lentiviral constructs lentiviral constructs  (New England Biolabs)


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    Name:
    AgeI HF
    Description:
    AgeI HF 1 500 units
    Catalog Number:
    r3552l
    Price:
    298
    Size:
    1 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs lentiviral constructs lentiviral constructs
    AgeI HF
    AgeI HF 1 500 units
    https://www.bioz.com/result/lentiviral constructs lentiviral constructs/product/New England Biolabs
    Average 91 stars, based on 3770 article reviews
    Price from $9.99 to $1999.99
    lentiviral constructs lentiviral constructs - by Bioz Stars, 2021-02
    91/100 stars

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    Images

    1) Product Images from "Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface"

    Article Title: Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface

    Journal: The Journal of Allergy and Clinical Immunology

    doi: 10.1016/j.jaci.2018.04.033

    Assessment of p.W655 NLRC4 in oligomerized form. A, Ribbon representation of NLRC4 in the oligomerized state (PDB Code 6B5B 24 ) with magnified inset areas highlighting 2 potential regions of interaction with p.W655 (LRR1 and LRR2). NLRC4 KO THP-1 cells transduced with lentiviral constructs expressing WT or mutant NLRC4 combined with LRR1 or LRR2 mutations. B-D, Twenty-four hours after transduction, cells were primed with Pam3CSK4 for 24 hours, and then propidium iodine (PI) staining was assessed by using flow cytometry to quantify cell death (Fig 7, B ), and IL-1β (Fig 7, C ) and IL-18 (Fig 7, D ) secretion were measured by ELISA. NLRC4 KO THP-1 cells were transduced with 1 mL of WT NLRC4 , LRR1 NLRC4 , or LRR2 NLRC4 virus or 2 mL of LRR1 NLRC4 virus (LRR1 [×2]). E-G, After 24 hours, cells were infected with a retroviral PrgI construct and cell death (Fig 7, E ), and IL-1β (Fig 7, F ) and IL-18 (Fig 7, G ) secretion assessed the following day. H and I , NLRC4 expression was assessed by using Western blot analysis. Data were pooled from at least 3 independent experiments. * P
    Figure Legend Snippet: Assessment of p.W655 NLRC4 in oligomerized form. A, Ribbon representation of NLRC4 in the oligomerized state (PDB Code 6B5B 24 ) with magnified inset areas highlighting 2 potential regions of interaction with p.W655 (LRR1 and LRR2). NLRC4 KO THP-1 cells transduced with lentiviral constructs expressing WT or mutant NLRC4 combined with LRR1 or LRR2 mutations. B-D, Twenty-four hours after transduction, cells were primed with Pam3CSK4 for 24 hours, and then propidium iodine (PI) staining was assessed by using flow cytometry to quantify cell death (Fig 7, B ), and IL-1β (Fig 7, C ) and IL-18 (Fig 7, D ) secretion were measured by ELISA. NLRC4 KO THP-1 cells were transduced with 1 mL of WT NLRC4 , LRR1 NLRC4 , or LRR2 NLRC4 virus or 2 mL of LRR1 NLRC4 virus (LRR1 [×2]). E-G, After 24 hours, cells were infected with a retroviral PrgI construct and cell death (Fig 7, E ), and IL-1β (Fig 7, F ) and IL-18 (Fig 7, G ) secretion assessed the following day. H and I , NLRC4 expression was assessed by using Western blot analysis. Data were pooled from at least 3 independent experiments. * P

    Techniques Used: Transduction, Construct, Expressing, Mutagenesis, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Infection, Western Blot

    2) Product Images from "Generation of a New Frizzled 2 Flox Mouse Model to Clarify Its Role in Development"

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

    Journal: bioRxiv

    doi: 10.1101/2021.01.27.428341

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction

    3) Product Images from "The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1018

    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.
    Figure Legend 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.

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

    Related Articles

    In Vitro:

    Article Title: SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin
    Article Snippet: .. For mRNA generation, Daxx plasmids were first linearized with AgeI‐HF (New England Biolabs), and plasmids for H3.2 , H3.3, and H3.3 mutations with XhoI (New England Biolabs), and subsequently subjected to in vitro transcription using the mMessage Machine T7 Kit (Thermo Fisher Scientific, Cat# AM1344). .. siRNA microinjection and intracytoplasmic sperm injection Fully grown GV oocytes were collected from ovaries of 8‐week‐old C57BL/6J female mice by puncturing ovarian follicles with needles (22G × 1 ¼”).

    Ligation:

    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.

    Isolation:

    Article Title: The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli
    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. .. The restriction fragments were determined using PAA gel-electrophoresis and visualized by EtBr-staining.

    Construct:

    Article Title: An opaque cell-specific expression program of secreted proteases and transporters allows cell-type cooperation in Candida albicans
    Article Snippet: .. The mCherry reporter strains were constructed using AgeI-HF (NEB R3552L) linearized reporter plasmids transformed into the SNY250-derived a /Δ wild type strain. .. Colonies were selected for growth on Yeast Extract Peptone Dextrose (YEPD) plates supplemented with 400 μg/mL nourseothricin (clonNAT, WERNER BioAgents, Jena, Germany).

    Reverse Transcription Polymerase Chain Reaction:

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

    other:

    Article Title: Targeted removal of epigenetic barriers during transcriptional reprogramming
    Article Snippet: The backbone was digested with AgeI-HF.

    Polymerase Chain Reaction:

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

    Transformation Assay:

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research). .. Transformed cells were spread on several SCD-ura plates in a way that assures a moderate colony density which simplifies picking clones.

    Article Title: An opaque cell-specific expression program of secreted proteases and transporters allows cell-type cooperation in Candida albicans
    Article Snippet: .. The mCherry reporter strains were constructed using AgeI-HF (NEB R3552L) linearized reporter plasmids transformed into the SNY250-derived a /Δ wild type strain. .. Colonies were selected for growth on Yeast Extract Peptone Dextrose (YEPD) plates supplemented with 400 μg/mL nourseothricin (clonNAT, WERNER BioAgents, Jena, Germany).

    Plasmid Preparation:

    Article Title: Expression and clinical significance of basic transcription factor 3 in nasopharyngeal carcinoma
    Article Snippet: .. siRNA viral vector construction The BTF3 siRNA transcript template (sense, 5′-GCCGAAGAAGCCTGGGAATCA-3′) was used to generate the viral vector, digested with Age I/Eco RI enzymes (cat. no. R3552L/R3101L; New England BioLabs, Inc., Ipswich, MA, USA), and then ligated with pGCSIL-GFP vector (New England Biolabs). ..

    Article Title: Riboswitching with ciprofloxacin—development and characterization of a novel RNA regulator
    Article Snippet: .. Target vector pWHE601* was digested using AgeI-HF and NheI-HF (both NEB) and transformed into RS463α with a 10-molar excess of insert using Frozen-EZ Yeast Transformation II Kit (Zymo Research). .. Transformed cells were spread on several SCD-ura plates in a way that assures a moderate colony density which simplifies picking clones.

    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.

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    • agei hf  (New England Biolabs)
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    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: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/agei hf/product/New England Biolabs
    Average 94 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    agei hf - by Bioz Stars, 2021-02
    94/100 stars
    		  Buy from Supplier

    Image Search Results


    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