dsdna donor cassettes  (New England Biolabs)


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    New England Biolabs dsdna donor cassettes
    Modification of 5’ ends of long <t>dsDNA</t> fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo <t>PCR</t> genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.
    Dsdna Donor Cassettes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dsdna donor cassettes/product/New England Biolabs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    dsdna donor cassettes - by Bioz Stars, 2020-04
    95/100 stars

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    Images

    1) Product Images from "Efficient single-copy HDR by 5’ modified long dsDNA donors"

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    Journal: eLife

    doi: 10.7554/eLife.39468

    Modification of 5’ ends of long dsDNA fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo PCR genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.
    Figure Legend Snippet: Modification of 5’ ends of long dsDNA fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo PCR genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.

    Techniques Used: Modification, Expressing, Injection, Polymerase Chain Reaction, Amplification

    Single-copy integration of long dsDNA donor establishes stably transmitted gfp-rx2 fusion gene. ( A ) F2 homozygous embryos exhibit GFP-Rx2 fusion protein expression in the pattern of the endogenous gene in the retina. ( B ) Southern Blot analysis of F2 gfp-rx2 embryos reveals a single band for a digestion scheme cutting outside the donor cassette (BglII/HindIII) or within the 5’ donor cassette and in intron 2 (ScaI/HindIII) indicating precise single-copy donor integration. ( B’ ) Schematic representation of the modified locus indicating the restriction sites and the domain complementary to the probe used in ( B ). ( C ) RT-PCR analysis on mRNA isolated from individual homozygous F3 embryos indicates the transcription of a single gfp-rx2 fusion mRNA in comparison to the shorter wild-type rx2 mRNA as schematically represented in ( C’ ).
    Figure Legend Snippet: Single-copy integration of long dsDNA donor establishes stably transmitted gfp-rx2 fusion gene. ( A ) F2 homozygous embryos exhibit GFP-Rx2 fusion protein expression in the pattern of the endogenous gene in the retina. ( B ) Southern Blot analysis of F2 gfp-rx2 embryos reveals a single band for a digestion scheme cutting outside the donor cassette (BglII/HindIII) or within the 5’ donor cassette and in intron 2 (ScaI/HindIII) indicating precise single-copy donor integration. ( B’ ) Schematic representation of the modified locus indicating the restriction sites and the domain complementary to the probe used in ( B ). ( C ) RT-PCR analysis on mRNA isolated from individual homozygous F3 embryos indicates the transcription of a single gfp-rx2 fusion mRNA in comparison to the shorter wild-type rx2 mRNA as schematically represented in ( C’ ).

    Techniques Used: Stable Transfection, Expressing, Southern Blot, Modification, Reverse Transcription Polymerase Chain Reaction, Isolation

    Modification of 5’ ends of long dsDNA fragments prevents in vivo multimerization. ( A ) Schematic representation of long dsDNA donor cassette PCR amplification with universal primers (black arrows) complementary to the cloning vector backbone outside of the assembled donor cassette (e. g. gfp with homology flanks). Bulky moieties like Biotin at the 5’ ends of both modified primers (red octagon) prevent multimerization/NHEJ of dsDNA, providing optimal conditions for HDR-mediated single-copy integration following CRISPR/Cas9-introduced DSB at the target locus (grey scissors). Representation of locus (Lf/Lr) and internal gfp (Gf/Gr) primers for PCR genotyping of putative HDR-mediated gfp integration events. ( B ) Southern blot analysis reveals the monomeric state of injected dsDNA fragments in vivo for 5’ modification with Biotin or Spacer C3. Long dsDNAs generated with control unmodified primers or Amino-dT attached primers multimerize as indicated by a high molecular weight ladder apparent already within two hours post-injection (hpi). Note: 5’ moieties did not enhance the stability of injected DNA.
    Figure Legend Snippet: Modification of 5’ ends of long dsDNA fragments prevents in vivo multimerization. ( A ) Schematic representation of long dsDNA donor cassette PCR amplification with universal primers (black arrows) complementary to the cloning vector backbone outside of the assembled donor cassette (e. g. gfp with homology flanks). Bulky moieties like Biotin at the 5’ ends of both modified primers (red octagon) prevent multimerization/NHEJ of dsDNA, providing optimal conditions for HDR-mediated single-copy integration following CRISPR/Cas9-introduced DSB at the target locus (grey scissors). Representation of locus (Lf/Lr) and internal gfp (Gf/Gr) primers for PCR genotyping of putative HDR-mediated gfp integration events. ( B ) Southern blot analysis reveals the monomeric state of injected dsDNA fragments in vivo for 5’ modification with Biotin or Spacer C3. Long dsDNAs generated with control unmodified primers or Amino-dT attached primers multimerize as indicated by a high molecular weight ladder apparent already within two hours post-injection (hpi). Note: 5’ moieties did not enhance the stability of injected DNA.

