xbai  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    XbaI 10 U µL
    Description:
    5 T ↓C T A G A 3 3 A G A T C ↑T 5 Thermo Scientific XbaI restriction enzyme recognizes T CTAGA sites and cuts best at 37°C in Tango buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Note Also available as a FastDigest enzyme for rapid DNA digestion Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    Catalog Number:
    er0681
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher xbai
    Southern blot analysis of the four STS markers- CtR-431, CtR-594., CtR-496, and AFLP-376. Restriction enzymes used are <t>EcoRI,</t> HinDIII and <t>XbaI</t> . M, molecular weight marker; R resistant genotype Punjab Lal; S susceptible genotype Arka Lohit. Hybridization
    5 T ↓C T A G A 3 3 A G A T C ↑T 5 Thermo Scientific XbaI restriction enzyme recognizes T CTAGA sites and cuts best at 37°C in Tango buffer See Reaction Conditions for Restriction Enzymes for a table of enzyme activity conditions for double digestion and heat inactivation for this and other restriction enzymes Note Also available as a FastDigest enzyme for rapid DNA digestion Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes optimized to work in one of the buffers of the Five Buffer System In addition the universal Tango buffer is provided for convenience in double digestions All of the enzymes exhibit 100 activity in the recommended buffer and reaction conditions To ensure consistent performance Thermo Scientific restriction enzyme reaction buffers contain premixed BSA which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations Features• Superior quality stringent quality control and industry leading manufacturing process• Convenient color coded Five Buffer System• Includes universal Tango buffer for double digestions• BSA premixed in reaction buffers• Wide selection of restriction endonuclease specificitiesApplications• Molecular cloning• Restriction site mapping• Genotyping• Southern blotting• Restriction fragment length polymorphism RFLP • SNPNote For methylation sensitivity refer to product specifications
    https://www.bioz.com/result/xbai/product/Thermo Fisher
    Average 99 stars, based on 466 article reviews
    Price from $9.99 to $1999.99
    xbai - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Sequence-tagged site-based diagnostic markers linked to a novel anthracnose resistance gene RCt1 in chili pepper (Capsicum annuum L.)"

    Article Title: Sequence-tagged site-based diagnostic markers linked to a novel anthracnose resistance gene RCt1 in chili pepper (Capsicum annuum L.)

    Journal: 3 Biotech

    doi: 10.1007/s13205-018-1552-0

    Southern blot analysis of the four STS markers- CtR-431, CtR-594., CtR-496, and AFLP-376. Restriction enzymes used are EcoRI, HinDIII and XbaI . M, molecular weight marker; R resistant genotype Punjab Lal; S susceptible genotype Arka Lohit. Hybridization
    Figure Legend Snippet: Southern blot analysis of the four STS markers- CtR-431, CtR-594., CtR-496, and AFLP-376. Restriction enzymes used are EcoRI, HinDIII and XbaI . M, molecular weight marker; R resistant genotype Punjab Lal; S susceptible genotype Arka Lohit. Hybridization

    Techniques Used: Southern Blot, Molecular Weight, Marker, Hybridization

    2) Product Images from "Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application"

    Article Title: Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-016-0309-2

    Map of the vector constructed for the expression of TrxIA-2 ic in E. coli . The pTrxFus vector was used to create a C-terminal fusion to E. coli thioredoxin. The IA-2 ic optimised sequence was inserted into the multiple cloning site of the expression vector and expressed as amino terminal fusion to the E. coli protein thioredoxin. This vector includes an enterokinase (EK) cleavage site that allows release of the native protein from Trx. To drive expression of thioredoxin fusions, pTrxFus uses the pL promoter from the λ bacteriophage and the AspA transcription terminator. Plasmid selection and maintenance is ensured by the presence of a beta-lactamase gene ( BLA ) that provides ampicillin resistance. SmaI and XbaI sites are indicated at the 3’ and 5’ ends of the IA-2 ic sequence. RBS: ribosome binding site
    Figure Legend Snippet: Map of the vector constructed for the expression of TrxIA-2 ic in E. coli . The pTrxFus vector was used to create a C-terminal fusion to E. coli thioredoxin. The IA-2 ic optimised sequence was inserted into the multiple cloning site of the expression vector and expressed as amino terminal fusion to the E. coli protein thioredoxin. This vector includes an enterokinase (EK) cleavage site that allows release of the native protein from Trx. To drive expression of thioredoxin fusions, pTrxFus uses the pL promoter from the λ bacteriophage and the AspA transcription terminator. Plasmid selection and maintenance is ensured by the presence of a beta-lactamase gene ( BLA ) that provides ampicillin resistance. SmaI and XbaI sites are indicated at the 3’ and 5’ ends of the IA-2 ic sequence. RBS: ribosome binding site

    Techniques Used: Plasmid Preparation, Construct, Expressing, IA, Sequencing, Clone Assay, Selection, Binding Assay

    3) Product Images from "Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum"

    Article Title: Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni037

    Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the BamHI fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the XbaI fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.
    Figure Legend Snippet: Example of a single BIBAC fingerprint generated with five restriction enzymes (the HaeIII fragment ends were not labeled) and four fluorescent-ddNTPs of the SNaPshot Multiplex Ready Reaction Mix on the ABI 3100 DNA analyzer. The blue peaks show the BamHI fragments labeled with ddGTP-dR110; the green peaks show the HindIII fragments labeled with ddATP-dR6G; the black peaks show the XbaI fragments labeled with ddCTP–dTAMRA; the red peaks show the XhoI fragments labeled with ddTTP–dROX; and the orange peaks show the internal size standard LIZ-500.

    Techniques Used: Generated, Labeling, Multiplex Assay

    4) Product Images from "Characterization of diverse homoserine lactone synthases in Escherichia coli"

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202294

    Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI -flanked synthase open reading frame (ORF) and co-expression with mCherry (mCh) from a single bicistronic mRNA. The vector is represented as SBOL [ 44 ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.
    Figure Legend Snippet: Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI -flanked synthase open reading frame (ORF) and co-expression with mCherry (mCh) from a single bicistronic mRNA. The vector is represented as SBOL [ 44 ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.

    Techniques Used: Plasmid Preparation, Clone Assay, Expressing

    5) Product Images from "Impact of UGT1A9 Polymorphism on Mycophenolic Acid Pharmacokinetic Parameters in Stable Renal Transplant Patients "

    Article Title: Impact of UGT1A9 Polymorphism on Mycophenolic Acid Pharmacokinetic Parameters in Stable Renal Transplant Patients

    Journal: Iranian Journal of Pharmaceutical Research : IJPR

    doi:

    The PCR-RFPL analysis of UGT1A9 T-275A SNP in patients. The DNA from patients was subjected to PCR followed by RFPL using XbaI digest. The reactions were resolved on 2% agarose gel electrophoresis. Lanes 1-3 and 4-6 are from two different patient hetrozygote for T-275A SNP. Lane 1, 4 are PCR products and lane 2, 3 and 5, 6 are XbaI digest of corresponding samples. The reactions were compared to 100 bp DNA ladder. (PCR-RFLP :poly chain reaction-restricted fragment length polymorphism, UGT: Uridine diphosphate glucuronosyl transferase, SNP: Single nucleotide polymorphism).
    Figure Legend Snippet: The PCR-RFPL analysis of UGT1A9 T-275A SNP in patients. The DNA from patients was subjected to PCR followed by RFPL using XbaI digest. The reactions were resolved on 2% agarose gel electrophoresis. Lanes 1-3 and 4-6 are from two different patient hetrozygote for T-275A SNP. Lane 1, 4 are PCR products and lane 2, 3 and 5, 6 are XbaI digest of corresponding samples. The reactions were compared to 100 bp DNA ladder. (PCR-RFLP :poly chain reaction-restricted fragment length polymorphism, UGT: Uridine diphosphate glucuronosyl transferase, SNP: Single nucleotide polymorphism).

