GRK1 or CAR promoter sequences.

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: Construction of Cas9 expression cassettes The DNA sequence of the Cas9 gene was synthesized from the MLM3613 plasmid (Addgene, Catalog no. 42251). .. For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions.

    Binding Assay:

    Article Title: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease
    Article Snippet: MagBead binding was performed using the PacBio protocol for kit 100–125-900. .. Genome complexity reduction was then performed by incubating each sample with high fidelity restriction enzymes KpnI-HF and SpeI-HF (New England Biolabs, PN R3142S and R3133S, respectively) and Exonuclease III and VII (Pacific Biosciences, part of SMRTbell Template Prep Kit 1.0, PN 100–259-100).

    Article Title: Origin plasticity during budding yeast DNA replication in vitro
    Article Snippet: pARS1 was digested with AfeI, NdeI, SpeI-HF, and XmnI (all DNA modifying enzymes were from NEB); pARS305 was digested with BglI, Bsu36I, and PstI. .. To identify ORC binding sites, restriction fragment mixtures containing 11 nM of each fragment were incubated with ORC as indicated in 40 μl of 25 mM Hepes-KOH pH 7.6/0.12 M KOAc/10 mM Mg(OAc)2 /0.02% NP-40/5% glycerol/1 mM DTT/5 mM ATP for 30 min at 30°C.

    DNA Extraction:

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: The PCR conditions are set out below: Denature 98 °C 30 s 1X Denature 98 °C 8 s Anneal 57–60 °C 20 s 35X Extension 72 °C 15 s Final Extension 72 °C 5 min 1X The second PCR was performed using 120 ng DNA generated in the first PCR reaction and 70 ng of the pEF1α-V5/His vector as the destination vector using the following PCR conditions: Denature 98 °C 30 s 1X Denature 98 °C 8 s Extension 72°2 min 15 s 15X Final extension 72 °C 5 min 1X Competent cells were transformed with 1 µl of the final PCR product using One Shot® MAX Efficiency® DH5α™-T1R chemically-competent cells (Invitrogen) and single colonies grown for plasmid DNA extraction. .. The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes.

    Mutagenesis:

    Article Title: Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain
    Article Snippet: .. This was done by PCR mutagenesis with Q5 polymerase (M0491S; NEB), primers i75 and i76 , digestion with SpeI (R3133S; NEB), and subsequent religation. .. Constitutive promoter-mRFP1 constructs (BBa_J23100–Bba_J23117 by iGEM 2006 Berkeley) were received from the iGEM Registry of Standard Biological Parts ( ) and subcloned into pSEVA331Bb.

    Isolation:

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: .. Positive clones were confirmed by restriction digestion of the isolated plasmid with EcoRI HF and SpeI-HF (New England Biolabs, Massachusetts, USA) sequentially, at 37°C overnight and electrophoresing the digested products on 0.8% agarose gels. .. Overlapping primers were designed for hdac6 ORF using Primer 3 software to amplify and sequence the 3.56 kb gene by Sanger sequencing ( ).

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: .. Plasmids were isolated from the positive colonies and double digested with EcoRI HF and SpeI-HF. .. Two bands of 2.9 Kb and 3.5 kb were observed for vector and hdac6 gene, respectively (data not shown).

    Purification:

    Article Title: Structure of a human translation termination complex
    Article Snippet: .. In vitro transcription Plasmids were linearized with SpeI-HF (NEB) in CutSmart (NEB) buffer at 37°C overnight and purified (QIAquick PCR purification kit (Qiagen)) before in vitro transcription (tc) to ensure the right length of the mRNA product. .. The final mRNA construct encoded a CrPV IGR IRES sequence for translation initiation, an N-terminal HA- and (His)6 -tag, the well characterized DP75 dipeptidyl-aminopeptidase B (DPAPB) , the hCMV uORF2 stalling sequence and a polyA-tail.

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: .. The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes. .. The pEF1α-GFP backbone and FH insert were purified, ligated and used to transform bacterial competent cells as described above.

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions. .. The purified product was used in PCR with the aforementioned Cas9 expression cassette to produce the Cas9 transgenic cassette: HRL-Ef1α -Cas9-NLS-bGH poly (A)-HRR.

