cpd containing plasmid dna  (New England Biolabs)


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    Name:
    SapI
    Description:
    SapI 1 250 units
    Catalog Number:
    r0569l
    Price:
    282
    Size:
    1 250 units
    Category:
    Restriction Enzymes
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    New England Biolabs cpd containing plasmid dna
    SapI
    SapI 1 250 units
    https://www.bioz.com/result/cpd containing plasmid dna/product/New England Biolabs
    Average 91 stars, based on 151 article reviews
    Price from $9.99 to $1999.99
    cpd containing plasmid dna - by Bioz Stars, 2021-02
    91/100 stars

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    Images

    1) Product Images from "The Initial Response of a Eukaryotic Replisome to DNA Damage"

    Article Title: The Initial Response of a Eukaryotic Replisome to DNA Damage

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.04.022

    Evidence of Leading-Strand Re-priming by the Core Eukaryotic Replisome (A) Schematic of the 3 kb CPD LEAD template and the predicted replication products of leading-strand lesion bypass by re-priming followed by post-replicative SmaI digestion. (B) SmaI-digested replication products (60 min) were run in a native gel (analytical, 2%, one lane; preparative, 98%, two lanes). Full-length products were electroeluted from excised preparative lane gel slices. (C and D) Electroeluted DNA from (B) was digested with BamHI and DpnI and analyzed by native (C) and two-dimensional electrophoresis (BamHI + DpnI-digested sample) (D). (E and F) Electroeluted full-length products from CPD LEAD (E) and undamaged (F) templates in Figure S4 C were digested with BamHI and DpnI and separated through two-dimensional gels. In (D) and (E), products arising from leading-strand re-priming are shown in red.
    Figure Legend Snippet: Evidence of Leading-Strand Re-priming by the Core Eukaryotic Replisome (A) Schematic of the 3 kb CPD LEAD template and the predicted replication products of leading-strand lesion bypass by re-priming followed by post-replicative SmaI digestion. (B) SmaI-digested replication products (60 min) were run in a native gel (analytical, 2%, one lane; preparative, 98%, two lanes). Full-length products were electroeluted from excised preparative lane gel slices. (C and D) Electroeluted DNA from (B) was digested with BamHI and DpnI and analyzed by native (C) and two-dimensional electrophoresis (BamHI + DpnI-digested sample) (D). (E and F) Electroeluted full-length products from CPD LEAD (E) and undamaged (F) templates in Figure S4 C were digested with BamHI and DpnI and separated through two-dimensional gels. In (D) and (E), products arising from leading-strand re-priming are shown in red.

    Techniques Used: Electrophoresis

    The Replisome Rapidly and Efficiently Bypasses a Lagging-Strand CPD (A) Schematic of AhdI- and BamHI-linearized undamaged templates and the predicted replication products. In this and all subsequent figures; red: leading-strands; blue: lagging-strands; the position of the ARS306 origin of replication is marked, Ori. The location of the SmaI restriction site is indicated. (B) Standard replication reactions performed on the templates illustrated in (A). Unless stated otherwise, this and all subsequent standard replication assays contained 217 mM potassium glutamate. Templates were prepared by linearizing maxiprep DNA (Maxi) or undamaged plasmids prepared using the same method used to generate CPD containing plasmids (Ligated). (C) Schematic of the 6.7 kb CPD LAG template and the predicted replication products of lagging-strand lesion bypass. (D) Replication reaction comparing undamaged and 6.7 kb CPD LAG templates. (E) Schematic of the 5.1 kb CPD LAG template. (F) Pulse-chase experiment on undamaged and 5.1 kb CPD LAG templates. The chase was added at 2 min 50 s. (G) Quantitation of pulse-chase experiments performed as in (F). Error bars represent the SEM from four experiments. Data were fit to a linear regression. Dashed line indicates the distance from Ori to CPD LAG (5.1 kb).
    Figure Legend Snippet: The Replisome Rapidly and Efficiently Bypasses a Lagging-Strand CPD (A) Schematic of AhdI- and BamHI-linearized undamaged templates and the predicted replication products. In this and all subsequent figures; red: leading-strands; blue: lagging-strands; the position of the ARS306 origin of replication is marked, Ori. The location of the SmaI restriction site is indicated. (B) Standard replication reactions performed on the templates illustrated in (A). Unless stated otherwise, this and all subsequent standard replication assays contained 217 mM potassium glutamate. Templates were prepared by linearizing maxiprep DNA (Maxi) or undamaged plasmids prepared using the same method used to generate CPD containing plasmids (Ligated). (C) Schematic of the 6.7 kb CPD LAG template and the predicted replication products of lagging-strand lesion bypass. (D) Replication reaction comparing undamaged and 6.7 kb CPD LAG templates. (E) Schematic of the 5.1 kb CPD LAG template. (F) Pulse-chase experiment on undamaged and 5.1 kb CPD LAG templates. The chase was added at 2 min 50 s. (G) Quantitation of pulse-chase experiments performed as in (F). Error bars represent the SEM from four experiments. Data were fit to a linear regression. Dashed line indicates the distance from Ori to CPD LAG (5.1 kb).

