xbai  (New England Biolabs)


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    New England Biolabs xbai
    Generation of a series of amx genomic rescue constructs to test a disease-associated variant in TM2D3 . (A) Schematic diagram of the amx locus on the Drosophila X -chromosome. The coding region of genes are shown as orange boxes and the untranslated regions (UTR) are shown in grey. Two primers (Oligo 1 and 2) were designed to amplify a ~3.3kb region that contains the amx gene and neighboring sequences. (B) Construction of the amx[+]-attB genomic rescue construct by subcloning the PCR amplicon from (A) into pattB using XhoI and <t>XbaI</t> digestion and ligation. pattB contains a mini-white + cassette (red box) as a dominant visual marker that can be used to track the transgenesis event in a white − mutant background. It also carries an Ampicillin resistance (Amp R ) cassette (green box) for selection in bacteria. (C) Construction of a humanized amx genomic rescue construct (TM2D3[+]-attB) based on amx[+]-attB and two intermediate vectors. Human reference TM2D3 coding sequence was PCR amplified from a cDNA plasmid and integrated into a plasmid in which the amx coding sequence was replaced by a spacer sequence flanking two BsaI recognition sites (Intermediate Vector 2). A construct in which the upstream region of the amx genomic rescue amplicon with a BsaI site was cloned into pattB (Intermediate Vector 1) as a precursor of Intermediate Vector 2. Golden Gate cloning with BsaI and overhangs ①/② allows swapping of the spacer sequence in Intermediate Vector 2 with human TM2D3 coding sequence to generate the humanized genomic rescue construct. (D) Generation of the humanized amx genomic rescue construct carrying the variant of interest (TM2D3[P155L]-attB) through site-directed mutagenesis. The variant of interest is shown using an asterisk.
    Xbai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xbai/product/New England Biolabs
    Average 98 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    xbai - by Bioz Stars, 2022-12
    98/100 stars

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    1) Product Images from "Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila"

    Article Title: Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-0716-2201-8_19

    Generation of a series of amx genomic rescue constructs to test a disease-associated variant in TM2D3 . (A) Schematic diagram of the amx locus on the Drosophila X -chromosome. The coding region of genes are shown as orange boxes and the untranslated regions (UTR) are shown in grey. Two primers (Oligo 1 and 2) were designed to amplify a ~3.3kb region that contains the amx gene and neighboring sequences. (B) Construction of the amx[+]-attB genomic rescue construct by subcloning the PCR amplicon from (A) into pattB using XhoI and XbaI digestion and ligation. pattB contains a mini-white + cassette (red box) as a dominant visual marker that can be used to track the transgenesis event in a white − mutant background. It also carries an Ampicillin resistance (Amp R ) cassette (green box) for selection in bacteria. (C) Construction of a humanized amx genomic rescue construct (TM2D3[+]-attB) based on amx[+]-attB and two intermediate vectors. Human reference TM2D3 coding sequence was PCR amplified from a cDNA plasmid and integrated into a plasmid in which the amx coding sequence was replaced by a spacer sequence flanking two BsaI recognition sites (Intermediate Vector 2). A construct in which the upstream region of the amx genomic rescue amplicon with a BsaI site was cloned into pattB (Intermediate Vector 1) as a precursor of Intermediate Vector 2. Golden Gate cloning with BsaI and overhangs ①/② allows swapping of the spacer sequence in Intermediate Vector 2 with human TM2D3 coding sequence to generate the humanized genomic rescue construct. (D) Generation of the humanized amx genomic rescue construct carrying the variant of interest (TM2D3[P155L]-attB) through site-directed mutagenesis. The variant of interest is shown using an asterisk.
    Figure Legend Snippet: Generation of a series of amx genomic rescue constructs to test a disease-associated variant in TM2D3 . (A) Schematic diagram of the amx locus on the Drosophila X -chromosome. The coding region of genes are shown as orange boxes and the untranslated regions (UTR) are shown in grey. Two primers (Oligo 1 and 2) were designed to amplify a ~3.3kb region that contains the amx gene and neighboring sequences. (B) Construction of the amx[+]-attB genomic rescue construct by subcloning the PCR amplicon from (A) into pattB using XhoI and XbaI digestion and ligation. pattB contains a mini-white + cassette (red box) as a dominant visual marker that can be used to track the transgenesis event in a white − mutant background. It also carries an Ampicillin resistance (Amp R ) cassette (green box) for selection in bacteria. (C) Construction of a humanized amx genomic rescue construct (TM2D3[+]-attB) based on amx[+]-attB and two intermediate vectors. Human reference TM2D3 coding sequence was PCR amplified from a cDNA plasmid and integrated into a plasmid in which the amx coding sequence was replaced by a spacer sequence flanking two BsaI recognition sites (Intermediate Vector 2). A construct in which the upstream region of the amx genomic rescue amplicon with a BsaI site was cloned into pattB (Intermediate Vector 1) as a precursor of Intermediate Vector 2. Golden Gate cloning with BsaI and overhangs ①/② allows swapping of the spacer sequence in Intermediate Vector 2 with human TM2D3 coding sequence to generate the humanized genomic rescue construct. (D) Generation of the humanized amx genomic rescue construct carrying the variant of interest (TM2D3[P155L]-attB) through site-directed mutagenesis. The variant of interest is shown using an asterisk.

    Techniques Used: Construct, Variant Assay, Subcloning, Polymerase Chain Reaction, Amplification, Ligation, Marker, Mutagenesis, Selection, Sequencing, Plasmid Preparation, Clone Assay

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    New England Biolabs xbai
    Xbai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xbai/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xbai - by Bioz Stars, 2022-12
    98/100 stars
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