    Article Title: Origin plasticity during budding yeast DNA replication in vitro
    Article Snippet: pARS1 was digested with AfeI, NdeI, SpeI-HF, and XmnI (all DNA modifying enzymes were from NEB); pARS305 was digested with BglI, Bsu36I, and PstI. .. Restriction fragments were 5′-end-labeled with γ-32 P-ATP (Perkin Elmer) and T4 polynucleotide kinase, and purified by phenol/chloroform extraction and ethanol precipitation.

    Polymerase Chain Reaction:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease
    Article Snippet: The PacBio No-Amp targeted sequencing procedure, currently in development, uses the CRISPR-Cas9 system to target and enrich a region of interest without PCR amplification (Fig. ) [ ]. .. Genome complexity reduction was then performed by incubating each sample with high fidelity restriction enzymes KpnI-HF and SpeI-HF (New England Biolabs, PN R3142S and R3133S, respectively) and Exonuclease III and VII (Pacific Biosciences, part of SMRTbell Template Prep Kit 1.0, PN 100–259-100).

    Article Title: Structure of a human translation termination complex
    Article Snippet: .. In vitro transcription Plasmids were linearized with SpeI-HF (NEB) in CutSmart (NEB) buffer at 37°C overnight and purified (QIAquick PCR purification kit (Qiagen)) before in vitro transcription (tc) to ensure the right length of the mRNA product. .. The final mRNA construct encoded a CrPV IGR IRES sequence for translation initiation, an N-terminal HA- and (His)6 -tag, the well characterized DP75 dipeptidyl-aminopeptidase B (DPAPB) , the hCMV uORF2 stalling sequence and a polyA-tail.

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: .. The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes. .. The pEF1α-GFP backbone and FH insert were purified, ligated and used to transform bacterial competent cells as described above.

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions. .. The purified product was used in PCR with the aforementioned Cas9 expression cassette to produce the Cas9 transgenic cassette: HRL-Ef1α -Cas9-NLS-bGH poly (A)-HRR.

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain
    Article Snippet: .. This was done by PCR mutagenesis with Q5 polymerase (M0491S; NEB), primers i75 and i76 , digestion with SpeI (R3133S; NEB), and subsequent religation. .. Constitutive promoter-mRFP1 constructs (BBa_J23100–Bba_J23117 by iGEM 2006 Berkeley) were received from the iGEM Registry of Standard Biological Parts ( ) and subcloned into pSEVA331Bb.

    CRISPR:

    Article Title: Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease
    Article Snippet: The PacBio No-Amp targeted sequencing procedure, currently in development, uses the CRISPR-Cas9 system to target and enrich a region of interest without PCR amplification (Fig. ) [ ]. .. Genome complexity reduction was then performed by incubating each sample with high fidelity restriction enzymes KpnI-HF and SpeI-HF (New England Biolabs, PN R3142S and R3133S, respectively) and Exonuclease III and VII (Pacific Biosciences, part of SMRTbell Template Prep Kit 1.0, PN 100–259-100).

    Plasmid Preparation:

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: .. Positive clones were confirmed by restriction digestion of the isolated plasmid with EcoRI HF and SpeI-HF (New England Biolabs, Massachusetts, USA) sequentially, at 37°C overnight and electrophoresing the digested products on 0.8% agarose gels. .. Overlapping primers were designed for hdac6 ORF using Primer 3 software to amplify and sequence the 3.56 kb gene by Sanger sequencing ( ).

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: .. The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes. .. The pEF1α-GFP backbone and FH insert were purified, ligated and used to transform bacterial competent cells as described above.

    Article Title: CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters
    Article Snippet: Paragraph title: Plasmid Generation ... In addition, the CMV promoter cassette was removed by digestion with NheI-HF and SpeI-HF (NEB) and replaced with the GRK1 or CAR promoter sequences.

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: .. For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions. .. The expression cassette consists of the following elements: Ef1α -Cas9-NLS-bGH poly (A).