    Techniques Used: Pulse Chase, Quantitation Assay

    2) Product Images from "SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors"

    Article Title: SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.119.400822

    SapTrap assembly of MosSCI targeting vectors using the four-cassette system. A. The MosSCI targeting vector pXF87 was derived from pCFJ350 by mutating two SapI restriction sites (indicated by arrowheads in the “Left” (L) and “Right” (R) homology arms) and introducing two SapI sites (blue text) between the XhoI and SpeI sites (green text). SapI cleavage sites are in red text. The SapI recognition sites are oriented such that upon digestion they are removed from the vector backbone. The cbr- unc-119 gene is used as a positive selection marker to facilitate the identification of transgenic animals. B. Design of the donor cassette vectors used for the 4-cassette cloning strategy. C. The curved dotted lines indicate the overhangs that anneal during the ligation reaction. D. Overview of the assembly protocol. For a detailed protocol, see the Materials and Methods section. E. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids.
    Figure Legend Snippet: SapTrap assembly of MosSCI targeting vectors using the four-cassette system. A. The MosSCI targeting vector pXF87 was derived from pCFJ350 by mutating two SapI restriction sites (indicated by arrowheads in the “Left” (L) and “Right” (R) homology arms) and introducing two SapI sites (blue text) between the XhoI and SpeI sites (green text). SapI cleavage sites are in red text. The SapI recognition sites are oriented such that upon digestion they are removed from the vector backbone. The cbr- unc-119 gene is used as a positive selection marker to facilitate the identification of transgenic animals. B. Design of the donor cassette vectors used for the 4-cassette cloning strategy. C. The curved dotted lines indicate the overhangs that anneal during the ligation reaction. D. Overview of the assembly protocol. For a detailed protocol, see the Materials and Methods section. E. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids.

    Techniques Used: Plasmid Preparation, Derivative Assay, Selection, Marker, Transgenic Assay, Clone Assay, Ligation

    SapTrap assembly of MosSCI targeting vectors using the five-cassette system. A. Schematic of pXF87 and the donor cassettes following SapI digestion. The dotted lines indicate the overhangs that anneal during ligation. B. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids for the five-cassette system.
    Figure Legend Snippet: SapTrap assembly of MosSCI targeting vectors using the five-cassette system. A. Schematic of pXF87 and the donor cassettes following SapI digestion. The dotted lines indicate the overhangs that anneal during ligation. B. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids for the five-cassette system.

    Techniques Used: Ligation

    3) Product Images from "CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo"

    Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo

    Journal: Current protocols in molecular biology

    doi: 10.1002/cpmb.46

    Insertion of gRNA sequences into AAV vector gRNAs are inserted into AAV plasmid containing U6 promoters and cardiac troponin T (cTNT) promoter-driven Cre recombinase. Annealed oligos corresponding to the targeting portions of gRNA1 and gRNA2 are cloned between AarI and SapI sites, respectively. The entire construct is contained within AAV inverted terminal repeat sequences, allowing for packaging into AAV capsids. Examples of gRNAs designed against Gata4 are shown.
    Figure Legend Snippet: Insertion of gRNA sequences into AAV vector gRNAs are inserted into AAV plasmid containing U6 promoters and cardiac troponin T (cTNT) promoter-driven Cre recombinase. Annealed oligos corresponding to the targeting portions of gRNA1 and gRNA2 are cloned between AarI and SapI sites, respectively. The entire construct is contained within AAV inverted terminal repeat sequences, allowing for packaging into AAV capsids. Examples of gRNAs designed against Gata4 are shown.