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    6) Product Images from "Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains"

    Article Title: Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00076-13

    Restriction enzyme analysis of the DNA from Ln. mesenteroides phages using XbaI (A and B) and Ln. pseudomesenteroides phages using HaeIII (C to G). (A and B) Ln. mesenteroides phages were isolated from buttermilk samples (Bm) obtained from dairy L4 (A) or 3 phage collections (C1 to C3) (B). (C to G) Ln. pseudomesenteroides phages were obtained from phage collections C1 and C2 (C) or isolated from various dairy products: whey (W), hard cheese (C), brine bath (S), butter cream (B), and acid curd cheese (aC). Phages were isolated from dairies L1 (D), S6 (E), S3 (F), and S7 (G). HindIII digests of phage λ DNA were used as size references (lanes M).
    Figure Legend Snippet: Restriction enzyme analysis of the DNA from Ln. mesenteroides phages using XbaI (A and B) and Ln. pseudomesenteroides phages using HaeIII (C to G). (A and B) Ln. mesenteroides phages were isolated from buttermilk samples (Bm) obtained from dairy L4 (A) or 3 phage collections (C1 to C3) (B). (C to G) Ln. pseudomesenteroides phages were obtained from phage collections C1 and C2 (C) or isolated from various dairy products: whey (W), hard cheese (C), brine bath (S), butter cream (B), and acid curd cheese (aC). Phages were isolated from dairies L1 (D), S6 (E), S3 (F), and S7 (G). HindIII digests of phage λ DNA were used as size references (lanes M).

    Techniques Used: Isolation

    7) Product Images from "Shifts in targeting of class switch recombination sites in mice that lack ? switch region tandem repeats or Msh2"

    Article Title: Shifts in targeting of class switch recombination sites in mice that lack ? switch region tandem repeats or Msh2

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20042491

    CSR within sequences upstream of the S μ TR are reduced in Msh2 − / − mice. (a) Twofold dilutions of genomic DNAs were assayed by Sμ/Sγ1 EcoRI DC-PCR and EcoRI–BsrGI DC-PCR. Samples were normalized using nAChR DC-PCR. (b) CSR sites in wild-type or Msh2 −/− mice measured relative to total wild-type CSR (set to 100%). Mean values and SEM of at least four independent experiments are presented. (c) CSR events in Msh2 −/− mice measured by EcoRI, HindIII, and XbaI real-time DC-PCR and compared with wild-type mice. Mean values and SEM are from six independent experiments with two Msh2 −/− mice and seven independent experiments with four wild-type mice.
    Figure Legend Snippet: CSR within sequences upstream of the S μ TR are reduced in Msh2 − / − mice. (a) Twofold dilutions of genomic DNAs were assayed by Sμ/Sγ1 EcoRI DC-PCR and EcoRI–BsrGI DC-PCR. Samples were normalized using nAChR DC-PCR. (b) CSR sites in wild-type or Msh2 −/− mice measured relative to total wild-type CSR (set to 100%). Mean values and SEM of at least four independent experiments are presented. (c) CSR events in Msh2 −/− mice measured by EcoRI, HindIII, and XbaI real-time DC-PCR and compared with wild-type mice. Mean values and SEM are from six independent experiments with two Msh2 −/− mice and seven independent experiments with four wild-type mice.

    Techniques Used: Mouse Assay, Polymerase Chain Reaction

    S μ TR − / − mice have increased levels of CSR events targeted to downstream sequences. (a) DC-PCR measuring CSR junction sites downstream of the SμTR region. HindIII and XbaI sites downstream of the SμTR region were used in DC-PCR assays to measure CSR junctions in sequences downstream of each site. Total CSR sites were determined by EcoRI DC-PCR (E, EcoRI; H1 and H2, HindIII; X, XbaI). Primer pairs used for the different DC-PCR assays are indicated by small arrows of different types. (b) Wild-type and SμTR −/− CSR events measured by EcoRI, HindIII, and XbaI real-time DC-PCR. Copy numbers for CSR events were derived from pSμ/Sγ3 standard curves. Rag-1 PCR normalized CSR events relative to Rag-1 copy number. Mean values and SEM are from seven independent experiments with four mice per group. (c) Percentage of SμTR −/− switch events downstream of the H2 relative to events downstream of H1 (set to 100%). Mean values and SEM are from three independent experiments per group.
    Figure Legend Snippet: S μ TR − / − mice have increased levels of CSR events targeted to downstream sequences. (a) DC-PCR measuring CSR junction sites downstream of the SμTR region. HindIII and XbaI sites downstream of the SμTR region were used in DC-PCR assays to measure CSR junctions in sequences downstream of each site. Total CSR sites were determined by EcoRI DC-PCR (E, EcoRI; H1 and H2, HindIII; X, XbaI). Primer pairs used for the different DC-PCR assays are indicated by small arrows of different types. (b) Wild-type and SμTR −/− CSR events measured by EcoRI, HindIII, and XbaI real-time DC-PCR. Copy numbers for CSR events were derived from pSμ/Sγ3 standard curves. Rag-1 PCR normalized CSR events relative to Rag-1 copy number. Mean values and SEM are from seven independent experiments with four mice per group. (c) Percentage of SμTR −/− switch events downstream of the H2 relative to events downstream of H1 (set to 100%). Mean values and SEM are from three independent experiments per group.

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Derivative Assay

    8) Product Images from "Reevaluating the Serotype II Capsular Locus of Streptococcus agalactiae ▿"

    Article Title: Reevaluating the Serotype II Capsular Locus of Streptococcus agalactiae ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01296-07

    RFLP-specific band patterns of the capsular loci of serotypes Ia, Ib, II to V, and VII. A fragment encompassing the cpsG - neuA region was digested simultaneously with MspI and XbaI. “1Kb Plus” denotes the 1-kb Plus DNA ladder (Invitrogen,
    Figure Legend Snippet: RFLP-specific band patterns of the capsular loci of serotypes Ia, Ib, II to V, and VII. A fragment encompassing the cpsG - neuA region was digested simultaneously with MspI and XbaI. “1Kb Plus” denotes the 1-kb Plus DNA ladder (Invitrogen,

    Techniques Used: IA

    9) Product Images from "Sequence-tagged site-based diagnostic markers linked to a novel anthracnose resistance gene RCt1 in chili pepper (Capsicum annuum L.)"

    Article Title: Sequence-tagged site-based diagnostic markers linked to a novel anthracnose resistance gene RCt1 in chili pepper (Capsicum annuum L.)

