xba i  (New England Biolabs)


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    Structured Review

    New England Biolabs xba i
    Xba I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xba i/product/New England Biolabs
    Average 98 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    xba i - by Bioz Stars, 2020-04
    98/100 stars

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    Related Articles

    Clone Assay:

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: The eGFP, IRES2, and synGAP segments were subcloned in tandem into the multiple cloning site of pBSK II (-) (eGFP-IRES2-flag-synGAP). .. It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S.

    Transfection:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs). .. All constructs were sequenced before use in cell transfection or transformation.

    Amplification:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs). .. For the pTiger-SogN1, N2 and N3 constructs, SogN1, N2 and N3 sequences were amplified by PCR from pBS-SogN1, N2 and N3, digested with Kpn I and Xba I and introduced into Kpn I/ Xba I-digested pTiger using T4 DNA ligase (New England Biolabs). pTiger-Sog constructs were sent to Bestgene for injection and integrated into the attP VK00027-docking site using the ΦC31 system ( ).

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: .. For the pTiger-SogN1, N2 and N3 constructs, SogN1, N2 and N3 sequences were amplified by PCR from pBS-SogN1, N2 and N3, digested with Kpn I and Xba I and introduced into Kpn I/ Xba I-digested pTiger using T4 DNA ligase (New England Biolabs). pTiger-Sog constructs were sent to Bestgene for injection and integrated into the attP VK00027-docking site using the ΦC31 system ( ). .. All constructs were sequenced before use in cell transfection or transformation.

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: For construction of the virus expressing wild-type synGAP, a cDNA encoding flag-synGAP (gift from Dr. Jeong Oh, California Institute of Technology, Pasadena, CA) was amplified by PCR with a 5′-oligo containing a Not I site and a 3′-oligo containing a Sac II site. .. It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S.

    Transgenic Assay:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: Paragraph title: Transgenic constructs ... For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs).

    Mutagenesis:

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S. .. The ΔSXV mutant was created by amplifying the flag-synGAP cDNA with a 5′-oligo containing a Not I site and a 3′-oligo designed to substitute a stop codon followed by a Sac II site for the terminal 5 amino acids (QQTRV) and incorporated into nsp2S as described above.

    Construct:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: .. For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs). .. For the pTiger-SogN1, N2 and N3 constructs, SogN1, N2 and N3 sequences were amplified by PCR from pBS-SogN1, N2 and N3, digested with Kpn I and Xba I and introduced into Kpn I/ Xba I-digested pTiger using T4 DNA ligase (New England Biolabs). pTiger-Sog constructs were sent to Bestgene for injection and integrated into the attP VK00027-docking site using the ΦC31 system ( ).

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: .. For the pTiger-SogN1, N2 and N3 constructs, SogN1, N2 and N3 sequences were amplified by PCR from pBS-SogN1, N2 and N3, digested with Kpn I and Xba I and introduced into Kpn I/ Xba I-digested pTiger using T4 DNA ligase (New England Biolabs). pTiger-Sog constructs were sent to Bestgene for injection and integrated into the attP VK00027-docking site using the ΦC31 system ( ). .. All constructs were sequenced before use in cell transfection or transformation.

    Produced:

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S. .. Viral particles encoding eGFP-IRES2-wtSynGAP were produced according to the procedures provided by Invitrogen.

    Plasmid Purification:

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: Similarly, DNA sequence encoding an IRES2 was obtained from pLP-IRES2-eGFP by PCR with a 5′-oligo upstream of a Bam HI site in the vector and a 3′-oligo containing a Not I site. .. It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S.

    Generated:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: Constructs for Gal4/UAS mediated expression in flies were generated in pTiger, a derivative of pUASp ( ). .. For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs).