    Techniques Used: Plasmid Preparation, Clone Assay, Construct

    4) Product Images from "Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts"

    Article Title: Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts

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

    doi: 10.1007/978-1-61779-998-3_16

    Examination of dual incisions during ICL repair. HincII digested repair intermediates are separated on a denaturing gel and visualized using Southern blotting (land 5–14) (Subheading 3.4). Undigested and HincII and/or SapI digested pControl and
    Figure Legend Snippet: Examination of dual incisions during ICL repair. HincII digested repair intermediates are separated on a denaturing gel and visualized using Southern blotting (land 5–14) (Subheading 3.4). Undigested and HincII and/or SapI digested pControl and

    Techniques Used: Southern Blot

    Determination of ICL repair efficiency. (a) Analysis of HincII (lane 1–10) and HincII/SapI (lane 11–20) digested repair intermediates on a native agarose gel (Subheading 3.5). (b) Calculated repair efficiency plotted against time.
    Figure Legend Snippet: Determination of ICL repair efficiency. (a) Analysis of HincII (lane 1–10) and HincII/SapI (lane 11–20) digested repair intermediates on a native agarose gel (Subheading 3.5). (b) Calculated repair efficiency plotted against time.

    Techniques Used: Agarose Gel Electrophoresis

    5) Product Images from "CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo"

    Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo

    Journal: Current protocols in molecular biology

    doi: 10.1002/cpmb.46

    Insertion of gRNA sequences into AAV vector gRNAs are inserted into AAV plasmid containing U6 promoters and cardiac troponin T (cTNT) promoter-driven Cre recombinase. Annealed oligos corresponding to the targeting portions of gRNA1 and gRNA2 are cloned between AarI and SapI sites, respectively. The entire construct is contained within AAV inverted terminal repeat sequences, allowing for packaging into AAV capsids. Examples of gRNAs designed against Gata4 are shown.
    Figure Legend Snippet: Insertion of gRNA sequences into AAV vector gRNAs are inserted into AAV plasmid containing U6 promoters and cardiac troponin T (cTNT) promoter-driven Cre recombinase. Annealed oligos corresponding to the targeting portions of gRNA1 and gRNA2 are cloned between AarI and SapI sites, respectively. The entire construct is contained within AAV inverted terminal repeat sequences, allowing for packaging into AAV capsids. Examples of gRNAs designed against Gata4 are shown.

    Techniques Used: Plasmid Preparation, Clone Assay, Construct

    6) Product Images from "The Initial Response of a Eukaryotic Replisome to DNA Damage"

    Article Title: The Initial Response of a Eukaryotic Replisome to DNA Damage

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.04.022

    Evidence of Leading-Strand Re-priming by the Core Eukaryotic Replisome (A) Schematic of the 3 kb CPD LEAD template and the predicted replication products of leading-strand lesion bypass by re-priming followed by post-replicative SmaI digestion. (B) SmaI-digested replication products (60 min) were run in a native gel (analytical, 2%, one lane; preparative, 98%, two lanes). Full-length products were electroeluted from excised preparative lane gel slices. (C and D) Electroeluted DNA from (B) was digested with BamHI and DpnI and analyzed by native (C) and two-dimensional electrophoresis (BamHI + DpnI-digested sample) (D). (E and F) Electroeluted full-length products from CPD LEAD C were digested with BamHI and DpnI and separated through two-dimensional gels. In (D) and (E), products arising from leading-strand re-priming are shown in red.
    Figure Legend Snippet: Evidence of Leading-Strand Re-priming by the Core Eukaryotic Replisome (A) Schematic of the 3 kb CPD LEAD template and the predicted replication products of leading-strand lesion bypass by re-priming followed by post-replicative SmaI digestion. (B) SmaI-digested replication products (60 min) were run in a native gel (analytical, 2%, one lane; preparative, 98%, two lanes). Full-length products were electroeluted from excised preparative lane gel slices. (C and D) Electroeluted DNA from (B) was digested with BamHI and DpnI and analyzed by native (C) and two-dimensional electrophoresis (BamHI + DpnI-digested sample) (D). (E and F) Electroeluted full-length products from CPD LEAD C were digested with BamHI and DpnI and separated through two-dimensional gels. In (D) and (E), products arising from leading-strand re-priming are shown in red.