    Techniques Used: Modification, In Vivo, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Non-Homologous End Joining, CRISPR, Southern Blot, Injection, Generated, Molecular Weight

    2) Product Images from "Efficient single-copy HDR by 5’ modified long dsDNA donors"

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    Journal: eLife

    doi: 10.7554/eLife.39468

    Schematic representation of the donor plasmids. ( A–D ) Schematic to-scale representation of respective target locus (A, rx2 ; B, rx1 ; C, actb ; D, dnmt1 ) with UTR (white boxes with red outlines) and exons (red boxes) highlighted. Homology flanks (HF, grey), sgRNA target sites (white scissors) and respective locus primers (black arrows, Lf, Lr, see Supplementary file 2 ) are indicated. Respective assembled donor plasmid (A, B, C Golden GATEway ( Kirchmaier et al., 2013 ) or D, conventional cloning) that served as a template for PCR amplification of the unmodified/modified long dsDNA gfp donor cassette is depicted below (FL, flexible linker; backbone sequence in blue). Entry vectors (EV) or restriction enzyme sites for cloning are indicated. The position of primers flanking the donor cassette is indicated, modification highlighted by a red octagon.
    Figure Legend Snippet: Schematic representation of the donor plasmids. ( A–D ) Schematic to-scale representation of respective target locus (A, rx2 ; B, rx1 ; C, actb ; D, dnmt1 ) with UTR (white boxes with red outlines) and exons (red boxes) highlighted. Homology flanks (HF, grey), sgRNA target sites (white scissors) and respective locus primers (black arrows, Lf, Lr, see Supplementary file 2 ) are indicated. Respective assembled donor plasmid (A, B, C Golden GATEway ( Kirchmaier et al., 2013 ) or D, conventional cloning) that served as a template for PCR amplification of the unmodified/modified long dsDNA gfp donor cassette is depicted below (FL, flexible linker; backbone sequence in blue). Entry vectors (EV) or restriction enzyme sites for cloning are indicated. The position of primers flanking the donor cassette is indicated, modification highlighted by a red octagon.

    Techniques Used: Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Amplification, Modification, Sequencing

    5’Biotin modification of long dsDNA donors strongly enhances HDR-mediated integration. ( A ) rx2 locus PCR genotyping of individual GFP-Rx2 positive embryos injected with unmodified or 5’Biotin or Spacer C3 modified long dsDNA gfp donor cassettes (green asterisk, single-copy HDR-mediated integration of gfp , 2547 bp, open arrowhead, rx2 allele without gfp integration, 1719 bp;). Horizontal bar, individual embryo; L, rx2 locus PCR with rx2 Lf/rx2 Lr; G, gfp internal PCR for control with Gf/Gr. ( B ) Qualitative summary of band spectrum (single-copy HDR-mediated gfp integration, rx2 allele without gfp integration, other) resulting from PCR genotyping in ( A ). n, number of genotyped GFP-Rx2 expressing embryos.
    Figure Legend Snippet: 5’Biotin modification of long dsDNA donors strongly enhances HDR-mediated integration. ( A ) rx2 locus PCR genotyping of individual GFP-Rx2 positive embryos injected with unmodified or 5’Biotin or Spacer C3 modified long dsDNA gfp donor cassettes (green asterisk, single-copy HDR-mediated integration of gfp , 2547 bp, open arrowhead, rx2 allele without gfp integration, 1719 bp;). Horizontal bar, individual embryo; L, rx2 locus PCR with rx2 Lf/rx2 Lr; G, gfp internal PCR for control with Gf/Gr. ( B ) Qualitative summary of band spectrum (single-copy HDR-mediated gfp integration, rx2 allele without gfp integration, other) resulting from PCR genotyping in ( A ). n, number of genotyped GFP-Rx2 expressing embryos.