    Polymerase Chain Reaction:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs). .. For the pTiger-SogN1, N2 and N3 constructs, SogN1, N2 and N3 sequences were amplified by PCR from pBS-SogN1, N2 and N3, digested with Kpn I and Xba I and introduced into Kpn I/ Xba I-digested pTiger using T4 DNA ligase (New England Biolabs). pTiger-Sog constructs were sent to Bestgene for injection and integrated into the attP VK00027-docking site using the ΦC31 system ( ).

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: .. For the pTiger-SogN1, N2 and N3 constructs, SogN1, N2 and N3 sequences were amplified by PCR from pBS-SogN1, N2 and N3, digested with Kpn I and Xba I and introduced into Kpn I/ Xba I-digested pTiger using T4 DNA ligase (New England Biolabs). pTiger-Sog constructs were sent to Bestgene for injection and integrated into the attP VK00027-docking site using the ΦC31 system ( ). .. All constructs were sequenced before use in cell transfection or transformation.

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: For construction of the virus expressing wild-type synGAP, a cDNA encoding flag-synGAP (gift from Dr. Jeong Oh, California Institute of Technology, Pasadena, CA) was amplified by PCR with a 5′-oligo containing a Not I site and a 3′-oligo containing a Sac II site. .. It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S.

    Expressing:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: Constructs for Gal4/UAS mediated expression in flies were generated in pTiger, a derivative of pUASp ( ). .. For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs).

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: For construction of the virus expressing wild-type synGAP, a cDNA encoding flag-synGAP (gift from Dr. Jeong Oh, California Institute of Technology, Pasadena, CA) was amplified by PCR with a 5′-oligo containing a Not I site and a 3′-oligo containing a Sac II site. .. It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S.

    Sequencing:

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: This procedure resulted in inclusion of a Kozak sequence at the 3′-end of the IRES. .. It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S.

    Injection:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs). .. For the pTiger-SogN1, N2 and N3 constructs, SogN1, N2 and N3 sequences were amplified by PCR from pBS-SogN1, N2 and N3, digested with Kpn I and Xba I and introduced into Kpn I/ Xba I-digested pTiger using T4 DNA ligase (New England Biolabs). pTiger-Sog constructs were sent to Bestgene for injection and integrated into the attP VK00027-docking site using the ΦC31 system ( ).

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: .. For the pTiger-SogN1, N2 and N3 constructs, SogN1, N2 and N3 sequences were amplified by PCR from pBS-SogN1, N2 and N3, digested with Kpn I and Xba I and introduced into Kpn I/ Xba I-digested pTiger using T4 DNA ligase (New England Biolabs). pTiger-Sog constructs were sent to Bestgene for injection and integrated into the attP VK00027-docking site using the ΦC31 system ( ). .. All constructs were sequenced before use in cell transfection or transformation.

    Transformation Assay:

    Article Title: N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs
    Article Snippet: For the pTiger-SogWT construct, pBS-SogWT and pTiger were digested with Kpn I and Xba I and ligated using T4 DNA ligase (New England Biolabs). .. All constructs were sequenced before use in cell transfection or transformation.

    Plasmid Preparation:

    Article Title: SynGAP Regulates Spine Formation
    Article Snippet: Similarly, DNA sequence encoding an IRES2 was obtained from pLP-IRES2-eGFP by PCR with a 5′-oligo upstream of a Bam HI site in the vector and a 3′-oligo containing a Not I site. .. It was digested with Xba I and Sac II, and the Sac II site was blunted with ExoT (New England Biolabs, Beverly, MA) and ligated into the Xba I and Stu I sites of pSinRep5 (Invitrogen) or nsp2S.