    Techniques Used: Electrophoresis

    The Replisome Rapidly and Efficiently Bypasses a Lagging-Strand CPD (A) Schematic of AhdI- and BamHI-linearized undamaged templates and the predicted replication products. In this and all subsequent figures; red: leading-strands; blue: lagging-strands; the position of the ARS306 origin of replication is marked, Ori. The location of the SmaI restriction site is indicated. (B) Standard replication reactions performed on the templates illustrated in (A). Unless stated otherwise, this and all subsequent standard replication assays contained 217 mM potassium glutamate. Templates were prepared by linearizing maxiprep DNA (Maxi) or undamaged plasmids prepared using the same method used to generate CPD containing plasmids (Ligated). (C) Schematic of the 6.7 kb CPD LAG template and the predicted replication products of lagging-strand lesion bypass. (D) Replication reaction comparing undamaged and 6.7 kb CPD LAG templates. (E) Schematic of the 5.1 kb CPD LAG template. (F) Pulse-chase experiment on undamaged and 5.1 kb CPD LAG templates. The chase was added at 2 min 50 s. (G) Quantitation of pulse-chase experiments performed as in (F). Error bars represent the SEM from four experiments. Data were fit to a linear regression. Dashed line indicates the distance from Ori to CPD LAG (5.1 kb).
    Figure Legend Snippet: The Replisome Rapidly and Efficiently Bypasses a Lagging-Strand CPD (A) Schematic of AhdI- and BamHI-linearized undamaged templates and the predicted replication products. In this and all subsequent figures; red: leading-strands; blue: lagging-strands; the position of the ARS306 origin of replication is marked, Ori. The location of the SmaI restriction site is indicated. (B) Standard replication reactions performed on the templates illustrated in (A). Unless stated otherwise, this and all subsequent standard replication assays contained 217 mM potassium glutamate. Templates were prepared by linearizing maxiprep DNA (Maxi) or undamaged plasmids prepared using the same method used to generate CPD containing plasmids (Ligated). (C) Schematic of the 6.7 kb CPD LAG template and the predicted replication products of lagging-strand lesion bypass. (D) Replication reaction comparing undamaged and 6.7 kb CPD LAG templates. (E) Schematic of the 5.1 kb CPD LAG template. (F) Pulse-chase experiment on undamaged and 5.1 kb CPD LAG templates. The chase was added at 2 min 50 s. (G) Quantitation of pulse-chase experiments performed as in (F). Error bars represent the SEM from four experiments. Data were fit to a linear regression. Dashed line indicates the distance from Ori to CPD LAG (5.1 kb).

    Techniques Used: Pulse Chase, Quantitation Assay

    7) Product Images from "SapTrap assembly of C. elegans MosSCI transgene vectors"

    Article Title: SapTrap assembly of C. elegans MosSCI transgene vectors

    Journal: bioRxiv

    doi: 10.1101/805507

    SapTrap assembly of MosSCI targeting vectors using the five-cassette system. A. Schematic of pXF87 and the donor cassettes following SapI digestion. The dotted lines indicate the overhangs that anneal during ligation. B. Summary of available promoter, gene tag and 3’UTR donor cassette plasmids for the five-cassette system.
    Figure Legend Snippet: SapTrap assembly of MosSCI targeting vectors using the five-cassette system. A. Schematic of pXF87 and the donor cassettes following SapI digestion. The dotted lines indicate the overhangs that anneal during ligation. B. Summary of available promoter, gene tag and 3’UTR donor cassette plasmids for the five-cassette system.

    Techniques Used: Ligation

    SapTrap assembly of MosSCI targeting vectors using the four-cassette system. A. The MosSCI targeting vector pXF87 was derived from pCFJ350 by mutating two SapI restriction sites (indicated by arrowheads in the “Left” (L) and “Right” (R) homology arms) and introducing two SapI sites (blue text) between the XhoI and SpeI sites (green text). SapI cleavage sites are in red text. The SapI recognition sites are oriented such that upon digestion they are removed from the vector backbone. The cbr- unc-119 gene is used as a positive selection marker to facilitate the identification of transgenic animals. B. Design of the donor cassette vectors used for the 4- cassette cloning strategy. C. The curved dotted lines indicate the overhangs that anneal during the ligation reaction. D. Overview of the assembly protocol. For a detailed protocol, see the Materials and Methods section. E. Summary of available promoter, gene tag and 3’UTR donor cassette plasmids.
    Figure Legend Snippet: SapTrap assembly of MosSCI targeting vectors using the four-cassette system. A. The MosSCI targeting vector pXF87 was derived from pCFJ350 by mutating two SapI restriction sites (indicated by arrowheads in the “Left” (L) and “Right” (R) homology arms) and introducing two SapI sites (blue text) between the XhoI and SpeI sites (green text). SapI cleavage sites are in red text. The SapI recognition sites are oriented such that upon digestion they are removed from the vector backbone. The cbr- unc-119 gene is used as a positive selection marker to facilitate the identification of transgenic animals. B. Design of the donor cassette vectors used for the 4- cassette cloning strategy. C. The curved dotted lines indicate the overhangs that anneal during the ligation reaction. D. Overview of the assembly protocol. For a detailed protocol, see the Materials and Methods section. E. Summary of available promoter, gene tag and 3’UTR donor cassette plasmids.

