sapi  (New England Biolabs)


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    • 95
    Name:
    SapI
    Description:
    SapI 1 250 units
    Catalog Number:
    R0569L
    Price:
    282
    Category:
    Restriction Enzymes
    Size:
    1 250 units
    		Buy from Supplier

    Structured Review

    New England Biolabs sapi
    SapI
    SapI 1 250 units
    https://www.bioz.com/result/sapi/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sapi - by Bioz Stars, 2021-07
    95/100 stars

    Images

    1) 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

    2) 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

    Related Articles

    Plasmid Preparation:

    Article Title: Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides
    Article Snippet: .. We then combined 4 μg of vector and 140 ng of barcode fragments in a 50 µl reaction with 5 µl 10x T4 ligase buffer, 5 µl 10x NEB CutSmart buffer (NEB, B7204S, Ipswich, MA), 2.5 µl T7 ligase (NEB, M0318L), and 2.5 µl of SapI (NEB, R0569S). .. Without cooling the product, we added 1 µl SapI and incubated for 30 min at 37°C to digest any uncut vector, then cooled to 10°C.

    Article Title: Plug and Play Protein Modification using Homology-independent Universal Genome Engineering
    Article Snippet: For each gene of interest (GOI), GS-gRNA sequences targeting either the N-term or the C-term of the GOI were designed using an online gRNA evaluation tool “CRISPOR” , and cloned into the SapI site using a highly efficient (typically > 90% positive rate of colony picking, - ) restriction digestion / ligation cloning method. .. In brief, 10ng (~2fmol) of the GS-gRNA backbone plasmid was digested with SapI enzyme (NEB R0569, 1uL) and simultaneously ligated with 50fmol of annealed 23-24mer (including SapI sticky ends) GS-gRNA oligos with T4 DNA ligase (NEB M0202, 1uL) in a 10uL reaction, by 10 repeats of thermocycling between 37°C (5 min) and 21°C (5 min). .. Colony PCR was performed to detect GS-gRNA integration into the backbone plasmid using a forward primer in the upstream U6 promoter region, paired with the reverse GS-gRNA oligo as the reverse primer to amplify an ~100bp amplicon.

    Article Title: Development of the SapI/AarI Incision Mediated Plasmid Editing Method
    Article Snippet: All PCR reactions were performed using the Phusion High-Fidelity DNA Polymerase (NEB), using the manufacturer’s recommended specifications. .. 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: The protocol is written for cloning of a single gRNA, but the protocol can be scaled to conveniently clone multiple gRNAs in parallel. .. 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). ..

    Functional Assay:

    Article Title: High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR/Cas9 in yeast
    Article Snippet: .. To clone in the additional functional components between the guide and donor, we amplified and cloned in the sgtail and terminator sequences following the same Golden Gate cloning method as described above, but this time using SapI (NEB R0569S) and T4 ligase. .. The resulting Golden Gate reactions were then PCR purified and electroporated into 5-alpha Electrocompetent E.coli cells to create a final guide+donor library.

    Amplification:

    Article Title: High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR/Cas9 in yeast
    Article Snippet: .. To clone in the additional functional components between the guide and donor, we amplified and cloned in the sgtail and terminator sequences following the same Golden Gate cloning method as described above, but this time using SapI (NEB R0569S) and T4 ligase. .. The resulting Golden Gate reactions were then PCR purified and electroporated into 5-alpha Electrocompetent E.coli cells to create a final guide+donor library.

    Article Title: Development of the SapI/AarI Incision Mediated Plasmid Editing Method
    Article Snippet: All PCR reactions were performed using the Phusion High-Fidelity DNA Polymerase (NEB), using the manufacturer’s recommended specifications. .. 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).

    Clone Assay:

    Article Title: High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR/Cas9 in yeast
    Article Snippet: .. To clone in the additional functional components between the guide and donor, we amplified and cloned in the sgtail and terminator sequences following the same Golden Gate cloning method as described above, but this time using SapI (NEB R0569S) and T4 ligase. .. The resulting Golden Gate reactions were then PCR purified and electroporated into 5-alpha Electrocompetent E.coli cells to create a final guide+donor library.

