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    XhoI
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    5 C ↓T C G A G 3 3 G A G C T ↑C 5 Thermo Scientific XhoI restriction enzyme recognizes C TCGAG sites and cuts best at 37°C in R 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 Isoschizomers PaeR7I Sfr274I SlaI StrI TliI 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:
    ER0693
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    Category:
    Proteins Enzymes Peptides
    Applications:
    Cloning|Restriction Enzyme Cloning
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    Structured Review

    Thermo Fisher xhoi
    Assessment of def1 Δ in Meiosis and Mitosis (A) Immunocytology of nuclear spreads of SK1 wild-type and def1Δ strains after 8 h of sporulation. The meiosis-specific subunit of cohesin, Rec8, was tagged with multiple Haemagglutinin (HA) epitopes. Using antibodies for HA and Zip1 allowed analysis of sister chormatid cohesion and synaptonemal complex formation, respectively. It can be seen in the wild-type example that all 16 chromosomes have long cohesin axes and close to full chromosome synapsis except for the rDNA region on Chromosome XII. Whereas from the first panel for def1Δ it can be seen that axes are aligned but synapsis is minimal. The second and third panels for def1Δ again show aligned axes, but homologues are only partially synapsed. However, as shown in the final panel, synapsis was observed in some meiotic nuclei of the def1Δ strain. Polycomplexes (PCs) of Zip1 were observed in 20% of the nuclei counted for def1Δ at this time point whereas less than 1% PCs were observed for the wild type. (B) Time course of the meiotic nuclei counted using immunocytology for both wild type and def1Δ during meiosis. The def1Δ mutant synapsis phenotype represented in (A) was counted as “aligned” axes in the Rec8 analysis graph. At least 200 nuclei were counted per time point. (C) Ectopic URA3 - ARG4 interval on Chromosome III described in Figure 3 . <t>XhoI</t> and EcoRI restriction sites are indicated by “X” and “E,” respectively. To detect NCOs, COs, and DSBs, <t>DNA</t> is digested with XhoI and EcoRI then probed with HIS4 sequences (hisU; [ 44 ]). For graphs (D–F), wild-type and def1Δ are represented by blue squares and pink diamonds, respectively. The corresponding XhoI and EcoRI double digest Southern blots, the XhoI single digest Southern blots, together with the molecular analyses, are presented in Figure S5 . (D) Molecular analysis for DSB (DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (E) Molecular analysis for NCO (NCO1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO (CO1') signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Southern blot of DNA isolated from wild-type and def1Δ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. DNA was digested with XhoI and EcoRI then probed to detect NCOs, COs, and DSBs; mw represents the 1-kb molecular weight marker (Fermentas).
    5 C ↓T C G A G 3 3 G A G C T ↑C 5 Thermo Scientific XhoI restriction enzyme recognizes C TCGAG sites and cuts best at 37°C in R 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 Isoschizomers PaeR7I Sfr274I SlaI StrI TliI 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/xhoi/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xhoi - by Bioz Stars, 2021-05
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    Images

    1) Product Images from "Novel Roles for Selected Genes in Meiotic DNA Processing"