    Techniques Used: Plasmid Preparation, Derivative Assay, Selection, Marker, Transgenic Assay, Clone Assay, Ligation

    8) Product Images from "A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology"

    Article Title: A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2013.00339

    Golden Gate assembly of an insert into the destination vector. (A) Recognition sequences for the Type IIS restriction endonucleases BsaI and SapI . 5′ overhang sequences used for annealing fragments are shown in bold magenta lettering. (B) Example of a Golden Gate-compatible destination vector. Note the orientation of the BsaI sites cause excision of the lacZ α gene. (C) Example of a Golden Gate compatible vector containing a gene of interest (GOI) that will be released after digestion with BsaI . (D) A typical Golden Gate Cloning reaction would involve mixing the destination vector and insert storage vector together into one tube at equal molar ratio with BsaI and T4 DNA ligase. The final vector produced would lack BsaI recognition sequences and be resistant to digestion.
    Figure Legend Snippet: Golden Gate assembly of an insert into the destination vector. (A) Recognition sequences for the Type IIS restriction endonucleases BsaI and SapI . 5′ overhang sequences used for annealing fragments are shown in bold magenta lettering. (B) Example of a Golden Gate-compatible destination vector. Note the orientation of the BsaI sites cause excision of the lacZ α gene. (C) Example of a Golden Gate compatible vector containing a gene of interest (GOI) that will be released after digestion with BsaI . (D) A typical Golden Gate Cloning reaction would involve mixing the destination vector and insert storage vector together into one tube at equal molar ratio with BsaI and T4 DNA ligase. The final vector produced would lack BsaI recognition sequences and be resistant to digestion.

    Techniques Used: Plasmid Preparation, Clone Assay, Produced

    9) Product Images from "Development of the SapI/AarI Incision Mediated Plasmid Editing Method"

    Article Title: Development of the SapI/AarI Incision Mediated Plasmid Editing Method

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2018.03.030

    Primer design for introducing point mutations using AarI and SapI. (A) Recognition sites and cutting patterns for the SapI and AarI restriction enzymes. (B) Hypothetical sequence that will be targeted for mutagenesis. (C) Primers that can be used to mutate the sequence shown in (B) with the SIMPLE method, using the SapI enzyme.
    Figure Legend Snippet: Primer design for introducing point mutations using AarI and SapI. (A) Recognition sites and cutting patterns for the SapI and AarI restriction enzymes. (B) Hypothetical sequence that will be targeted for mutagenesis. (C) Primers that can be used to mutate the sequence shown in (B) with the SIMPLE method, using the SapI enzyme.

    Techniques Used: Sequencing, Mutagenesis

    Related Articles

    Clone Assay:

    Article Title: CC-Chemokine Receptor 5 on Pulmonary Mesenchymal Cells Promotes Experimental Metastasis via the Induction of Erythroid Differentiation Regulator 1
    Article Snippet: .. After cloning, Erdr1 and LacZ control plasmids were linearized with SAPI (NEB, Ipswich, MA). .. One μg of plasmid DNA was transfected using Qiagen Attractene reagent according to the manufacturer's instructions (Qiagen, Germantown, MD).

    Article Title: SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors
    Article Snippet: .. Cloning To generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 ( ) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs (NEB)) with the oligo pairs XF30F/XF30R and XF31F/XF31R. .. In addition, the annealed oligos Eg717 and Eg718 were cloned between the XhoI and SpeI sites of pCFJ350.

    Amplification:

    Article Title: Development of the SapI/AarI Incision Mediated Plasmid Editing Method
    Article Snippet: .. After amplification, PCR products were first digested with DpnI (NEB) to eliminate the parental plasmid, cleaned up over columns (Qiagen), and then digested with either the SapI (NEB) or AarI (Thermo) restriction enzyme, following the manufacturer’s specifications. .. After digestion, DNA products were cleaned up again as before, and 50μg of the DNA was self-ligated using T4 DNA ligase (NEB).