    Techniques Used: Modification, Polymerase Chain Reaction, Injection, Expressing

    Related Articles

    In Vivo:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs). .. The LacZ cassette of the pGGDestSC-ATG ( ) (addgene #49322) which served as DIG labelled dsDNA fragment to test in vivo multimerization was amplified via Q5-PCR as above using a mixture of 200 µM dATP, dCTP, dGTP, 170 µM dTTP and 30 µM DIG-dUTP and purified as detailed.

    Amplification:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: .. The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs). ..

    Purification:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs). .. The PCR reaction was treated with 20 units of DpnI (New England Biolabs) to remove any plasmid template following gel purified using the QIAquick Gel Extraction Kit (Qiagen, 28706) and elution with 20 µl nuclease-free water.

    Polymerase Chain Reaction:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: .. The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs). ..

    Modification:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: Modified primers obtained from Sigma-Aldrich (pDest f mod, pDest r mod, pCS2 f mod, pCS2 r mod) consist of the same sequences with phosphorothioate bonds in the first five nucleotides and 5’moiety extension: 5’Biotin, 5’Amino-dT or 5’Spacer C3. .. The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Gel Extraction:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs). .. The PCR reaction was treated with 20 units of DpnI (New England Biolabs) to remove any plasmid template following gel purified using the QIAquick Gel Extraction Kit (Qiagen, 28706) and elution with 20 µl nuclease-free water.

    Plasmid Preparation:

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors
    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs). .. The PCR reaction was treated with 20 units of DpnI (New England Biolabs) to remove any plasmid template following gel purified using the QIAquick Gel Extraction Kit (Qiagen, 28706) and elution with 20 µl nuclease-free water.

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  • Press Release
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  • Contact
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  • 95
    New England Biolabs dsdna donor cassettes
    Modification of 5’ ends of long <t>dsDNA</t> fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo <t>PCR</t> genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.
    Dsdna Donor Cassettes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dsdna donor cassettes/product/New England Biolabs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    dsdna donor cassettes - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

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    Modification of 5’ ends of long dsDNA fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo PCR genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.

    Journal: eLife

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    doi: 10.7554/eLife.39468

    Figure Lengend Snippet: Modification of 5’ ends of long dsDNA fragments promotes HDR-mediated single-copy integration. ( A ) GFP expression in the respective expression domain after HDR-mediated integration of modified dsDNA gfp donor cassettes into rx2 , rx1 , actb and dnmt1 ORFs in the injected generation. ( B ) Individual embryo PCR genotyping highlights efficient HDR-mediated single-copy integration of 5’Biotin modified long dsDNA donors, but not unmodified donor cassettes. Locus PCR reveals band size indicative of single-copy gfp integration (asterisk) besides alleles without gfp integration (open arrowhead). Amplification of gfp donor (white arrow) for control.