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030222

    Assessment of def1 Δ in Meiosis and Mitosis (A) Immunocytology of nuclear spreads of SK1 wild-type and def1Δ strains after 8 h of sporulation. The meiosis-specific subunit of cohesin, Rec8, was tagged with multiple Haemagglutinin (HA) epitopes. Using antibodies for HA and Zip1 allowed analysis of sister chormatid cohesion and synaptonemal complex formation, respectively. It can be seen in the wild-type example that all 16 chromosomes have long cohesin axes and close to full chromosome synapsis except for the rDNA region on Chromosome XII. Whereas from the first panel for def1Δ it can be seen that axes are aligned but synapsis is minimal. The second and third panels for def1Δ again show aligned axes, but homologues are only partially synapsed. However, as shown in the final panel, synapsis was observed in some meiotic nuclei of the def1Δ strain. Polycomplexes (PCs) of Zip1 were observed in 20% of the nuclei counted for def1Δ at this time point whereas less than 1% PCs were observed for the wild type. (B) Time course of the meiotic nuclei counted using immunocytology for both wild type and def1Δ during meiosis. The def1Δ mutant synapsis phenotype represented in (A) was counted as “aligned” axes in the Rec8 analysis graph. At least 200 nuclei were counted per time point. (C) Ectopic URA3 - ARG4 interval on Chromosome III described in Figure 3 . XhoI and EcoRI restriction sites are indicated by “X” and “E,” respectively. To detect NCOs, COs, and DSBs, DNA is digested with XhoI and EcoRI then probed with HIS4 sequences (hisU; [ 44 ]). For graphs (D–F), wild-type and def1Δ are represented by blue squares and pink diamonds, respectively. The corresponding XhoI and EcoRI double digest Southern blots, the XhoI single digest Southern blots, together with the molecular analyses, are presented in Figure S5 . (D) Molecular analysis for DSB (DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (E) Molecular analysis for NCO (NCO1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO (CO1') signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Southern blot of DNA isolated from wild-type and def1Δ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. DNA was digested with XhoI and EcoRI then probed to detect NCOs, COs, and DSBs; mw represents the 1-kb molecular weight marker (Fermentas).
    Figure Legend Snippet: Assessment of def1 Δ in Meiosis and Mitosis (A) Immunocytology of nuclear spreads of SK1 wild-type and def1Δ strains after 8 h of sporulation. The meiosis-specific subunit of cohesin, Rec8, was tagged with multiple Haemagglutinin (HA) epitopes. Using antibodies for HA and Zip1 allowed analysis of sister chormatid cohesion and synaptonemal complex formation, respectively. It can be seen in the wild-type example that all 16 chromosomes have long cohesin axes and close to full chromosome synapsis except for the rDNA region on Chromosome XII. Whereas from the first panel for def1Δ it can be seen that axes are aligned but synapsis is minimal. The second and third panels for def1Δ again show aligned axes, but homologues are only partially synapsed. However, as shown in the final panel, synapsis was observed in some meiotic nuclei of the def1Δ strain. Polycomplexes (PCs) of Zip1 were observed in 20% of the nuclei counted for def1Δ at this time point whereas less than 1% PCs were observed for the wild type. (B) Time course of the meiotic nuclei counted using immunocytology for both wild type and def1Δ during meiosis. The def1Δ mutant synapsis phenotype represented in (A) was counted as “aligned” axes in the Rec8 analysis graph. At least 200 nuclei were counted per time point. (C) Ectopic URA3 - ARG4 interval on Chromosome III described in Figure 3 . XhoI and EcoRI restriction sites are indicated by “X” and “E,” respectively. To detect NCOs, COs, and DSBs, DNA is digested with XhoI and EcoRI then probed with HIS4 sequences (hisU; [ 44 ]). For graphs (D–F), wild-type and def1Δ are represented by blue squares and pink diamonds, respectively. The corresponding XhoI and EcoRI double digest Southern blots, the XhoI single digest Southern blots, together with the molecular analyses, are presented in Figure S5 . (D) Molecular analysis for DSB (DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (E) Molecular analysis for NCO (NCO1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO (CO1') signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Southern blot of DNA isolated from wild-type and def1Δ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. DNA was digested with XhoI and EcoRI then probed to detect NCOs, COs, and DSBs; mw represents the 1-kb molecular weight marker (Fermentas).