    Nucleic Acid Electrophoresis:

    Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo
    Article Snippet: .. AAV-U6gRNA1-U6gRNA2-TnT-Cre Vector (Addgene #87682) gRNA sequences from Basic Protocol 1/DNA oligos Thermocycler AarI enzyme and buffer (ThermoFisher ER1581) SapI enzyme and buffer (NEB R0569L) 37°C incubator 37°C shaking incubator Agarose (GeneMate E-3120-500) 50× TAE (Boston BioProducts BM250) Ethidium bromide (Sigma E7637) DNA loading dye (NEB B7024S) Gel electrophoresis chamber UV transilluminator (365 nm) Gel purification kit (Invitrogen K210012) Quick ligation kit (NEB M2200L) Chemically competent cells such as ThermoFisher MAX Efficiency Stbl2 Competent Cells (Cat# 10268019). ..

    Ligation:

    Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo
    Article Snippet: .. AAV-U6gRNA1-U6gRNA2-TnT-Cre Vector (Addgene #87682) gRNA sequences from Basic Protocol 1/DNA oligos Thermocycler AarI enzyme and buffer (ThermoFisher ER1581) SapI enzyme and buffer (NEB R0569L) 37°C incubator 37°C shaking incubator Agarose (GeneMate E-3120-500) 50× TAE (Boston BioProducts BM250) Ethidium bromide (Sigma E7637) DNA loading dye (NEB B7024S) Gel electrophoresis chamber UV transilluminator (365 nm) Gel purification kit (Invitrogen K210012) Quick ligation kit (NEB M2200L) Chemically competent cells such as ThermoFisher MAX Efficiency Stbl2 Competent Cells (Cat# 10268019). ..

    Mutagenesis:

    Article Title: SapTrap assembly of C. elegans MosSCI transgene vectors
    Article Snippet: .. CloningTo generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 (Frøkjær-Jensen et al., 2012) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs) with the oligo pairs XF30F/XF30R and XF31F/XF31R. .. In addition, the annealed oligos Eg717 and Eg718 were cloned between the XhoI and SpeI sites of pCFJ350.

    Article Title: SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors
    Article Snippet: .. Cloning To generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 ( ) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs (NEB)) with the oligo pairs XF30F/XF30R and XF31F/XF31R. .. In addition, the annealed oligos Eg717 and Eg718 were cloned between the XhoI and SpeI sites of pCFJ350.

    other:

    Article Title: Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts
    Article Snippet: HincII and SapI restriction enzymes (NEB).

    Expressing:

    Article Title: SapTrap assembly of C. elegans MosSCI transgene vectors
    Article Snippet: .. CloningTo generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 (Frøkjær-Jensen et al., 2012) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs) with the oligo pairs XF30F/XF30R and XF31F/XF31R. .. In addition, the annealed oligos Eg717 and Eg718 were cloned between the XhoI and SpeI sites of pCFJ350.

    Article Title: SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors
    Article Snippet: .. Cloning To generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 ( ) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs (NEB)) with the oligo pairs XF30F/XF30R and XF31F/XF31R. .. In addition, the annealed oligos Eg717 and Eg718 were cloned between the XhoI and SpeI sites of pCFJ350.

    Polymerase Chain Reaction:

    Article Title: Development of the SapI/AarI Incision Mediated Plasmid Editing Method
    Article Snippet: .. After amplification, PCR products were first digested with DpnI (NEB) to eliminate the parental plasmid, cleaned up over columns (Qiagen), and then digested with either the SapI (NEB) or AarI (Thermo) restriction enzyme, following the manufacturer’s specifications. .. After digestion, DNA products were cleaned up again as before, and 50μg of the DNA was self-ligated using T4 DNA ligase (NEB).

    Plasmid Preparation:

    Article Title: SapTrap assembly of C. elegans MosSCI transgene vectors
    Article Snippet: .. CloningTo generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 (Frøkjær-Jensen et al., 2012) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs) with the oligo pairs XF30F/XF30R and XF31F/XF31R. .. In addition, the annealed oligos Eg717 and Eg718 were cloned between the XhoI and SpeI sites of pCFJ350.

    Article Title: SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors
    Article Snippet: .. Cloning To generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 ( ) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs (NEB)) with the oligo pairs XF30F/XF30R and XF31F/XF31R. .. In addition, the annealed oligos Eg717 and Eg718 were cloned between the XhoI and SpeI sites of pCFJ350.

    Article Title: Development of the SapI/AarI Incision Mediated Plasmid Editing Method
    Article Snippet: .. After amplification, PCR products were first digested with DpnI (NEB) to eliminate the parental plasmid, cleaned up over columns (Qiagen), and then digested with either the SapI (NEB) or AarI (Thermo) restriction enzyme, following the manufacturer’s specifications. .. After digestion, DNA products were cleaned up again as before, and 50μg of the DNA was self-ligated using T4 DNA ligase (NEB).

    Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo
    Article Snippet: .. AAV-U6gRNA1-U6gRNA2-TnT-Cre Vector (Addgene #87682) gRNA sequences from Basic Protocol 1/DNA oligos Thermocycler AarI enzyme and buffer (ThermoFisher ER1581) SapI enzyme and buffer (NEB R0569L) 37°C incubator 37°C shaking incubator Agarose (GeneMate E-3120-500) 50× TAE (Boston BioProducts BM250) Ethidium bromide (Sigma E7637) DNA loading dye (NEB B7024S) Gel electrophoresis chamber UV transilluminator (365 nm) Gel purification kit (Invitrogen K210012) Quick ligation kit (NEB M2200L) Chemically competent cells such as ThermoFisher MAX Efficiency Stbl2 Competent Cells (Cat# 10268019). ..

    Gel Purification:

    Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo
    Article Snippet: .. AAV-U6gRNA1-U6gRNA2-TnT-Cre Vector (Addgene #87682) gRNA sequences from Basic Protocol 1/DNA oligos Thermocycler AarI enzyme and buffer (ThermoFisher ER1581) SapI enzyme and buffer (NEB R0569L) 37°C incubator 37°C shaking incubator Agarose (GeneMate E-3120-500) 50× TAE (Boston BioProducts BM250) Ethidium bromide (Sigma E7637) DNA loading dye (NEB B7024S) Gel electrophoresis chamber UV transilluminator (365 nm) Gel purification kit (Invitrogen K210012) Quick ligation kit (NEB M2200L) Chemically competent cells such as ThermoFisher MAX Efficiency Stbl2 Competent Cells (Cat# 10268019). ..

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    • sapi restriction sites  (New England Biolabs)
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    New England Biolabs sapi restriction sites
    SapTrap assembly of MosSCI targeting vectors using the four-cassette system. A. The MosSCI targeting vector <t>pXF87</t> was derived from pCFJ350 by mutating two <t>SapI</t> restriction sites (indicated by arrowheads in the “Left” (L) and “Right” (R) homology arms) and introducing two SapI sites (blue text) between the XhoI and SpeI sites (green text). SapI cleavage sites are in red text. The SapI recognition sites are oriented such that upon digestion they are removed from the vector backbone. The cbr- unc-119 gene is used as a positive selection marker to facilitate the identification of transgenic animals. B. Design of the donor cassette vectors used for the 4-cassette cloning strategy. C. The curved dotted lines indicate the overhangs that anneal during the ligation reaction. D. Overview of the assembly protocol. For a detailed protocol, see the Materials and Methods section. E. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids.
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    SapTrap assembly of MosSCI targeting vectors using the four-cassette system. A. The MosSCI targeting vector pXF87 was derived from pCFJ350 by mutating two SapI restriction sites (indicated by arrowheads in the “Left” (L) and “Right” (R) homology arms) and introducing two SapI sites (blue text) between the XhoI and SpeI sites (green text). SapI cleavage sites are in red text. The SapI recognition sites are oriented such that upon digestion they are removed from the vector backbone. The cbr- unc-119 gene is used as a positive selection marker to facilitate the identification of transgenic animals. B. Design of the donor cassette vectors used for the 4-cassette cloning strategy. C. The curved dotted lines indicate the overhangs that anneal during the ligation reaction. D. Overview of the assembly protocol. For a detailed protocol, see the Materials and Methods section. E. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids.

    Journal: G3: Genes|Genomes|Genetics

    Article Title: SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors

    doi: 10.1534/g3.119.400822

    Figure Lengend Snippet: SapTrap assembly of MosSCI targeting vectors using the four-cassette system. A. The MosSCI targeting vector pXF87 was derived from pCFJ350 by mutating two SapI restriction sites (indicated by arrowheads in the “Left” (L) and “Right” (R) homology arms) and introducing two SapI sites (blue text) between the XhoI and SpeI sites (green text). SapI cleavage sites are in red text. The SapI recognition sites are oriented such that upon digestion they are removed from the vector backbone. The cbr- unc-119 gene is used as a positive selection marker to facilitate the identification of transgenic animals. B. Design of the donor cassette vectors used for the 4-cassette cloning strategy. C. The curved dotted lines indicate the overhangs that anneal during the ligation reaction. D. Overview of the assembly protocol. For a detailed protocol, see the Materials and Methods section. E. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids.

    Article Snippet: Cloning To generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 ( ) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs (NEB)) with the oligo pairs XF30F/XF30R and XF31F/XF31R.

