r3552l  (New England Biolabs)


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    Name:
    AgeI HF
    Description:
    AgeI HF 1 500 units
    Catalog Number:
    R3552L
    Price:
    298
    Category:
    Restriction Enzymes
    Size:
    1 500 units
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    Structured Review

    New England Biolabs r3552l
    AgeI HF
    AgeI HF 1 500 units
    https://www.bioz.com/result/r3552l/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    r3552l - by Bioz Stars, 2021-06
    99/100 stars

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    Related Articles

    Polymerase Chain Reaction:

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: For cloning, two 30 bp overlapping oligonucleotides were designed and amplified using Q5® High-Fidelity DNA polymerase (NEB) according to the supplier's instructions. .. The resulting PCR product was purified (QIAquick PCR Purification Kit, Qiagen), digested with AgeI-HF and NheI-HF (NEB) and ligated into equally digested pWHE601* with T4 DNA Ligase (NEB). .. Doped pools were generated using the oligonucleotides AgeI_doped_fwd and NheI_[3.0/4.5/9.0/30.0]_doped_rev ( , Microsynth AG), respectively, with the construct ΔATG as template and amplified using Q5® High-Fidelity DNA polymerase (NEB) according to the supplier's instructions.

    Article Title: Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development
    Article Snippet: Restriction Digestion of PCR Products PCR products for the 5’ loxP , 3’ loxP , and Cre mediated KO recombination allele were amplified from the Fzd2 tm1Vari flox model, column purified (T1030S, New England Biolabs) and then used for a restriction endonuclease digest. .. Digestion reactions consisted of 500ng of PCR product, 20 units of either XhoI (R1046S, NEB) or AgeI (R3552S, NEB) in their specified buffers. ..

    Purification:

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: For cloning, two 30 bp overlapping oligonucleotides were designed and amplified using Q5® High-Fidelity DNA polymerase (NEB) according to the supplier's instructions. .. The resulting PCR product was purified (QIAquick PCR Purification Kit, Qiagen), digested with AgeI-HF and NheI-HF (NEB) and ligated into equally digested pWHE601* with T4 DNA Ligase (NEB). .. Doped pools were generated using the oligonucleotides AgeI_doped_fwd and NheI_[3.0/4.5/9.0/30.0]_doped_rev ( , Microsynth AG), respectively, with the construct ΔATG as template and amplified using Q5® High-Fidelity DNA polymerase (NEB) according to the supplier's instructions.

    Plasmid Preparation:

    Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain
    Article Snippet: The resulting plasmid, pT2-CAG-ZEB2-GFP-Flag-2A-Puro, was used as template in a PCR reaction with primers ZEB2_GFP_Flag_IndOE_F & _R. .. The resulting fragment encoding ZEB2-GFP-Flag was then digested with restriction enzymes AgeI (NEB, R3552S) and KpnI (NEB, R3142S) and ligated into the CAGs-MCS-Enhanced Episomal Vector (EEV) (System Biosciences, EEV600A-1) linearized with AgeI and KpnI, using T4 ligase. .. Next, using primers ZEB2_AAVS1 IndOE_F & _R a fragment containing ZEB2-GFP-Flag-WPRE-poly(A) and overhangs containing MluI and FseI restriction sites at the 5′ and 3′ end, respectively, was generated.

    Article Title: Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance
    Article Snippet: .. Immunohistochemical analysis of αMSH and AgRP projections Human wild-type NRP1 and NRP2 along with the NRP1 R237Q and NRP2 A506V variants were cloned into the pCMV6-Entry vector (Origene), which was then linearized using the AgeI-HF restriction enzyme (New England Biolabs, #R3552), column cleaned (Zymo Research Clean & Concentrator, #D4013) and RNA transcribed using the mMessage mMachine T7 Transcription Kit as according to the manufacturers protocol (Thermo Fisher, #AM1344). .. Capped RNA was then cleaned using Zymo RNA Clean & Concentrator columns (Zymo Research Clean & Concentrator, #R1013) and 100 pg injected into one-cell stage wild-type (WIK) zebrafish embryos.