    Techniques Used: Mutagenesis, Southern Blot, Isolation, Molecular Weight, Marker

    Assessment of YGL250W , SOH1 , and BRE5 (A) Schematic representation of Chromosome II from the diploid W303 background which consists of two LYS2 heteroalleles ( lys2–5′nde I − and lys2–3′nde I − ). These were used to measure meiotic gene conversion (see Materials and Methods ). (B) Spot test of wild type, ygl250wΔ , soh1Δ , and bre5Δ on haploid selection plates and haploid selection plates without lysine to measure meiotic gene conversion. The reduction in meiotic gene conversion of ygl250wΔ , soh1Δ , and bre5Δ was further assessed by random spore analysis ( Table 1 ). (C) Southern blot of DNA isolated from wild type, soh1Δ , and ygl250wΔ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). The full-sized Southern blots are presented in Figure S1 . For graphs (D–G), wild type, ygl250wΔ , and soh1Δ are represented by black diamonds, black circles, and white squares, respectively. The corresponding XhoI-digested Southern blots are presented in Figure 3 C. (D) Pre-meiotic DNA replication was assessed for synchronized meiotic cultures by fluorescence-activated cell sorting (FACS) and the change from 2c to 4c DNA content was plotted over time. (E) Nuclear divisions (MI and MII) of the synchronized meiotic cultures in (E) were assessed with fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) staining to visualize nuclear division. (F) Molecular analysis for DSB (DSB1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO (CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures.
    Figure Legend Snippet: Assessment of YGL250W , SOH1 , and BRE5 (A) Schematic representation of Chromosome II from the diploid W303 background which consists of two LYS2 heteroalleles ( lys2–5′nde I − and lys2–3′nde I − ). These were used to measure meiotic gene conversion (see Materials and Methods ). (B) Spot test of wild type, ygl250wΔ , soh1Δ , and bre5Δ on haploid selection plates and haploid selection plates without lysine to measure meiotic gene conversion. The reduction in meiotic gene conversion of ygl250wΔ , soh1Δ , and bre5Δ was further assessed by random spore analysis ( Table 1 ). (C) Southern blot of DNA isolated from wild type, soh1Δ , and ygl250wΔ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). The full-sized Southern blots are presented in Figure S1 . For graphs (D–G), wild type, ygl250wΔ , and soh1Δ are represented by black diamonds, black circles, and white squares, respectively. The corresponding XhoI-digested Southern blots are presented in Figure 3 C. (D) Pre-meiotic DNA replication was assessed for synchronized meiotic cultures by fluorescence-activated cell sorting (FACS) and the change from 2c to 4c DNA content was plotted over time. (E) Nuclear divisions (MI and MII) of the synchronized meiotic cultures in (E) were assessed with fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) staining to visualize nuclear division. (F) Molecular analysis for DSB (DSB1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO (CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures.

    Techniques Used: Spot Test, Selection, Southern Blot, Isolation, Molecular Weight, Marker, Fluorescence, FACS, Microscopy, Staining

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
    Article Snippet: 2.1 Construction of expression plasmid To express the α-amylase protein with its fusion intein protein, the plasmid pTYB2 (New England BioLabs, Beverly, MA, USA) was used. .. P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania). .. The reaction was performed using 10 ng DNA, 2 μL (10 μM) of each primer, 5 μL (10mM) dNTPs, 5 μL 10× PCR buffer [100mM Tris-HCl (pH 8.9), 500mM KCl, 20mM MgCl2 , 1% Triton X-100] and High-fidelity PCR Enzyme Mix (Fermentas).

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System
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    Article Title: Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens
    Article Snippet: The SalI adapter contains a cohesive compatible end (with XhoI -digested product) and common complementary sequence for hybridizing to the Ion Torrent RP primer at a later step. .. For the ligation reaction, 1:3 molar ratio of XhoI -digested half-shRNA product and SalI adapter was used (600 ng of XhoI digested PCR product was mixed with 5 µl of SalI adapter (42.7 ng/µl)) with 5 µl of T4 DNA Ligase (Invitrogen), 200 µl of 5X Ligase Reaction Buffer (Invitrogen) and DNAase-free ddH2 O added to give a final volume of 1 ml, which was divided into 10 reactions. .. Reactions were incubated at 25 °C for 1 hour using an IsotempTM Incubator.

    Amplification:

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei
    Article Snippet: 2.1 Construction of expression plasmid To express the α-amylase protein with its fusion intein protein, the plasmid pTYB2 (New England BioLabs, Beverly, MA, USA) was used. .. P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania). .. The reaction was performed using 10 ng DNA, 2 μL (10 μM) of each primer, 5 μL (10mM) dNTPs, 5 μL 10× PCR buffer [100mM Tris-HCl (pH 8.9), 500mM KCl, 20mM MgCl2 , 1% Triton X-100] and High-fidelity PCR Enzyme Mix (Fermentas).