    Journal: 3 Biotech

    doi: 10.1007/s13205-018-1552-0

    Southern blot analysis of the four STS markers- CtR-431, CtR-594., CtR-496, and AFLP-376. Restriction enzymes used are EcoRI, HinDIII and XbaI . M, molecular weight marker; R resistant genotype Punjab Lal; S susceptible genotype Arka Lohit. Hybridization
    Figure Legend Snippet: Southern blot analysis of the four STS markers- CtR-431, CtR-594., CtR-496, and AFLP-376. Restriction enzymes used are EcoRI, HinDIII and XbaI . M, molecular weight marker; R resistant genotype Punjab Lal; S susceptible genotype Arka Lohit. Hybridization

    Techniques Used: Southern Blot, Molecular Weight, Marker, Hybridization

    10) Product Images from "Characterization of diverse homoserine lactone synthases in Escherichia coli"

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202294

    Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.
    Figure Legend Snippet: Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.

    Techniques Used: Plasmid Preparation, Clone Assay, Expressing

    11) Product Images from "Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle "

    Article Title: Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00873-09

    Dendrogram of PFGE patterns of E. coli O157 bovine strains. DNA samples were digested with XbaI, and the patterns were calculated with the Dice coefficient as described in Materials and Methods. A, B, C, and D are the main PFGE clusters.
    Figure Legend Snippet: Dendrogram of PFGE patterns of E. coli O157 bovine strains. DNA samples were digested with XbaI, and the patterns were calculated with the Dice coefficient as described in Materials and Methods. A, B, C, and D are the main PFGE clusters.

    Techniques Used:

    12) Product Images from "First Nosocomial Outbreak of Pseudomonas aeruginosa Producing an Integron-Borne Metallo-?-Lactamase (VIM-2) in the United States"

    Article Title: First Nosocomial Outbreak of Pseudomonas aeruginosa Producing an Integron-Borne Metallo-?-Lactamase (VIM-2) in the United States

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.49.8.3538-3540.2005

    PFGE of XbaI-digested chromosomal DNA. Lanes 1 and 8, lambda markers; lane 2, P. aeruginosa 7052; lane 3, case no. 2; lane 4, case no. 3; lane 5, case no. 4; lane 6, case no. 5; lane 7, case no. 6.
    Figure Legend Snippet: PFGE of XbaI-digested chromosomal DNA. Lanes 1 and 8, lambda markers; lane 2, P. aeruginosa 7052; lane 3, case no. 2; lane 4, case no. 3; lane 5, case no. 4; lane 6, case no. 5; lane 7, case no. 6.

    Techniques Used:

    13) Product Images from "Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes"

    Article Title: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-016-0234-4

    Coinjection of Cas9 mRNA and Cas9 protein into zygotes. a Strategy for insertion of a Venus reporter into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 0.8 kb fragments used as homology arms in the pR26-Venus targeting vector. The locations of PCR primers in the targeted and wildtype Rosa26 alleles are indicated. SA- splice acceptor, pA polyadenylation site. b Mouse zygotes were microinjected with pR26-Venus, sgRosa26-1 and Cas9 mRNA or Cas9 mRNA and protein. The embryos were cultured for 4 days and genomic DNA was isolated from 12 blastocysts each, for PCR-based detection of HDR or deletion events. Top panel: gel electrophoresis of PCR products. Targeted alleles (KI) are detected by amplification of a 1.3 kb genomic segment using the vector-specific primer VenusF and the R26R3 primer, located downstream of the vector homology region. The presence of integrated or nonintegrated vector DNA was tested using the R26F2/R2 primer pair, amplifying a 1.4 kb vector segment as well as 0.2 kb of the Rosa26 target region (middle panel). Lower panel: Rosa26 alleles with sequence deletions were detected by 0.2 kb of the target region (R26F2/R26R2 primers), followed by XbaI digestion and gel separation. XbaI resistant PCR products indicate the presence of sequence deletions (mut, 0.2 kb) whereas wildtype products are reduced to 0.12 kb fragments (wt). c Sequencing of PCR products amplified with primers VenusF and R26R3 (from B, top) showed the predicted recombination between the targeting vectors homology region and adjacent downstream genomic sequence
    Figure Legend Snippet: Coinjection of Cas9 mRNA and Cas9 protein into zygotes. a Strategy for insertion of a Venus reporter into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 0.8 kb fragments used as homology arms in the pR26-Venus targeting vector. The locations of PCR primers in the targeted and wildtype Rosa26 alleles are indicated. SA- splice acceptor, pA polyadenylation site. b Mouse zygotes were microinjected with pR26-Venus, sgRosa26-1 and Cas9 mRNA or Cas9 mRNA and protein. The embryos were cultured for 4 days and genomic DNA was isolated from 12 blastocysts each, for PCR-based detection of HDR or deletion events. Top panel: gel electrophoresis of PCR products. Targeted alleles (KI) are detected by amplification of a 1.3 kb genomic segment using the vector-specific primer VenusF and the R26R3 primer, located downstream of the vector homology region. The presence of integrated or nonintegrated vector DNA was tested using the R26F2/R2 primer pair, amplifying a 1.4 kb vector segment as well as 0.2 kb of the Rosa26 target region (middle panel). Lower panel: Rosa26 alleles with sequence deletions were detected by 0.2 kb of the target region (R26F2/R26R2 primers), followed by XbaI digestion and gel separation. XbaI resistant PCR products indicate the presence of sequence deletions (mut, 0.2 kb) whereas wildtype products are reduced to 0.12 kb fragments (wt). c Sequencing of PCR products amplified with primers VenusF and R26R3 (from B, top) showed the predicted recombination between the targeting vectors homology region and adjacent downstream genomic sequence

    Techniques Used: Introduce, Plasmid Preparation, Polymerase Chain Reaction, Cell Culture, Isolation, Nucleic Acid Electrophoresis, Amplification, Sequencing

