xbai  (Thermo Fisher)

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

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

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

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

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

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

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