    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Techniques: Modification, Expressing, Injection, Polymerase Chain Reaction, Amplification

    Single-copy integration of long dsDNA donor establishes stably transmitted gfp-rx2 fusion gene. ( A ) F2 homozygous embryos exhibit GFP-Rx2 fusion protein expression in the pattern of the endogenous gene in the retina. ( B ) Southern Blot analysis of F2 gfp-rx2 embryos reveals a single band for a digestion scheme cutting outside the donor cassette (BglII/HindIII) or within the 5’ donor cassette and in intron 2 (ScaI/HindIII) indicating precise single-copy donor integration. ( B’ ) Schematic representation of the modified locus indicating the restriction sites and the domain complementary to the probe used in ( B ). ( C ) RT-PCR analysis on mRNA isolated from individual homozygous F3 embryos indicates the transcription of a single gfp-rx2 fusion mRNA in comparison to the shorter wild-type rx2 mRNA as schematically represented in ( C’ ).

    Journal: eLife

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    doi: 10.7554/eLife.39468

    Figure Lengend Snippet: Single-copy integration of long dsDNA donor establishes stably transmitted gfp-rx2 fusion gene. ( A ) F2 homozygous embryos exhibit GFP-Rx2 fusion protein expression in the pattern of the endogenous gene in the retina. ( B ) Southern Blot analysis of F2 gfp-rx2 embryos reveals a single band for a digestion scheme cutting outside the donor cassette (BglII/HindIII) or within the 5’ donor cassette and in intron 2 (ScaI/HindIII) indicating precise single-copy donor integration. ( B’ ) Schematic representation of the modified locus indicating the restriction sites and the domain complementary to the probe used in ( B ). ( C ) RT-PCR analysis on mRNA isolated from individual homozygous F3 embryos indicates the transcription of a single gfp-rx2 fusion mRNA in comparison to the shorter wild-type rx2 mRNA as schematically represented in ( C’ ).

    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Techniques: Stable Transfection, Expressing, Southern Blot, Modification, Reverse Transcription Polymerase Chain Reaction, Isolation

    Modification of 5’ ends of long dsDNA fragments prevents in vivo multimerization. ( A ) Schematic representation of long dsDNA donor cassette PCR amplification with universal primers (black arrows) complementary to the cloning vector backbone outside of the assembled donor cassette (e. g. gfp with homology flanks). Bulky moieties like Biotin at the 5’ ends of both modified primers (red octagon) prevent multimerization/NHEJ of dsDNA, providing optimal conditions for HDR-mediated single-copy integration following CRISPR/Cas9-introduced DSB at the target locus (grey scissors). Representation of locus (Lf/Lr) and internal gfp (Gf/Gr) primers for PCR genotyping of putative HDR-mediated gfp integration events. ( B ) Southern blot analysis reveals the monomeric state of injected dsDNA fragments in vivo for 5’ modification with Biotin or Spacer C3. Long dsDNAs generated with control unmodified primers or Amino-dT attached primers multimerize as indicated by a high molecular weight ladder apparent already within two hours post-injection (hpi). Note: 5’ moieties did not enhance the stability of injected DNA.

    Journal: eLife

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    doi: 10.7554/eLife.39468

    Figure Lengend Snippet: Modification of 5’ ends of long dsDNA fragments prevents in vivo multimerization. ( A ) Schematic representation of long dsDNA donor cassette PCR amplification with universal primers (black arrows) complementary to the cloning vector backbone outside of the assembled donor cassette (e. g. gfp with homology flanks). Bulky moieties like Biotin at the 5’ ends of both modified primers (red octagon) prevent multimerization/NHEJ of dsDNA, providing optimal conditions for HDR-mediated single-copy integration following CRISPR/Cas9-introduced DSB at the target locus (grey scissors). Representation of locus (Lf/Lr) and internal gfp (Gf/Gr) primers for PCR genotyping of putative HDR-mediated gfp integration events. ( B ) Southern blot analysis reveals the monomeric state of injected dsDNA fragments in vivo for 5’ modification with Biotin or Spacer C3. Long dsDNAs generated with control unmodified primers or Amino-dT attached primers multimerize as indicated by a high molecular weight ladder apparent already within two hours post-injection (hpi). Note: 5’ moieties did not enhance the stability of injected DNA.