    Gel Purification:

    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)). .. 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 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)). .. 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.

    Ligation:

    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)). .. 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 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)). .. 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.

    Immunohistochemistry:

    Article Title: Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance
    Article Snippet: .. Immunohistochemical analysis of αMSH and AgRP projections Human wild-type NRP1 and NRP2 along with the NRP1 R237Q and NRP2 A506V variants were cloned into the pCMV6-Entry vector (Origene), which was then linearized using the AgeI-HF restriction enzyme (New England Biolabs, #R3552), column cleaned (Zymo Research Clean & Concentrator, #D4013) and RNA transcribed using the mMessage mMachine T7 Transcription Kit as according to the manufacturers protocol (Thermo Fisher, #AM1344). .. Capped RNA was then cleaned using Zymo RNA Clean & Concentrator columns (Zymo Research Clean & Concentrator, #R1013) and 100 pg injected into one-cell stage wild-type (WIK) zebrafish embryos.

    Clone Assay:

    Article Title: Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance
    Article Snippet: .. Immunohistochemical analysis of αMSH and AgRP projections Human wild-type NRP1 and NRP2 along with the NRP1 R237Q and NRP2 A506V variants were cloned into the pCMV6-Entry vector (Origene), which was then linearized using the AgeI-HF restriction enzyme (New England Biolabs, #R3552), column cleaned (Zymo Research Clean & Concentrator, #D4013) and RNA transcribed using the mMessage mMachine T7 Transcription Kit as according to the manufacturers protocol (Thermo Fisher, #AM1344). .. Capped RNA was then cleaned using Zymo RNA Clean & Concentrator columns (Zymo Research Clean & Concentrator, #R1013) and 100 pg injected into one-cell stage wild-type (WIK) zebrafish embryos.

    Isolation:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: The RT-PCR product was analyzed on 10% PAA gels in TBE buffer, and purified using ‘PCR clean up kit’ (Qiagen, Hilden, Germany). .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C. .. The restriction fragments were determined using PAA gel-electrophoresis and visualized by EtBr-staining.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: The RT-PCR product was analyzed on 10% PAA gels in TBE buffer, and purified using ‘PCR clean up kit’ (Qiagen, Hilden, Germany). .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C. .. The restriction fragments were determined using PAA gel-electrophoresis and visualized by EtBr-staining.

    Incubation:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    Article Snippet: The RT-PCR product was analyzed on 10% PAA gels in TBE buffer, and purified using ‘PCR clean up kit’ (Qiagen, Hilden, Germany). .. For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C. .. The restriction fragments were determined using PAA gel-electrophoresis and visualized by EtBr-staining.