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  • 99
    New England Biolabs xba i
    Southern blot showing copy numbers of the 16D10i-2 RNAi transgene in transformed potato lines and controls. For each plant line, 15 µg <t>DNA</t> was digested with 50 U <t>Xba</t> I and separated on a 0.8% agarose gel. 82-4, PA99N82-4 advanced breeding line;
    Xba I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xba i/product/New England Biolabs
    Average 99 stars, based on 34 article reviews
    Price from $9.99 to $1999.99
    xba i - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    93
    New England Biolabs xba i mcs p2a sfgfp xba i pcr
    Cloning strategy for producing mutation reporter constructs using <t>pCS2+MCS-P2A-sfGFP</t> vector. (A) Mutation reporter vector structure and cloning strategy. <t>PCR</t> products are amplified from cDNA with the primers designed to introduce the T3 polymerase promoter and Pac I and Asc I sites for cloning and to amplify the 5′ UTR and 200 codons of coding sequence for insertion into pCS2+MCS-P2A-sfGFP vector. (B) The multiple cloning site sequence (MCS) of pCS2+MCS-P2A-sfGFP contains Bst BI, Eco RI, Pac I, Sph I and Asc I restriction sites followed by the P2A sequence and sfGFP coding sequence. (C) Gene mutations in cloned cDNA fragments. Mutations identified at the level of cDNA in chd7 (deletion of 2 nt), hace1 (insertion of 8 nt) and pycr1a (deletion of 71 nt) are shown using alignment of mutant sequences to wild-type ones.
    Xba I Mcs P2a Sfgfp Xba I Pcr, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xba i mcs p2a sfgfp xba i pcr/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xba i mcs p2a sfgfp xba i pcr - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    Image Search Results


    Southern blot showing copy numbers of the 16D10i-2 RNAi transgene in transformed potato lines and controls. For each plant line, 15 µg DNA was digested with 50 U Xba I and separated on a 0.8% agarose gel. 82-4, PA99N82-4 advanced breeding line;

    Journal: Journal of Nematology

    Article Title: Broad Meloidogyne Resistance in Potato Based on RNA Interference of Effector Gene 16D10

    doi:

    Figure Lengend Snippet: Southern blot showing copy numbers of the 16D10i-2 RNAi transgene in transformed potato lines and controls. For each plant line, 15 µg DNA was digested with 50 U Xba I and separated on a 0.8% agarose gel. 82-4, PA99N82-4 advanced breeding line;

    Article Snippet: For each plant line, 15 µg DNA was digested with 50 U Xba I (New England Biolabs, Ipswich, MA) for 16 hr at 37 ° C, then separated on a 0.8% agarose gel at 70 V for 16 hr and transferred to a GeneScreen Plus nylon membrane (PerkinElmer, Boston, MA) in 10× saline sodium citrate (SSC) buffer (1.5 M NaCl, 0.15 M sodium citrate, pH 7).

    Techniques: Southern Blot, Transformation Assay, Agarose Gel Electrophoresis

    Mapping and tandem arrangement of DXZ4 in primates . (a) Direct-labeled fluorescence in situ hybridization (FISH) of human DXZ4 BAC clone (2272M5; red) and human PLS3 BAC clone (268A15; green) to male and female rhesus macaque metaphase chromosomes. White arrows point to the hybridizing X chromosome. Metaphase chromosomes were counterstained with DAPI, and converted to gray-scale to assist in visualizing the FISH signals. (b) Southern blot of Xba I digested primate genomic DNA separated by pulsed field gel electrophoresis, hybridized with a human digoxigenin-labeled DXZ4 probe. Primates and group are listed along the top and gender indicated by M (male) or F (female), including rhesus macaque (R. Macaque), pig-tailed macaque (P-T. Macaque), common squirrel monkey (Sq. Monkey) and black-handed spider monkey (Sp. Monkey). Size in kilobases is given to the left. (c) Ethidium bromide stained 0.9% agarose gel showing green monkey and macaque BAC DNA digested with the restriction endonuclease Hin dIII and separated by gel electrophoresis. The sizes of the molecular weight marker are given to the left in kilobases.