    CRISPR/Cas9 induced DSBs at the Rosa26 intronic XbaI site in mouse zygotes. a : Diagram of the mouse Rosa26 locus. The sgRosa26-1 target sequence upstream of the protospacer adjacent motif (PAM) and the XbaI site within the first intron are indicated. The locations of primers used for nested PCR are shown (1. PCR: R26F1/R26R1, 2. PCR: R26F2/R26R2). b : In vitro blastocyst assay: zygotes microinjected with Cas9 mRNA and sgRosa26-1 RNA were cultured for 4 days to blastocysts. Genomic DNA was extracted from each blastocyst and used for PCR amplification of the target region and genotyping by XbaI or T7 endonuclease I (T7EI). c : Agarose gel electrophoresis of 0.2 kb PCR products amplified with the R26F2/R26R2 primer pair from blastocysts derived from microinjected zygotes (25 ng/μl sgRosa26-1 and 50 ng/μl Cas9 mRNA) (top). PCR products were either digested with XbaI (middle) or with T7EI (bottom). XbaI resistant 0.2 kb or T7EI sensitive 0.1 kb bands (arrows) indicate the presence of modified Rosa26 alleles harboring sequence deletions. WT – wildtype control, M – size marker. d : Sequence comparison of cloned PCR products (from c ) amplified from blastocysts #4 - #7 (from B). Deleted nucleotides are shown as dashes, the sgRosa26-1 PAM sequence is shown in red. e : Frequency of blastocysts showing NHEJ-based mutagenesis as indicated by the presence of XbaI resistant Rosa26 PCR products, in relation to the concentrations of Cas9 and sgRosa26-1 RNAs used for the microinjection of zygotes
    Figure Legend Snippet: CRISPR/Cas9 induced DSBs at the Rosa26 intronic XbaI site in mouse zygotes. a : Diagram of the mouse Rosa26 locus. The sgRosa26-1 target sequence upstream of the protospacer adjacent motif (PAM) and the XbaI site within the first intron are indicated. The locations of primers used for nested PCR are shown (1. PCR: R26F1/R26R1, 2. PCR: R26F2/R26R2). b : In vitro blastocyst assay: zygotes microinjected with Cas9 mRNA and sgRosa26-1 RNA were cultured for 4 days to blastocysts. Genomic DNA was extracted from each blastocyst and used for PCR amplification of the target region and genotyping by XbaI or T7 endonuclease I (T7EI). c : Agarose gel electrophoresis of 0.2 kb PCR products amplified with the R26F2/R26R2 primer pair from blastocysts derived from microinjected zygotes (25 ng/μl sgRosa26-1 and 50 ng/μl Cas9 mRNA) (top). PCR products were either digested with XbaI (middle) or with T7EI (bottom). XbaI resistant 0.2 kb or T7EI sensitive 0.1 kb bands (arrows) indicate the presence of modified Rosa26 alleles harboring sequence deletions. WT – wildtype control, M – size marker. d : Sequence comparison of cloned PCR products (from c ) amplified from blastocysts #4 - #7 (from B). Deleted nucleotides are shown as dashes, the sgRosa26-1 PAM sequence is shown in red. e : Frequency of blastocysts showing NHEJ-based mutagenesis as indicated by the presence of XbaI resistant Rosa26 PCR products, in relation to the concentrations of Cas9 and sgRosa26-1 RNAs used for the microinjection of zygotes

    Techniques Used: CRISPR, Sequencing, Nested PCR, Polymerase Chain Reaction, In Vitro, Cell Culture, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Modification, Marker, Clone Assay, Non-Homologous End Joining, Mutagenesis

    Knock-in of a conditional Cas9 transgene into Rosa26 of C57BL/6 zygotes. a : Strategy for insertion of the CAG-loxPSTOPloxP-Cas9-IRES-EGFP cassette into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 4 kb fragments used as homology arms in the targeting vector. Homology-directed repair (HDR) leads to the insertion of the cassette into the genome. The locations of PCR primers, restriction sites and the Rosa26 hybridisation probe in the targeted and wildtype alleles are indicated. b : Gel electrophoresis of XbaI digested Rosa26 PCR products (R26F2/R2 primers) amplified from pups (#7-#46) derived from microinjections of targeting vector, sgRosa26-1 and Cas9 RNAs. 0.2 kb bands of XbaI resistant products (mut) indicate sequence deletions, wildtype products (wt) are reduced to 0.1 kb. M - size marker, B6 - C57BL/6 wildtype control. c : PCR detection of an internal segment of Cas9 in pups derived from microinjections using primers Cas9F/Cas9R (top). Bottom: three primer PCR for the simultaneous detection of the Rosa26 target region (R26F2/R2 primers, 0.2 kb) and of vector sequences (R26F2-SAR, 0.12 kb), showing that all samples harbor at least one nonrecombined Rosa26 allele. V – vector positive control, H 2 O – negative control. d : Cas9-positive mice (from b ) were further tested for correct knock-in (KI) into Rosa26 using a PCR reaction with a forward primer located outside of the 5′-homology region (R26F3) and a reverse primer located in transgene (SAR); the predicted band has a size of 1.38 kb (top). Bottom: DNA quality was controlled with a Cas9 internal PCR (Cas9F/R primers,0.38 kb). H 2 O – negative control. e : Southern blot analysis of EcoRI digested tail DNA from Cas9-positive mice (from b ) using an external Rosa26 -specific hybridization. Knock-in alleles are predicted to show a 6 kb band. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control. f : Genotyping PCR of 15 F1 pups derived from founder mutants #35 or #39 using the Cas9 internal primer pair Cas9F/R. g : Southern blot analysis of EcoRI digested tail DNA from two F1 pups using an external Rosa26 -specific hybridization probe. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control
    Figure Legend Snippet: Knock-in of a conditional Cas9 transgene into Rosa26 of C57BL/6 zygotes. a : Strategy for insertion of the CAG-loxPSTOPloxP-Cas9-IRES-EGFP cassette into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 4 kb fragments used as homology arms in the targeting vector. Homology-directed repair (HDR) leads to the insertion of the cassette into the genome. The locations of PCR primers, restriction sites and the Rosa26 hybridisation probe in the targeted and wildtype alleles are indicated. b : Gel electrophoresis of XbaI digested Rosa26 PCR products (R26F2/R2 primers) amplified from pups (#7-#46) derived from microinjections of targeting vector, sgRosa26-1 and Cas9 RNAs. 0.2 kb bands of XbaI resistant products (mut) indicate sequence deletions, wildtype products (wt) are reduced to 0.1 kb. M - size marker, B6 - C57BL/6 wildtype control. c : PCR detection of an internal segment of Cas9 in pups derived from microinjections using primers Cas9F/Cas9R (top). Bottom: three primer PCR for the simultaneous detection of the Rosa26 target region (R26F2/R2 primers, 0.2 kb) and of vector sequences (R26F2-SAR, 0.12 kb), showing that all samples harbor at least one nonrecombined Rosa26 allele. V – vector positive control, H 2 O – negative control. d : Cas9-positive mice (from b ) were further tested for correct knock-in (KI) into Rosa26 using a PCR reaction with a forward primer located outside of the 5′-homology region (R26F3) and a reverse primer located in transgene (SAR); the predicted band has a size of 1.38 kb (top). Bottom: DNA quality was controlled with a Cas9 internal PCR (Cas9F/R primers,0.38 kb). H 2 O – negative control. e : Southern blot analysis of EcoRI digested tail DNA from Cas9-positive mice (from b ) using an external Rosa26 -specific hybridization. Knock-in alleles are predicted to show a 6 kb band. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control. f : Genotyping PCR of 15 F1 pups derived from founder mutants #35 or #39 using the Cas9 internal primer pair Cas9F/R. g : Southern blot analysis of EcoRI digested tail DNA from two F1 pups using an external Rosa26 -specific hybridization probe. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control

    Techniques Used: Knock-In, Introduce, Plasmid Preparation, Polymerase Chain Reaction, Hybridization, Nucleic Acid Electrophoresis, Amplification, Derivative Assay, Sequencing, Marker, Positive Control, Negative Control, Mouse Assay, Southern Blot, Generated