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  • 99
    New England Biolabs agei hf
    Confirmation of Cre-mediated recombination of Fzd2 tm1Vari allele. A. Map of proposed Fzd2 tm1Vari recombination product. Xho1 and Age1 restriction sites (purple) were incorporated to aid in the validation of the allele. LoxP sites are indicated by red triangles, and 5’and 3’ untranslated regions (UTR) are shown. Primer binding sites for genotyping are shown. B . Limb micromass cultures from Fzd2 tmVari flox/flox were infected with Ad5CMV:eGFP (control) or Ad5CMV:Cre-eGFP at an MOI of 100. Brightfield and fluorescent images show infection efficiency. Ad5CMV:eGFP is cytoplasmic and Ad5CMV:Cre-eGFP is nuclear. Scale = 200um C . <t>PCR-based</t> genotyping for Cre or Fzd2 knockout (KO). Ad5CMV:Cre-eGFP infected cells amplified a product for Cre and Fzd2-KO. D . Single-cell suspensions were prepared from Ad:eGFP or Ad:Cre-eGFP infected micromass cultures, stained for Fzd2, and analyzed by flow cytometry. Cells were gated to discard debris and doublets. Live cells were determined by DAPI exclusion followed by gating on eGFP. FZD2 median fluorescent intensity (MFI) was compared between Ad:eGFP or Ad:Cre-eGFP infected micromass cultures. E . PCR-based allele-specific genotyping products for wild-type (292bp) and KO (485bp) products. The third lane includes a PCR product using primer set for KO only to be used for restriction digestions. F . Wild-type product was digested with XhoI and <t>AgeI</t> resulting in a wild-type uncut product (292bp). G . KO product was digested with XhoI and AgeI resulting in the expected digestion products: XhoI (196 and 291bp), AgeI (239 and 246bp). undig = undigested
    Agei Hf, 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
    https://www.bioz.com/result/agei hf/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    agei hf - by Bioz Stars, 2021-06
    99/100 stars
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    Confirmation of Cre-mediated recombination of Fzd2 tm1Vari allele. A. Map of proposed Fzd2 tm1Vari recombination product. Xho1 and Age1 restriction sites (purple) were incorporated to aid in the validation of the allele. LoxP sites are indicated by red triangles, and 5’and 3’ untranslated regions (UTR) are shown. Primer binding sites for genotyping are shown. B . Limb micromass cultures from Fzd2 tmVari flox/flox were infected with Ad5CMV:eGFP (control) or Ad5CMV:Cre-eGFP at an MOI of 100. Brightfield and fluorescent images show infection efficiency. Ad5CMV:eGFP is cytoplasmic and Ad5CMV:Cre-eGFP is nuclear. Scale = 200um C . PCR-based genotyping for Cre or Fzd2 knockout (KO). Ad5CMV:Cre-eGFP infected cells amplified a product for Cre and Fzd2-KO. D . Single-cell suspensions were prepared from Ad:eGFP or Ad:Cre-eGFP infected micromass cultures, stained for Fzd2, and analyzed by flow cytometry. Cells were gated to discard debris and doublets. Live cells were determined by DAPI exclusion followed by gating on eGFP. FZD2 median fluorescent intensity (MFI) was compared between Ad:eGFP or Ad:Cre-eGFP infected micromass cultures. E . PCR-based allele-specific genotyping products for wild-type (292bp) and KO (485bp) products. The third lane includes a PCR product using primer set for KO only to be used for restriction digestions. F . Wild-type product was digested with XhoI and AgeI resulting in a wild-type uncut product (292bp). G . KO product was digested with XhoI and AgeI resulting in the expected digestion products: XhoI (196 and 291bp), AgeI (239 and 246bp). undig = undigested

    Journal: bioRxiv

    Article Title: Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development

    doi: 10.1101/2021.01.27.428341

    Figure Lengend Snippet: Confirmation of Cre-mediated recombination of Fzd2 tm1Vari allele. A. Map of proposed Fzd2 tm1Vari recombination product. Xho1 and Age1 restriction sites (purple) were incorporated to aid in the validation of the allele. LoxP sites are indicated by red triangles, and 5’and 3’ untranslated regions (UTR) are shown. Primer binding sites for genotyping are shown. B . Limb micromass cultures from Fzd2 tmVari flox/flox were infected with Ad5CMV:eGFP (control) or Ad5CMV:Cre-eGFP at an MOI of 100. Brightfield and fluorescent images show infection efficiency. Ad5CMV:eGFP is cytoplasmic and Ad5CMV:Cre-eGFP is nuclear. Scale = 200um C . PCR-based genotyping for Cre or Fzd2 knockout (KO). Ad5CMV:Cre-eGFP infected cells amplified a product for Cre and Fzd2-KO. D . Single-cell suspensions were prepared from Ad:eGFP or Ad:Cre-eGFP infected micromass cultures, stained for Fzd2, and analyzed by flow cytometry. Cells were gated to discard debris and doublets. Live cells were determined by DAPI exclusion followed by gating on eGFP. FZD2 median fluorescent intensity (MFI) was compared between Ad:eGFP or Ad:Cre-eGFP infected micromass cultures. E . PCR-based allele-specific genotyping products for wild-type (292bp) and KO (485bp) products. The third lane includes a PCR product using primer set for KO only to be used for restriction digestions. F . Wild-type product was digested with XhoI and AgeI resulting in a wild-type uncut product (292bp). G . KO product was digested with XhoI and AgeI resulting in the expected digestion products: XhoI (196 and 291bp), AgeI (239 and 246bp). undig = undigested

    Article Snippet: Digestion reactions consisted of 500ng of PCR product, 20 units of either XhoI (R1046S, NEB) or AgeI (R3552S, NEB) in their specified buffers.