    Southern Blot:

    Article Title: Live-Cell Imaging of Vaccinia Virus Recombination
    Article Snippet: .. Southern blotting DNA was purified from virus-infected BSC-40 cells by phenol/chloroform extraction and ethanol precipitation, digested with Xho I or Hin dIII (Fermentas), and size fractionated on 0.7% agarose gels. .. The DNA was denatured in situ in a solution containing 0.5M NaOH and 1.5M NaCl, transferred to a nylon membrane (Pall Corporation), and fixed by UV cross-linking.

    Article Title: Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut
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    Purification:

    Article Title: Live-Cell Imaging of Vaccinia Virus Recombination
    Article Snippet: .. Southern blotting DNA was purified from virus-infected BSC-40 cells by phenol/chloroform extraction and ethanol precipitation, digested with Xho I or Hin dIII (Fermentas), and size fractionated on 0.7% agarose gels. .. The DNA was denatured in situ in a solution containing 0.5M NaOH and 1.5M NaCl, transferred to a nylon membrane (Pall Corporation), and fixed by UV cross-linking.

    Ethanol Precipitation:

    Article Title: Live-Cell Imaging of Vaccinia Virus Recombination
    Article Snippet: .. Southern blotting DNA was purified from virus-infected BSC-40 cells by phenol/chloroform extraction and ethanol precipitation, digested with Xho I or Hin dIII (Fermentas), and size fractionated on 0.7% agarose gels. .. The DNA was denatured in situ in a solution containing 0.5M NaOH and 1.5M NaCl, transferred to a nylon membrane (Pall Corporation), and fixed by UV cross-linking.

    Transformation Assay:

    Article Title: Expression of Pennisetum glaucum Eukaryotic Translational Initiation Factor 4A (PgeIF4A) Confers Improved Drought, Salinity, and Oxidative Stress Tolerance in Groundnut
    Article Snippet: The amplified products were electrophoressed on 1% agarose gel and visualized on gel documentation system (G-Box Syngene, UK). .. Copy number detection by southern blotting Genomic DNA (20 μg) from control and transformed plants (T0 ) was digested using 30 U of Xho I (Fermentas life sciences), single cutter of T-DNA and non-cutter of PgeIF4A gene. .. The digested samples were size fractionated on agarose gel (0.8%) for 16 h at 30 V. The depurinated and denatured DNA fragments were subsequently transferred to positively charged Hybond N+ nylon membrane (GE Health Care, USA).

    Plasmid Preparation:

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System
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    Clone Assay:

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System
    Article Snippet: The eGFP gene was mutated at codon 70 ( C AG > T AG) creating a stop codon and, at the same time, eliminating a BtsI restriction site (in order to be able to screen the corrected clones). .. The mutated and the wild-type gene were extracted from pCR-2.1 vector by restriction with XhoI and HindIII and cloned inside the pCEP4 vector (Invitrogen, CA, USA), between pCMV promoter and SV40-pA, using the same restriction enzymes. .. To create stable cell clones, 3 µg of each SgrAI linearized plasmid were added to 1.7×106 cells and once transfected cells were plated in 75 cm2 flask in fresh medium containing 200 µg/ml of hygromycin (Sigma-Aldrich, Milan, Italy) for stable vector integration selection.

    Article Title: Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli
    Article Snippet: For cloning and expression, different vectors were utilized, i.e., pTZ57R/T (Fermentas Inc., Ontario, Canada) and pET-28a (+) (Novagen, Madison, USA), respectively. .. NcoI and XhoI (Fermentas Inc., Ontario, Canada) restriction enzymes were used for restriction of cloning and expression vectors. .. However, InsTAclone™, TransformAid™, DNA/plasmid extraction, and BigDye Terminator v3.1 Cycle Sequencing kits utilized in the present study were obtained from Thermo Scientific.