    Cas9 is functional in B cells of Rosa26 LSL-Cas9 knock-in mice. a : Scheme of genome editing in primary mouse B cells using CRISPR/Cas9. Naive B cells from spleens of three individual heterozygous Rosa26 LSL-Cas9 F1 mice were isolated using CD43 depletion, treated with TAT-Cre and stimulated with LPS for 24 h. TAT-Cre/LPS treated B cells were transduced with retroviral particles co-expressing sgRosa26-1 and BFP to target the Rosa26 locus. One day later, the transduced B cells were selected with puromycin until day 5. b : FACS analysis of B cells (from a ) before (day 2) and after puromycin selection (day 5). The gate indicates the fraction (percentage) of successfully transduced BFP + cells. c : Gel electrophoresis of T7EI or XbaI digested PCR products (R26T7F/R26T7R primers) amplified from DNA of FACS sorted BFP + cells (from b ), indicating sequence deletions by the presence of T7EI sensitive or XbaI resistant bands (arrows)
    Figure Legend Snippet: Cas9 is functional in B cells of Rosa26 LSL-Cas9 knock-in mice. a : Scheme of genome editing in primary mouse B cells using CRISPR/Cas9. Naive B cells from spleens of three individual heterozygous Rosa26 LSL-Cas9 F1 mice were isolated using CD43 depletion, treated with TAT-Cre and stimulated with LPS for 24 h. TAT-Cre/LPS treated B cells were transduced with retroviral particles co-expressing sgRosa26-1 and BFP to target the Rosa26 locus. One day later, the transduced B cells were selected with puromycin until day 5. b : FACS analysis of B cells (from a ) before (day 2) and after puromycin selection (day 5). The gate indicates the fraction (percentage) of successfully transduced BFP + cells. c : Gel electrophoresis of T7EI or XbaI digested PCR products (R26T7F/R26T7R primers) amplified from DNA of FACS sorted BFP + cells (from b ), indicating sequence deletions by the presence of T7EI sensitive or XbaI resistant bands (arrows)

    Techniques Used: Functional Assay, Knock-In, Mouse Assay, CRISPR, Isolation, Transduction, Expressing, FACS, Selection, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing

    14) Product Images from "Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients"

    Article Title: Application of Pulsed Field Gel Electrophoresis for Study of Genetic Diversity in Mycobacterium tuberculosis Strains Isolated From Tuberculosis Patients

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.9963

    PFGE Genomic Patterns After Digestion With DraI and XbaI Restriction Enzymes M. DNA weight marker, 1. BCG; 2. M. tuberculosis H37Rv; 3-6 clinical isolates: DraI digests (left); XbaI digests (right)
    Figure Legend Snippet: PFGE Genomic Patterns After Digestion With DraI and XbaI Restriction Enzymes M. DNA weight marker, 1. BCG; 2. M. tuberculosis H37Rv; 3-6 clinical isolates: DraI digests (left); XbaI digests (right)

    Techniques Used: Marker

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Shifts in targeting of class switch recombination sites in mice that lack ? switch region tandem repeats or Msh2
    Article Snippet: .. For switch events located downstream of the SμTR, genomic DNAs were digested with EcoRI, HindIII, or XbaI, and circularized for real-time PCR using fluorescent SYBR green dye (Applied Biosystems). .. The pSμ/Sγ3 control plasmid provided standard curves for each real-time DC-PCR.

    Multiplex Assay:

    Article Title: Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum
    Article Snippet: .. The DNA was digested and end-labeled using a five-enzyme, four-color labeling kit consisting of (i) four 6 bp restriction endonucleases, BamHI, Hind III, XbaI and XhoI, producing four different 3′ recessed ends to be labeled, (ii) one 4 bp restriction endonuclease, HaeIII, producing blunted ends and (iii) the SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems). .. The ends of the fragments produced by digestion with HindIII, BamHI, XbaI and XhoI were labeled with ddATP–dR6G, ddGTP–dR110, ddCTP–dTAMRA and ddTTP–dROX, respectively, and HaeIII was used to digest the labeled fragments for fractionation by CE.

    Agarose Gel Electrophoresis:

    Article Title: Reevaluating the Serotype II Capsular Locus of Streptococcus agalactiae ▿
    Article Snippet: .. PCR products were purified by using a high pure PCR product purification kit (Roche, Manheim, Germany), simultaneously digested with 10 U of MspI and 10 U of XbaI in Tango buffer (all from Fermentas, Vilnius, Lithuania) and separated in a 2% agarose gel. .. The RFLP patterns of three isolates of each serotype, representing distinct pulsed-field gel electrophoresis clusters, revealed the expected digestion profile discriminating the various serotypes, with the exception of isolates presenting serotype II (Fig. ), in which both the size of the cpsG - neuA region and the digestion pattern differed from those predicted.

    Labeling:

    Article Title: Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum
    Article Snippet: .. The DNA was digested and end-labeled using a five-enzyme, four-color labeling kit consisting of (i) four 6 bp restriction endonucleases, BamHI, Hind III, XbaI and XhoI, producing four different 3′ recessed ends to be labeled, (ii) one 4 bp restriction endonuclease, HaeIII, producing blunted ends and (iii) the SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems). .. The ends of the fragments produced by digestion with HindIII, BamHI, XbaI and XhoI were labeled with ddATP–dR6G, ddGTP–dR110, ddCTP–dTAMRA and ddTTP–dROX, respectively, and HaeIII was used to digest the labeled fragments for fractionation by CE.

    Purification:

    Article Title: Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains
    Article Snippet: .. Ln. mesenteroides phage P774 DNA was digested with XbaI according to the supplier's (Fermentas, St. Leon-Rot, Germany) recommendations and was subsequently purified by a PCR purification kit (Macherey-Nagel, Düren, Germany) (phage P774 XbaI profile; see ). .. Fragments were cloned into pSTBlue-1 (Novagen/Merck Millipore, Nottingham, United Kingdom).

    Article Title: Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application
    Article Snippet: .. After vector propagation and purification with QIAprep spin Miniprep Kit (QIAGEN, Hilden, Germany), the IA-2ic construct was released with SmaI and XbaI and ligated to the pTrxFus linearized vector (Invitrogen, Carlsbad, CA, USA) to yield pTrxIA-2ic (Fig. ). .. The quality of the new vector encoding the fusion protein TrxIA-2ic was corroborated by sequencing (Macrogen Inc, Seoul, Korea).

    Article Title: Reevaluating the Serotype II Capsular Locus of Streptococcus agalactiae ▿
    Article Snippet: .. PCR products were purified by using a high pure PCR product purification kit (Roche, Manheim, Germany), simultaneously digested with 10 U of MspI and 10 U of XbaI in Tango buffer (all from Fermentas, Vilnius, Lithuania) and separated in a 2% agarose gel. .. The RFLP patterns of three isolates of each serotype, representing distinct pulsed-field gel electrophoresis clusters, revealed the expected digestion profile discriminating the various serotypes, with the exception of isolates presenting serotype II (Fig. ), in which both the size of the cpsG - neuA region and the digestion pattern differed from those predicted.

    SYBR Green Assay:

    Article Title: Shifts in targeting of class switch recombination sites in mice that lack ? switch region tandem repeats or Msh2
    Article Snippet: .. For switch events located downstream of the SμTR, genomic DNAs were digested with EcoRI, HindIII, or XbaI, and circularized for real-time PCR using fluorescent SYBR green dye (Applied Biosystems). .. The pSμ/Sγ3 control plasmid provided standard curves for each real-time DC-PCR.