    Journal: Genome Biology

    Article Title: Characterization of DXZ4 conservation in primates implies important functional roles for CTCF binding, array expression and tandem repeat organization on the X chromosome

    doi: 10.1186/gb-2011-12-4-r37

    Figure Lengend Snippet: Mapping and tandem arrangement of DXZ4 in primates . (a) Direct-labeled fluorescence in situ hybridization (FISH) of human DXZ4 BAC clone (2272M5; red) and human PLS3 BAC clone (268A15; green) to male and female rhesus macaque metaphase chromosomes. White arrows point to the hybridizing X chromosome. Metaphase chromosomes were counterstained with DAPI, and converted to gray-scale to assist in visualizing the FISH signals. (b) Southern blot of Xba I digested primate genomic DNA separated by pulsed field gel electrophoresis, hybridized with a human digoxigenin-labeled DXZ4 probe. Primates and group are listed along the top and gender indicated by M (male) or F (female), including rhesus macaque (R. Macaque), pig-tailed macaque (P-T. Macaque), common squirrel monkey (Sq. Monkey) and black-handed spider monkey (Sp. Monkey). Size in kilobases is given to the left. (c) Ethidium bromide stained 0.9% agarose gel showing green monkey and macaque BAC DNA digested with the restriction endonuclease Hin dIII and separated by gel electrophoresis. The sizes of the molecular weight marker are given to the left in kilobases.

    Article Snippet: Agarose embedded DNA was digested with Xba I (NEB, Ipswich, MA, USA).

    Techniques: Labeling, Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, BAC Assay, Southern Blot, Pulsed-Field Gel, Electrophoresis, Staining, Agarose Gel Electrophoresis, Nucleic Acid Electrophoresis, Molecular Weight, Marker

    Dendrogram showing the cluster analysis of 76 S. Typhi isolates from Kolkata, India, 2009–2013, by Xba I-PFGE. Band comparison was performed by using the Dice coefficient with 1.5% optimization (Opt) and 1.5% position tolerance (Tol). Pan-susceptible, susceptible to all 17 drugs tested; A, ampicillin; Ac, amoxicillin/clavulanic acid; C, chloramphenicol; Q, co-trimoxazole; T, tetracycline; S, streptomycin; Na, nalidixic acid; Ci, ciprofloxacin; Of, ofloxacin; Le, levofloxacin.

    Journal: PLoS ONE

    Article Title: Antimicrobial Resistance, Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013

    doi: 10.1371/journal.pone.0101347

    Figure Lengend Snippet: Dendrogram showing the cluster analysis of 76 S. Typhi isolates from Kolkata, India, 2009–2013, by Xba I-PFGE. Band comparison was performed by using the Dice coefficient with 1.5% optimization (Opt) and 1.5% position tolerance (Tol). Pan-susceptible, susceptible to all 17 drugs tested; A, ampicillin; Ac, amoxicillin/clavulanic acid; C, chloramphenicol; Q, co-trimoxazole; T, tetracycline; S, streptomycin; Na, nalidixic acid; Ci, ciprofloxacin; Of, ofloxacin; Le, levofloxacin.

    Article Snippet: The DNA plugs were digested with 40 U of Xba I (New England Biolabs, MA) at 37°C for 18 h. The digested DNA was run on 1% pulsed field certified agarose gel (Bio-Rad, Hercules, Calif.) prepared in 0.5x TBE buffer (Sigma) using CHEF DRIII (Bio-Rad) apparatus with an initial switch time of 2.2 sec and a final switch time of 63.8 sec at 6 V/cm for 24 h. The gel was stained with ethidium bromide (1 µg/ml, Sigma), destained with deionized water and PFGE profiles were observed with the UV trans-illuminator using GelDoc (Bio-Rad).

    Techniques:

    Dendrogram showing the cluster analysis of 24 S. Paratyphi A isolates from Kolkata, India, 2009–2013, by Xba I-PFGE. Band comparison was performed by using the Dice coefficient with 1.5% optimization (Opt) and 1.5% position tolerance (Tol). Pan-susceptible, susceptible to all 17 drugs tested; Na, nalidixic acid; Ci, ciprofloxacin; Of, ofloxacin; Az, azithromycin.