    Techniques: Plasmid Preparation, Derivative Assay, Selection, Marker, Transgenic Assay, Clone Assay, Ligation

    SapTrap assembly of MosSCI targeting vectors using the five-cassette system. A. Schematic of pXF87 and the donor cassettes following SapI digestion. The dotted lines indicate the overhangs that anneal during ligation. B. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids for the five-cassette system.

    Journal: G3: Genes|Genomes|Genetics

    Article Title: SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors

    doi: 10.1534/g3.119.400822

    Figure Lengend Snippet: SapTrap assembly of MosSCI targeting vectors using the five-cassette system. A. Schematic of pXF87 and the donor cassettes following SapI digestion. The dotted lines indicate the overhangs that anneal during ligation. B. Summary of available promoter, gene tag and 3′UTR donor cassette plasmids for the five-cassette system.

    Article Snippet: Cloning To generate the expression vector pXF87, the two SapI restriction sites in pCFJ350 ( ) were mutated using Q5 Site-Directed Mutagenesis (New England Biolabs (NEB)) with the oligo pairs XF30F/XF30R and XF31F/XF31R.

    Techniques: Ligation

    Insertion of gRNA sequences into AAV vector gRNAs are inserted into AAV plasmid containing U6 promoters and cardiac troponin T (cTNT) promoter-driven Cre recombinase. Annealed oligos corresponding to the targeting portions of gRNA1 and gRNA2 are cloned between AarI and SapI sites, respectively. The entire construct is contained within AAV inverted terminal repeat sequences, allowing for packaging into AAV capsids. Examples of gRNAs designed against Gata4 are shown.

    Journal: Current protocols in molecular biology

    Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo

    doi: 10.1002/cpmb.46

    Figure Lengend Snippet: Insertion of gRNA sequences into AAV vector gRNAs are inserted into AAV plasmid containing U6 promoters and cardiac troponin T (cTNT) promoter-driven Cre recombinase. Annealed oligos corresponding to the targeting portions of gRNA1 and gRNA2 are cloned between AarI and SapI sites, respectively. The entire construct is contained within AAV inverted terminal repeat sequences, allowing for packaging into AAV capsids. Examples of gRNAs designed against Gata4 are shown.

    Article Snippet: AAV-U6gRNA1-U6gRNA2-TnT-Cre Vector (Addgene #87682) gRNA sequences from Basic Protocol 1/DNA oligos Thermocycler AarI enzyme and buffer (ThermoFisher ER1581) SapI enzyme and buffer (NEB R0569L) 37°C incubator 37°C shaking incubator Agarose (GeneMate E-3120-500) 50× TAE (Boston BioProducts BM250) Ethidium bromide (Sigma E7637) DNA loading dye (NEB B7024S) Gel electrophoresis chamber UV transilluminator (365 nm) Gel purification kit (Invitrogen K210012) Quick ligation kit (NEB M2200L) Chemically competent cells such as ThermoFisher MAX Efficiency Stbl2 Competent Cells (Cat# 10268019).

    Techniques: Plasmid Preparation, Clone Assay, Construct

    Examination of dual incisions during ICL repair. HincII digested repair intermediates are separated on a denaturing gel and visualized using Southern blotting (land 5–14) (Subheading 3.4). Undigested and HincII and/or SapI digested pControl and

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

    Article Title: Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts

    doi: 10.1007/978-1-61779-998-3_16

    Figure Lengend Snippet: Examination of dual incisions during ICL repair. HincII digested repair intermediates are separated on a denaturing gel and visualized using Southern blotting (land 5–14) (Subheading 3.4). Undigested and HincII and/or SapI digested pControl and

    Article Snippet: HincII and SapI restriction enzymes (NEB).

    Techniques: Southern Blot

    Determination of ICL repair efficiency. (a) Analysis of HincII (lane 1–10) and HincII/SapI (lane 11–20) digested repair intermediates on a native agarose gel (Subheading 3.5). (b) Calculated repair efficiency plotted against time.

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

    Article Title: Replication-coupled DNA Interstrand Crosslink repair in Xenopus egg extracts

    doi: 10.1007/978-1-61779-998-3_16

    Figure Lengend Snippet: Determination of ICL repair efficiency. (a) Analysis of HincII (lane 1–10) and HincII/SapI (lane 11–20) digested repair intermediates on a native agarose gel (Subheading 3.5). (b) Calculated repair efficiency plotted against time.

    Article Snippet: HincII and SapI restriction enzymes (NEB).

    Techniques: Agarose Gel Electrophoresis