    Polymerase Chain Reaction:

    Article Title: Classification of Lytic Bacteriophages Attacking Dairy Leuconostoc Starter Strains
    Article Snippet: .. Ln. mesenteroides phage P774 DNA was digested with XbaI according to the supplier's (Fermentas, St. Leon-Rot, Germany) recommendations and was subsequently purified by a PCR purification kit (Macherey-Nagel, Düren, Germany) (phage P774 XbaI profile; see ). .. Fragments were cloned into pSTBlue-1 (Novagen/Merck Millipore, Nottingham, United Kingdom).

    Article Title: Impact of UGT1A9 Polymorphism on Mycophenolic Acid Pharmacokinetic Parameters in Stable Renal Transplant Patients
    Article Snippet: .. The PCR products for T-275A, C-2152T and UGT1A9*3 were digested with XbaI (Fermentas), MseI (Fermentas) and StyI (Fermentas), respectively. .. Digestion conditions were as follows: 10 μL of PCR mixture, 3 μL of buffer (10X), 1 μL of enzyme and 16 μL of de-ionized water to reach the final volume of 30 μL.

    Article Title: Reevaluating the Serotype II Capsular Locus of Streptococcus agalactiae ▿
    Article Snippet: .. PCR products were purified by using a high pure PCR product purification kit (Roche, Manheim, Germany), simultaneously digested with 10 U of MspI and 10 U of XbaI in Tango buffer (all from Fermentas, Vilnius, Lithuania) and separated in a 2% agarose gel. .. The RFLP patterns of three isolates of each serotype, representing distinct pulsed-field gel electrophoresis clusters, revealed the expected digestion profile discriminating the various serotypes, with the exception of isolates presenting serotype II (Fig. ), in which both the size of the cpsG - neuA region and the digestion pattern differed from those predicted.

    Construct:

    Article Title: Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application
    Article Snippet: .. After vector propagation and purification with QIAprep spin Miniprep Kit (QIAGEN, Hilden, Germany), the IA-2ic construct was released with SmaI and XbaI and ligated to the pTrxFus linearized vector (Invitrogen, Carlsbad, CA, USA) to yield pTrxIA-2ic (Fig. ). .. The quality of the new vector encoding the fusion protein TrxIA-2ic was corroborated by sequencing (Macrogen Inc, Seoul, Korea).

    IA:

    Article Title: Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application
    Article Snippet: .. After vector propagation and purification with QIAprep spin Miniprep Kit (QIAGEN, Hilden, Germany), the IA-2ic construct was released with SmaI and XbaI and ligated to the pTrxFus linearized vector (Invitrogen, Carlsbad, CA, USA) to yield pTrxIA-2ic (Fig. ). .. The quality of the new vector encoding the fusion protein TrxIA-2ic was corroborated by sequencing (Macrogen Inc, Seoul, Korea).

    Plasmid Preparation:

    Article Title: Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application
    Article Snippet: .. After vector propagation and purification with QIAprep spin Miniprep Kit (QIAGEN, Hilden, Germany), the IA-2ic construct was released with SmaI and XbaI and ligated to the pTrxFus linearized vector (Invitrogen, Carlsbad, CA, USA) to yield pTrxIA-2ic (Fig. ). .. The quality of the new vector encoding the fusion protein TrxIA-2ic was corroborated by sequencing (Macrogen Inc, Seoul, Korea).

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli
    Article Snippet: .. The Modular Sender Vector and each HSL synthase oligo were cut with EcoRI and XbaI (Thermo Fisher Scientific) and ligated using T4 ligase (New England Biolabs). ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher xbai
    Southern blot analysis of the four STS markers- CtR-431, CtR-594., CtR-496, and AFLP-376. Restriction enzymes used are <t>EcoRI,</t> HinDIII and <t>XbaI</t> . M, molecular weight marker; R resistant genotype Punjab Lal; S susceptible genotype Arka Lohit. Hybridization
    Xbai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 534 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xbai/product/Thermo Fisher
    Average 99 stars, based on 534 article reviews
    Price from $9.99 to $1999.99
    xbai - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Southern blot analysis of the four STS markers- CtR-431, CtR-594., CtR-496, and AFLP-376. Restriction enzymes used are EcoRI, HinDIII and XbaI . M, molecular weight marker; R resistant genotype Punjab Lal; S susceptible genotype Arka Lohit. Hybridization

    Journal: 3 Biotech

    Article Title: Sequence-tagged site-based diagnostic markers linked to a novel anthracnose resistance gene RCt1 in chili pepper (Capsicum annuum L.)

    doi: 10.1007/s13205-018-1552-0

    Figure Lengend Snippet: Southern blot analysis of the four STS markers- CtR-431, CtR-594., CtR-496, and AFLP-376. Restriction enzymes used are EcoRI, HinDIII and XbaI . M, molecular weight marker; R resistant genotype Punjab Lal; S susceptible genotype Arka Lohit. Hybridization

    Article Snippet: Three different restriction endonucleases- EcoRI, HinDIII and XbaI (Thermo Fischer Scientific, USA) were used to digest the genomic DNA from the resistant line ‘Punjab Lal’ and the susceptible line ‘Arka Lohit’.

    Techniques: Southern Blot, Molecular Weight, Marker, Hybridization

    Coinjection of Cas9 mRNA and Cas9 protein into zygotes. a Strategy for insertion of a Venus reporter into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 0.8 kb fragments used as homology arms in the pR26-Venus targeting vector. The locations of PCR primers in the targeted and wildtype Rosa26 alleles are indicated. SA- splice acceptor, pA polyadenylation site. b Mouse zygotes were microinjected with pR26-Venus, sgRosa26-1 and Cas9 mRNA or Cas9 mRNA and protein. The embryos were cultured for 4 days and genomic DNA was isolated from 12 blastocysts each, for PCR-based detection of HDR or deletion events. Top panel: gel electrophoresis of PCR products. Targeted alleles (KI) are detected by amplification of a 1.3 kb genomic segment using the vector-specific primer VenusF and the R26R3 primer, located downstream of the vector homology region. The presence of integrated or nonintegrated vector DNA was tested using the R26F2/R2 primer pair, amplifying a 1.4 kb vector segment as well as 0.2 kb of the Rosa26 target region (middle panel). Lower panel: Rosa26 alleles with sequence deletions were detected by 0.2 kb of the target region (R26F2/R26R2 primers), followed by XbaI digestion and gel separation. XbaI resistant PCR products indicate the presence of sequence deletions (mut, 0.2 kb) whereas wildtype products are reduced to 0.12 kb fragments (wt). c Sequencing of PCR products amplified with primers VenusF and R26R3 (from B, top) showed the predicted recombination between the targeting vectors homology region and adjacent downstream genomic sequence