    Journal: PLoS ONE

    Article Title: Antimicrobial Resistance, Virulence Profiles and Molecular Subtypes of Salmonella enterica Serovars Typhi and Paratyphi A Blood Isolates from Kolkata, India during 2009-2013

    doi: 10.1371/journal.pone.0101347

    Figure Lengend Snippet: Dendrogram showing the cluster analysis of 24 S. Paratyphi A isolates from Kolkata, India, 2009–2013, by Xba I-PFGE. Band comparison was performed by using the Dice coefficient with 1.5% optimization (Opt) and 1.5% position tolerance (Tol). Pan-susceptible, susceptible to all 17 drugs tested; Na, nalidixic acid; Ci, ciprofloxacin; Of, ofloxacin; Az, azithromycin.

    Article Snippet: The DNA plugs were digested with 40 U of Xba I (New England Biolabs, MA) at 37°C for 18 h. The digested DNA was run on 1% pulsed field certified agarose gel (Bio-Rad, Hercules, Calif.) prepared in 0.5x TBE buffer (Sigma) using CHEF DRIII (Bio-Rad) apparatus with an initial switch time of 2.2 sec and a final switch time of 63.8 sec at 6 V/cm for 24 h. The gel was stained with ethidium bromide (1 µg/ml, Sigma), destained with deionized water and PFGE profiles were observed with the UV trans-illuminator using GelDoc (Bio-Rad).

    Techniques:

    Cloning strategy for producing mutation reporter constructs using pCS2+MCS-P2A-sfGFP vector. (A) Mutation reporter vector structure and cloning strategy. PCR products are amplified from cDNA with the primers designed to introduce the T3 polymerase promoter and Pac I and Asc I sites for cloning and to amplify the 5′ UTR and 200 codons of coding sequence for insertion into pCS2+MCS-P2A-sfGFP vector. (B) The multiple cloning site sequence (MCS) of pCS2+MCS-P2A-sfGFP contains Bst BI, Eco RI, Pac I, Sph I and Asc I restriction sites followed by the P2A sequence and sfGFP coding sequence. (C) Gene mutations in cloned cDNA fragments. Mutations identified at the level of cDNA in chd7 (deletion of 2 nt), hace1 (insertion of 8 nt) and pycr1a (deletion of 71 nt) are shown using alignment of mutant sequences to wild-type ones.

    Journal: Disease Models & Mechanisms

    Article Title: A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

    doi: 10.1242/dmm.026765

    Figure Lengend Snippet: Cloning strategy for producing mutation reporter constructs using pCS2+MCS-P2A-sfGFP vector. (A) Mutation reporter vector structure and cloning strategy. PCR products are amplified from cDNA with the primers designed to introduce the T3 polymerase promoter and Pac I and Asc I sites for cloning and to amplify the 5′ UTR and 200 codons of coding sequence for insertion into pCS2+MCS-P2A-sfGFP vector. (B) The multiple cloning site sequence (MCS) of pCS2+MCS-P2A-sfGFP contains Bst BI, Eco RI, Pac I, Sph I and Asc I restriction sites followed by the P2A sequence and sfGFP coding sequence. (C) Gene mutations in cloned cDNA fragments. Mutations identified at the level of cDNA in chd7 (deletion of 2 nt), hace1 (insertion of 8 nt) and pycr1a (deletion of 71 nt) are shown using alignment of mutant sequences to wild-type ones.

    Article Snippet: The resulting Xba I-MCS-P2A-sfGFP-Xba I PCR product was digested with Xba I and inserted into pCS2+ linearized with Xba I and dephosphorylated with CIP (New England Biolabs, M0290S).

    Techniques: Clone Assay, Mutagenesis, Construct, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Introduce, Sequencing