    Article Title: Oligomerized Pool ENgineering (OPEN): An “Open-Source” Protocol for Making Customized Zinc Finger Arrays
    Article Snippet: M13K07 helper phage (New England Biolabs, Cat #N0315S; store indefinitely at -20°C) < ! .. > CAUTION Care should be taken to avoid contaminating laboratory equipment and benches with bacteriophage Restriction enzymes (all from New England Biolabs): BamH I (cat. no. R0136S), Bbs I (cat. no R0539L), Bsa I (cat. no. R0533L), EcoR I (cat. no. R0101L), Hind III (cat. no. R0104L), Pst I (cat. no. R0140S), Sap I (cat. no. R0569L), Xba I (cat. no. R0145L) 10× restriction enzyme buffers (New England Biolabs, included with enzymes) T4 DNA Ligase and associated standard T4 DNA Ligase reaction buffer (New England Biolabs, cat. no. M0202S) Quick Ligation Kit (New England Biolabs, cat. no. M2200S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201S) Cloned Pfu polymerase and associated 10× reaction buffer (Stratagene, cat. no. 600159-81) Expand High-Fidelity thermostable polymerase and associated 10× Expand buffer with MgCl2 (Roche, cat. no. 11732641001) < ! .. > CRITICAL We have found the use of Expand enzyme to be critical for successful amplification of the zinc finger pools AccuGel 29:1 acrylamide:bis-acrylamide solution (National Diagnostics, cat. no. EC-852) < !

    Ligation:

    Article Title: Oligomerized Pool ENgineering (OPEN): An “Open-Source” Protocol for Making Customized Zinc Finger Arrays
    Article Snippet: M13K07 helper phage (New England Biolabs, Cat #N0315S; store indefinitely at -20°C) < ! .. > CAUTION Care should be taken to avoid contaminating laboratory equipment and benches with bacteriophage Restriction enzymes (all from New England Biolabs): BamH I (cat. no. R0136S), Bbs I (cat. no R0539L), Bsa I (cat. no. R0533L), EcoR I (cat. no. R0101L), Hind III (cat. no. R0104L), Pst I (cat. no. R0140S), Sap I (cat. no. R0569L), Xba I (cat. no. R0145L) 10× restriction enzyme buffers (New England Biolabs, included with enzymes) T4 DNA Ligase and associated standard T4 DNA Ligase reaction buffer (New England Biolabs, cat. no. M0202S) Quick Ligation Kit (New England Biolabs, cat. no. M2200S) T4 Polynucleotide Kinase (New England Biolabs, cat. no. M0201S) Cloned Pfu polymerase and associated 10× reaction buffer (Stratagene, cat. no. 600159-81) Expand High-Fidelity thermostable polymerase and associated 10× Expand buffer with MgCl2 (Roche, cat. no. 11732641001) < ! .. > CRITICAL We have found the use of Expand enzyme to be critical for successful amplification of the zinc finger pools AccuGel 29:1 acrylamide:bis-acrylamide solution (National Diagnostics, cat. no. EC-852) < !

    Article Title: CASAAV: a CRISPR based platform for rapid dissection of gene function in vivo
    Article Snippet: The protocol is written for cloning of a single gRNA, but the protocol can be scaled to conveniently clone multiple gRNAs in parallel. .. 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). ..

    other:

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

    Polymerase Chain Reaction:

    Article Title: Development of the SapI/AarI Incision Mediated Plasmid Editing Method
    Article Snippet: All PCR reactions were performed using the Phusion High-Fidelity DNA Polymerase (NEB), using the manufacturer’s recommended specifications. .. 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: The protocol is written for cloning of a single gRNA, but the protocol can be scaled to conveniently clone multiple gRNAs in parallel. .. 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: The protocol is written for cloning of a single gRNA, but the protocol can be scaled to conveniently clone multiple gRNAs in parallel. .. 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  (New England Biolabs)
    95
    New England Biolabs sapi
    Primer design for introducing point mutations using <t>AarI</t> and <t>SapI.</t> (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.
    Sapi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sapi/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sapi - by Bioz Stars, 2021-07
    95/100 stars
    		  Buy from Supplier

    Image Search Results


    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.

    Journal: Journal of molecular biology

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

    doi: 10.1016/j.jmb.2018.03.030

    Figure Lengend 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.

    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.

    Techniques: Sequencing, Mutagenesis

    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

    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