    Journal: BMC Biotechnology

    Article Title: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

    doi: 10.1186/s12896-016-0234-4

    Figure Lengend Snippet: Coinjection of Cas9 mRNA and Cas9 protein into zygotes. a Strategy for insertion of a Venus reporter into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 0.8 kb fragments used as homology arms in the pR26-Venus targeting vector. The locations of PCR primers in the targeted and wildtype Rosa26 alleles are indicated. SA- splice acceptor, pA polyadenylation site. b Mouse zygotes were microinjected with pR26-Venus, sgRosa26-1 and Cas9 mRNA or Cas9 mRNA and protein. The embryos were cultured for 4 days and genomic DNA was isolated from 12 blastocysts each, for PCR-based detection of HDR or deletion events. Top panel: gel electrophoresis of PCR products. Targeted alleles (KI) are detected by amplification of a 1.3 kb genomic segment using the vector-specific primer VenusF and the R26R3 primer, located downstream of the vector homology region. The presence of integrated or nonintegrated vector DNA was tested using the R26F2/R2 primer pair, amplifying a 1.4 kb vector segment as well as 0.2 kb of the Rosa26 target region (middle panel). Lower panel: Rosa26 alleles with sequence deletions were detected by 0.2 kb of the target region (R26F2/R26R2 primers), followed by XbaI digestion and gel separation. XbaI resistant PCR products indicate the presence of sequence deletions (mut, 0.2 kb) whereas wildtype products are reduced to 0.12 kb fragments (wt). c Sequencing of PCR products amplified with primers VenusF and R26R3 (from B, top) showed the predicted recombination between the targeting vectors homology region and adjacent downstream genomic sequence

    Article Snippet: For the RLFP assay, PCR products were digested with the restriction enzyme XbaI (Thermo Scientific).

    Techniques: Introduce, Plasmid Preparation, Polymerase Chain Reaction, Cell Culture, Isolation, Nucleic Acid Electrophoresis, Amplification, Sequencing

    CRISPR/Cas9 induced DSBs at the Rosa26 intronic XbaI site in mouse zygotes. a : Diagram of the mouse Rosa26 locus. The sgRosa26-1 target sequence upstream of the protospacer adjacent motif (PAM) and the XbaI site within the first intron are indicated. The locations of primers used for nested PCR are shown (1. PCR: R26F1/R26R1, 2. PCR: R26F2/R26R2). b : In vitro blastocyst assay: zygotes microinjected with Cas9 mRNA and sgRosa26-1 RNA were cultured for 4 days to blastocysts. Genomic DNA was extracted from each blastocyst and used for PCR amplification of the target region and genotyping by XbaI or T7 endonuclease I (T7EI). c : Agarose gel electrophoresis of 0.2 kb PCR products amplified with the R26F2/R26R2 primer pair from blastocysts derived from microinjected zygotes (25 ng/μl sgRosa26-1 and 50 ng/μl Cas9 mRNA) (top). PCR products were either digested with XbaI (middle) or with T7EI (bottom). XbaI resistant 0.2 kb or T7EI sensitive 0.1 kb bands (arrows) indicate the presence of modified Rosa26 alleles harboring sequence deletions. WT – wildtype control, M – size marker. d : Sequence comparison of cloned PCR products (from c ) amplified from blastocysts #4 - #7 (from B). Deleted nucleotides are shown as dashes, the sgRosa26-1 PAM sequence is shown in red. e : Frequency of blastocysts showing NHEJ-based mutagenesis as indicated by the presence of XbaI resistant Rosa26 PCR products, in relation to the concentrations of Cas9 and sgRosa26-1 RNAs used for the microinjection of zygotes

    Journal: BMC Biotechnology

    Article Title: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

    doi: 10.1186/s12896-016-0234-4

    Figure Lengend Snippet: CRISPR/Cas9 induced DSBs at the Rosa26 intronic XbaI site in mouse zygotes. a : Diagram of the mouse Rosa26 locus. The sgRosa26-1 target sequence upstream of the protospacer adjacent motif (PAM) and the XbaI site within the first intron are indicated. The locations of primers used for nested PCR are shown (1. PCR: R26F1/R26R1, 2. PCR: R26F2/R26R2). b : In vitro blastocyst assay: zygotes microinjected with Cas9 mRNA and sgRosa26-1 RNA were cultured for 4 days to blastocysts. Genomic DNA was extracted from each blastocyst and used for PCR amplification of the target region and genotyping by XbaI or T7 endonuclease I (T7EI). c : Agarose gel electrophoresis of 0.2 kb PCR products amplified with the R26F2/R26R2 primer pair from blastocysts derived from microinjected zygotes (25 ng/μl sgRosa26-1 and 50 ng/μl Cas9 mRNA) (top). PCR products were either digested with XbaI (middle) or with T7EI (bottom). XbaI resistant 0.2 kb or T7EI sensitive 0.1 kb bands (arrows) indicate the presence of modified Rosa26 alleles harboring sequence deletions. WT – wildtype control, M – size marker. d : Sequence comparison of cloned PCR products (from c ) amplified from blastocysts #4 - #7 (from B). Deleted nucleotides are shown as dashes, the sgRosa26-1 PAM sequence is shown in red. e : Frequency of blastocysts showing NHEJ-based mutagenesis as indicated by the presence of XbaI resistant Rosa26 PCR products, in relation to the concentrations of Cas9 and sgRosa26-1 RNAs used for the microinjection of zygotes

    Article Snippet: For the RLFP assay, PCR products were digested with the restriction enzyme XbaI (Thermo Scientific).

    Techniques: CRISPR, Sequencing, Nested PCR, Polymerase Chain Reaction, In Vitro, Cell Culture, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Modification, Marker, Clone Assay, Non-Homologous End Joining, Mutagenesis

    Knock-in of a conditional Cas9 transgene into Rosa26 of C57BL/6 zygotes. a : Strategy for insertion of the CAG-loxPSTOPloxP-Cas9-IRES-EGFP cassette into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 4 kb fragments used as homology arms in the targeting vector. Homology-directed repair (HDR) leads to the insertion of the cassette into the genome. The locations of PCR primers, restriction sites and the Rosa26 hybridisation probe in the targeted and wildtype alleles are indicated. b : Gel electrophoresis of XbaI digested Rosa26 PCR products (R26F2/R2 primers) amplified from pups (#7-#46) derived from microinjections of targeting vector, sgRosa26-1 and Cas9 RNAs. 0.2 kb bands of XbaI resistant products (mut) indicate sequence deletions, wildtype products (wt) are reduced to 0.1 kb. M - size marker, B6 - C57BL/6 wildtype control. c : PCR detection of an internal segment of Cas9 in pups derived from microinjections using primers Cas9F/Cas9R (top). Bottom: three primer PCR for the simultaneous detection of the Rosa26 target region (R26F2/R2 primers, 0.2 kb) and of vector sequences (R26F2-SAR, 0.12 kb), showing that all samples harbor at least one nonrecombined Rosa26 allele. V – vector positive control, H 2 O – negative control. d : Cas9-positive mice (from b ) were further tested for correct knock-in (KI) into Rosa26 using a PCR reaction with a forward primer located outside of the 5′-homology region (R26F3) and a reverse primer located in transgene (SAR); the predicted band has a size of 1.38 kb (top). Bottom: DNA quality was controlled with a Cas9 internal PCR (Cas9F/R primers,0.38 kb). H 2 O – negative control. e : Southern blot analysis of EcoRI digested tail DNA from Cas9-positive mice (from b ) using an external Rosa26 -specific hybridization. Knock-in alleles are predicted to show a 6 kb band. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control. f : Genotyping PCR of 15 F1 pups derived from founder mutants #35 or #39 using the Cas9 internal primer pair Cas9F/R. g : Southern blot analysis of EcoRI digested tail DNA from two F1 pups using an external Rosa26 -specific hybridization probe. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control