    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Techniques: Modification, In Vivo, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Non-Homologous End Joining, CRISPR, Southern Blot, Injection, Generated, Molecular Weight

    Schematic representation of the donor plasmids. ( A–D ) Schematic to-scale representation of respective target locus (A, rx2 ; B, rx1 ; C, actb ; D, dnmt1 ) with UTR (white boxes with red outlines) and exons (red boxes) highlighted. Homology flanks (HF, grey), sgRNA target sites (white scissors) and respective locus primers (black arrows, Lf, Lr, see Supplementary file 2 ) are indicated. Respective assembled donor plasmid (A, B, C Golden GATEway ( Kirchmaier et al., 2013 ) or D, conventional cloning) that served as a template for PCR amplification of the unmodified/modified long dsDNA gfp donor cassette is depicted below (FL, flexible linker; backbone sequence in blue). Entry vectors (EV) or restriction enzyme sites for cloning are indicated. The position of primers flanking the donor cassette is indicated, modification highlighted by a red octagon.

    Journal: eLife

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    doi: 10.7554/eLife.39468

    Figure Lengend Snippet: Schematic representation of the donor plasmids. ( A–D ) Schematic to-scale representation of respective target locus (A, rx2 ; B, rx1 ; C, actb ; D, dnmt1 ) with UTR (white boxes with red outlines) and exons (red boxes) highlighted. Homology flanks (HF, grey), sgRNA target sites (white scissors) and respective locus primers (black arrows, Lf, Lr, see Supplementary file 2 ) are indicated. Respective assembled donor plasmid (A, B, C Golden GATEway ( Kirchmaier et al., 2013 ) or D, conventional cloning) that served as a template for PCR amplification of the unmodified/modified long dsDNA gfp donor cassette is depicted below (FL, flexible linker; backbone sequence in blue). Entry vectors (EV) or restriction enzyme sites for cloning are indicated. The position of primers flanking the donor cassette is indicated, modification highlighted by a red octagon.

    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Techniques: Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Amplification, Modification, Sequencing

    5’Biotin modification of long dsDNA donors strongly enhances HDR-mediated integration. ( A ) rx2 locus PCR genotyping of individual GFP-Rx2 positive embryos injected with unmodified or 5’Biotin or Spacer C3 modified long dsDNA gfp donor cassettes (green asterisk, single-copy HDR-mediated integration of gfp , 2547 bp, open arrowhead, rx2 allele without gfp integration, 1719 bp;). Horizontal bar, individual embryo; L, rx2 locus PCR with rx2 Lf/rx2 Lr; G, gfp internal PCR for control with Gf/Gr. ( B ) Qualitative summary of band spectrum (single-copy HDR-mediated gfp integration, rx2 allele without gfp integration, other) resulting from PCR genotyping in ( A ). n, number of genotyped GFP-Rx2 expressing embryos.

    Journal: eLife

    Article Title: Efficient single-copy HDR by 5’ modified long dsDNA donors

    doi: 10.7554/eLife.39468

    Figure Lengend Snippet: 5’Biotin modification of long dsDNA donors strongly enhances HDR-mediated integration. ( A ) rx2 locus PCR genotyping of individual GFP-Rx2 positive embryos injected with unmodified or 5’Biotin or Spacer C3 modified long dsDNA gfp donor cassettes (green asterisk, single-copy HDR-mediated integration of gfp , 2547 bp, open arrowhead, rx2 allele without gfp integration, 1719 bp;). Horizontal bar, individual embryo; L, rx2 locus PCR with rx2 Lf/rx2 Lr; G, gfp internal PCR for control with Gf/Gr. ( B ) Qualitative summary of band spectrum (single-copy HDR-mediated gfp integration, rx2 allele without gfp integration, other) resulting from PCR genotyping in ( A ). n, number of genotyped GFP-Rx2 expressing embryos.

    Article Snippet: The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer, 200 µM dNTPs, 200 µM primer forward and reverse and 0.6 U/µl Q5 polymerase (New England Biolabs).

    Techniques: Modification, Polymerase Chain Reaction, Injection, Expressing