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dnmt1 gfp plasmid was cloned with homology flanks (5’ HF 402 bp, primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp, dnmt1 3’HF f/dnmt1 3’HF r) that were PCR amplified with Q5 polymerase (New England Biolabs, 30 cycles) from wild-type medaka genomic DNA. mgfp-flexible linker was amplified with primers mgfpf/mgfpr. .. The respective restriction enzyme was used to digest the amplicons (5’HF: SalI HF (New England Biolabs), AgeI HF (New England Biolabs); mgfp-flexible linker : AgeI HF (New England Biolabs), SpeI HF (New England Biolabs); 3’HF: SpeI HF (New England Biolabs), NotI HF (New England Biolabs)) followed by gel purification (Analytik Jena) and ligation into pCS2+ ( ) (digested with SalI HF (New England Biolabs), NotI HF (New England Biolabs)).

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: .. This was cloned into pJET cloning vector and transformed in TOP10 competent cells, and the plasmids from the positive colonies were double digested with EcoRI HF and SpeI-HF. .. Two bands of 2.9 Kb and 3.5 kb were observed for vector and hdac6 gene, respectively (data not shown).

    Software:

    Article Title: Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico
    Article Snippet: Positive clones were confirmed by restriction digestion of the isolated plasmid with EcoRI HF and SpeI-HF (New England Biolabs, Massachusetts, USA) sequentially, at 37°C overnight and electrophoresing the digested products on 0.8% agarose gels. .. Overlapping primers were designed for hdac6 ORF using Primer 3 software to amplify and sequence the 3.56 kb gene by Sanger sequencing ( ).

    In Vitro:

    Article Title: Structure of a human translation termination complex
    Article Snippet: .. In vitro transcription Plasmids were linearized with SpeI-HF (NEB) in CutSmart (NEB) buffer at 37°C overnight and purified (QIAquick PCR purification kit (Qiagen)) before in vitro transcription (tc) to ensure the right length of the mRNA product. .. The final mRNA construct encoded a CrPV IGR IRES sequence for translation initiation, an N-terminal HA- and (His)6 -tag, the well characterized DP75 dipeptidyl-aminopeptidase B (DPAPB) , the hCMV uORF2 stalling sequence and a polyA-tail.

    Transgenic Assay:

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions. .. The purified product was used in PCR with the aforementioned Cas9 expression cassette to produce the Cas9 transgenic cassette: HRL-Ef1α -Cas9-NLS-bGH poly (A)-HRR.

    Ethanol Precipitation:

    Article Title: Origin plasticity during budding yeast DNA replication in vitro
    Article Snippet: pARS1 was digested with AfeI, NdeI, SpeI-HF, and XmnI (all DNA modifying enzymes were from NEB); pARS305 was digested with BglI, Bsu36I, and PstI. .. Restriction fragments were 5′-end-labeled with γ-32 P-ATP (Perkin Elmer) and T4 polynucleotide kinase, and purified by phenol/chloroform extraction and ethanol precipitation.

    Concentration Assay:

    Article Title: Fumarate hydratase loss promotes mitotic entry in the presence of DNA damage after ionising radiation
    Article Snippet: The PCR was performed using the Phusion DNA polymerase and primers at a final concentration of 0.5 µM with the pAcGFP-N1 vector as a template to copy the GFP sequence. .. The purified FH PCR product and the recently generated pEF1α-GFP vector were then subject to digestion by Spe I (NEB, Cat. No. R3133S) and Eco RI (NEB, Cat. No. R3101S) HF restriction enzymes.

    Homologous Recombination:

    Article Title: Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model
    Article Snippet: For the construction of the Cas9 expression cassette, the Ef1α promoter was inserted into MLM3613 plasmid via Sep I (NEB, Catalog No. R3133S) and NheI digestion, followed by ligation with T4 ligase according to the manufacturers’ instructions. .. For locus-specific integration of the Cas9 expression cassette at the Mitfa locus in the zebrafish genome, Genewiz, Inc. (Suzhou, China), synthesized the 40-bp-long homologous recombination arms (HRL and HRR) around the Mitfα sgRNA identification site, together with the linker primers Cas9 cassette-F and Cas9 cassette-R (Supplemental Table 1).

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    New England Biolabs spei hf
    Spei Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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