    Journal: BMC Biotechnology

    Article Title: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

    doi: 10.1186/s12896-016-0234-4

    Figure Lengend Snippet: Knock-in of a conditional Cas9 transgene into Rosa26 of C57BL/6 zygotes. a : Strategy for insertion of the CAG-loxPSTOPloxP-Cas9-IRES-EGFP cassette into the mouse Rosa26 locus. sgRosa26-1 and Cas9 introduce a double-strand break between 1 kb and 4 kb fragments used as homology arms in the targeting vector. Homology-directed repair (HDR) leads to the insertion of the cassette into the genome. The locations of PCR primers, restriction sites and the Rosa26 hybridisation probe in the targeted and wildtype alleles are indicated. b : Gel electrophoresis of XbaI digested Rosa26 PCR products (R26F2/R2 primers) amplified from pups (#7-#46) derived from microinjections of targeting vector, sgRosa26-1 and Cas9 RNAs. 0.2 kb bands of XbaI resistant products (mut) indicate sequence deletions, wildtype products (wt) are reduced to 0.1 kb. M - size marker, B6 - C57BL/6 wildtype control. c : PCR detection of an internal segment of Cas9 in pups derived from microinjections using primers Cas9F/Cas9R (top). Bottom: three primer PCR for the simultaneous detection of the Rosa26 target region (R26F2/R2 primers, 0.2 kb) and of vector sequences (R26F2-SAR, 0.12 kb), showing that all samples harbor at least one nonrecombined Rosa26 allele. V – vector positive control, H 2 O – negative control. d : Cas9-positive mice (from b ) were further tested for correct knock-in (KI) into Rosa26 using a PCR reaction with a forward primer located outside of the 5′-homology region (R26F3) and a reverse primer located in transgene (SAR); the predicted band has a size of 1.38 kb (top). Bottom: DNA quality was controlled with a Cas9 internal PCR (Cas9F/R primers,0.38 kb). H 2 O – negative control. e : Southern blot analysis of EcoRI digested tail DNA from Cas9-positive mice (from b ) using an external Rosa26 -specific hybridization. Knock-in alleles are predicted to show a 6 kb band. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control. f : Genotyping PCR of 15 F1 pups derived from founder mutants #35 or #39 using the Cas9 internal primer pair Cas9F/R. g : Southern blot analysis of EcoRI digested tail DNA from two F1 pups using an external Rosa26 -specific hybridization probe. Control – DNA from a Rosa26 knock-in mouse generated from ES cells, C57BL/6 – wildtype control

    Article Snippet: For the RLFP assay, PCR products were digested with the restriction enzyme XbaI (Thermo Scientific).

    Techniques: Knock-In, Introduce, Plasmid Preparation, Polymerase Chain Reaction, Hybridization, Nucleic Acid Electrophoresis, Amplification, Derivative Assay, Sequencing, Marker, Positive Control, Negative Control, Mouse Assay, Southern Blot, Generated

    Cas9 is functional in B cells of Rosa26 LSL-Cas9 knock-in mice. a : Scheme of genome editing in primary mouse B cells using CRISPR/Cas9. Naive B cells from spleens of three individual heterozygous Rosa26 LSL-Cas9 F1 mice were isolated using CD43 depletion, treated with TAT-Cre and stimulated with LPS for 24 h. TAT-Cre/LPS treated B cells were transduced with retroviral particles co-expressing sgRosa26-1 and BFP to target the Rosa26 locus. One day later, the transduced B cells were selected with puromycin until day 5. b : FACS analysis of B cells (from a ) before (day 2) and after puromycin selection (day 5). The gate indicates the fraction (percentage) of successfully transduced BFP + cells. c : Gel electrophoresis of T7EI or XbaI digested PCR products (R26T7F/R26T7R primers) amplified from DNA of FACS sorted BFP + cells (from b ), indicating sequence deletions by the presence of T7EI sensitive or XbaI resistant bands (arrows)

    Journal: BMC Biotechnology

    Article Title: Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

    doi: 10.1186/s12896-016-0234-4

    Figure Lengend Snippet: Cas9 is functional in B cells of Rosa26 LSL-Cas9 knock-in mice. a : Scheme of genome editing in primary mouse B cells using CRISPR/Cas9. Naive B cells from spleens of three individual heterozygous Rosa26 LSL-Cas9 F1 mice were isolated using CD43 depletion, treated with TAT-Cre and stimulated with LPS for 24 h. TAT-Cre/LPS treated B cells were transduced with retroviral particles co-expressing sgRosa26-1 and BFP to target the Rosa26 locus. One day later, the transduced B cells were selected with puromycin until day 5. b : FACS analysis of B cells (from a ) before (day 2) and after puromycin selection (day 5). The gate indicates the fraction (percentage) of successfully transduced BFP + cells. c : Gel electrophoresis of T7EI or XbaI digested PCR products (R26T7F/R26T7R primers) amplified from DNA of FACS sorted BFP + cells (from b ), indicating sequence deletions by the presence of T7EI sensitive or XbaI resistant bands (arrows)

    Article Snippet: For the RLFP assay, PCR products were digested with the restriction enzyme XbaI (Thermo Scientific).

    Techniques: Functional Assay, Knock-In, Mouse Assay, CRISPR, Isolation, Transduction, Expressing, FACS, Selection, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing

    Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI -flanked synthase open reading frame (ORF) and co-expression with mCherry (mCh) from a single bicistronic mRNA. The vector is represented as SBOL [ 44 ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.

    Journal: PLoS ONE

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli

    doi: 10.1371/journal.pone.0202294

    Figure Lengend Snippet: Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI -flanked synthase open reading frame (ORF) and co-expression with mCherry (mCh) from a single bicistronic mRNA. The vector is represented as SBOL [ 44 ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.

    Article Snippet: The Modular Sender Vector and each HSL synthase oligo were cut with EcoRI and XbaI (Thermo Fisher Scientific) and ligated using T4 ligase (New England Biolabs).

    Techniques: Plasmid Preparation, Clone Assay, Expressing

    Southern blot of REFs and SRPPs in genomic DNA isolated from rubber tree clone RY 7-33-97. Endonucleases Eco R I, Hind III, Xba I, and Xho I were used for digestion of genomic DNA. DNA molecular mass markers are shown on the left.

    Journal: International Journal of Molecular Sciences

    Article Title: Comparative Proteomics of Rubber Latex Revealed Multiple Protein Species of REF/SRPP Family Respond Diversely to Ethylene Stimulation among Different Rubber Tree Clones

    doi: 10.3390/ijms18050958

    Figure Lengend Snippet: Southern blot of REFs and SRPPs in genomic DNA isolated from rubber tree clone RY 7-33-97. Endonucleases Eco R I, Hind III, Xba I, and Xho I were used for digestion of genomic DNA. DNA molecular mass markers are shown on the left.

    Article Snippet: H. brasiliensis genomic DNA was extracted from the leaves of rubber tree and digested with Bam H I, Eco R I, Hind III, and Xba I (Thermo Scientific, Guangzhou, China).

    Techniques: Southern Blot, Isolation