    Techniques: Binding Assay, Infection, Polymerase Chain Reaction, Knock-Out, Amplification, Staining, Flow Cytometry

    Generation and confirmation of Fzd2 tm1Vari allele. A. Map detailing the location of guide RNAs (indicated by the green and blue text on map and sequence) and regions of homology (ROH). B . Map of B6;C3H- Fzd2 tm1Vari intended modifications. LoxP sites were placed around the entire single exon Fzd2 gene. Xho1 and Age1 restriction sites (purple) were incorporated to aid in validation of the allele. LoxP sites are indicated by red triangles, and 5’ and 3’ untranslated regions (UTR) are shown. Primer binding sites for genotyping are shown in grey. C . PCR-based genotyping products for 5’ loxP site (top) and digestion with XhoI of flox product (bottom) results in expected products (193bp and 204 bp). D . PCR-based genotyping products for 3’ loxP site and digestion with AgeI of flox product results in the expected products (86bp and 238 bp). undig = undigested

    Journal: bioRxiv

    Article Title: Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development

    doi: 10.1101/2021.01.27.428341

    Figure Lengend Snippet: Generation and confirmation of Fzd2 tm1Vari allele. A. Map detailing the location of guide RNAs (indicated by the green and blue text on map and sequence) and regions of homology (ROH). B . Map of B6;C3H- Fzd2 tm1Vari intended modifications. LoxP sites were placed around the entire single exon Fzd2 gene. Xho1 and Age1 restriction sites (purple) were incorporated to aid in validation of the allele. LoxP sites are indicated by red triangles, and 5’ and 3’ untranslated regions (UTR) are shown. Primer binding sites for genotyping are shown in grey. C . PCR-based genotyping products for 5’ loxP site (top) and digestion with XhoI of flox product (bottom) results in expected products (193bp and 204 bp). D . PCR-based genotyping products for 3’ loxP site and digestion with AgeI of flox product results in the expected products (86bp and 238 bp). undig = undigested

    Article Snippet: Digestion reactions consisted of 500ng of PCR product, 20 units of either XhoI (R1046S, NEB) or AgeI (R3552S, NEB) in their specified buffers.

    Techniques: Sequencing, Binding Assay, Polymerase Chain Reaction

    RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Journal: Nucleic Acids Research

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli

    doi: 10.1093/nar/gkw1018

    Figure Lengend Snippet: RtcB re-ligates RNA43 to purified 70S Δ43 ribosomes in vitro . ( A ) Schematic showing the in vitro ribosome repair assay (see text for details). ( B ) RNA43 AgeI used in the assay. Nucleotides changed in helix 45 and the binding site for primers Y12 (gray) and H17 (green) are indicated. ( C ) RT-PCR performed on RNA purified from 70S Δ43 ribosomes incubated in the absence of RtcB and RNA43 AgeI (lane 1), in the presence of either RNA43 AgeI (lane 2) or RtcB (lane 3), or both (lane 4) employing primer pairs specific for the central region of 16S rRNA (S7/X15; panel a), the 3΄end of 16S rRNA (S7/Y12; panel b), and specific for the AU nucleotide change present in 16S AgeI rRNA upon successful ligation (S7/H17; panel c). ( D ) AgeI restriction analysis and ( E ), sequencing of the RT-PCR product obtained with primer pair S7/H17 (shown in C, panel c, lane 4). Sequencing analyses from position 1509–1517 of the 16S rRNA of (a) rrsB , and (b) the RT-PCR product are shown. Nucleotides changed in RNA43 AgeI are underlined.

    Article Snippet: For AgeI restriction analysis the isolated RT-PCR product was incubated with 4 units of AgeI-HF (New England Biolabs) for 30 min at 37°C.

    Techniques: Purification, In Vitro, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Ligation, Sequencing