    Ligation:

    Article Title: Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens
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    Molecular Weight:

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing
    Article Snippet: Southern blot of DNA isolated from SK1 wild-type (C) and def1Δ (D) strains containing the ectopic URA3 -ARG4 interval on Chromosome III described in . .. The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). (E) Molecular analysis for DSB (DSB1 + DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO1 signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO2 signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (H) Molecular analysis for CO (CO1 + CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (676 KB DOC) Click here for additional data file. .. Assessment of pmr1Δ (and hur1Δ ) in Mitosis and Meiosis (A) Diagram showing the 181-bp overlap between the open reading frames for HUR1 and PMR1 on Chromosome VII; two KANMX4 gene deletion mutants were constructed using cassettes that only interfere with either HUR1 (1–81 bp of HUR1 ORF, hur1Δ1–81 , represented by black region) or PMR1 (1–2,233 bp of PMR1 ORF, pmr1Δ1–2,233 , represented by grey region).

    Marker:

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing
    Article Snippet: Southern blot of DNA isolated from SK1 wild-type (C) and def1Δ (D) strains containing the ectopic URA3 -ARG4 interval on Chromosome III described in . .. The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). (E) Molecular analysis for DSB (DSB1 + DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO1 signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO2 signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (H) Molecular analysis for CO (CO1 + CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (676 KB DOC) Click here for additional data file. .. Assessment of pmr1Δ (and hur1Δ ) in Mitosis and Meiosis (A) Diagram showing the 181-bp overlap between the open reading frames for HUR1 and PMR1 on Chromosome VII; two KANMX4 gene deletion mutants were constructed using cassettes that only interfere with either HUR1 (1–81 bp of HUR1 ORF, hur1Δ1–81 , represented by black region) or PMR1 (1–2,233 bp of PMR1 ORF, pmr1Δ1–2,233 , represented by grey region).

    Expressing:

    Article Title: Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli
    Article Snippet: For cloning and expression, different vectors were utilized, i.e., pTZ57R/T (Fermentas Inc., Ontario, Canada) and pET-28a (+) (Novagen, Madison, USA), respectively. .. NcoI and XhoI (Fermentas Inc., Ontario, Canada) restriction enzymes were used for restriction of cloning and expression vectors. .. However, InsTAclone™, TransformAid™, DNA/plasmid extraction, and BigDye Terminator v3.1 Cycle Sequencing kits utilized in the present study were obtained from Thermo Scientific.

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    Thermo Fisher xhoi
    Experimental design for SDF and cell clone generation. A) SDF sequence is homologous to the entire wild type eGFP coding sequence. <t>SDF-PCR-WT,</t> 876 bp long was generated by PCR amplification with primer pair 1F/1R ( Table 1 ). SDF-DIG-WT, 752 bp long, was obtained by HindIII and <t>XhoI</t> digestion of pCR-2.1 vector. C/T transition, responsible of fluorescence switching off, is showed. B) Sequencing analysis showing wild type (WT; top panel) and mutated (Mut; bottom panel) pCEP4-eGFP in C1 and D1 cell clones, respectively. Arrows indicate the modified base (C→T). C) FACS density plot of C1 (WT; top) and D1 (Mut; bottom) respectively. D) pCEP4-eGFP copy number determination for each cell clone.
    Xhoi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xhoi/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xhoi - by Bioz Stars, 2021-05
    99/100 stars
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    Experimental design for SDF and cell clone generation. A) SDF sequence is homologous to the entire wild type eGFP coding sequence. SDF-PCR-WT, 876 bp long was generated by PCR amplification with primer pair 1F/1R ( Table 1 ). SDF-DIG-WT, 752 bp long, was obtained by HindIII and XhoI digestion of pCR-2.1 vector. C/T transition, responsible of fluorescence switching off, is showed. B) Sequencing analysis showing wild type (WT; top panel) and mutated (Mut; bottom panel) pCEP4-eGFP in C1 and D1 cell clones, respectively. Arrows indicate the modified base (C→T). C) FACS density plot of C1 (WT; top) and D1 (Mut; bottom) respectively. D) pCEP4-eGFP copy number determination for each cell clone.

    Journal: PLoS ONE

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System

    doi: 10.1371/journal.pone.0030851

    Figure Lengend Snippet: Experimental design for SDF and cell clone generation. A) SDF sequence is homologous to the entire wild type eGFP coding sequence. SDF-PCR-WT, 876 bp long was generated by PCR amplification with primer pair 1F/1R ( Table 1 ). SDF-DIG-WT, 752 bp long, was obtained by HindIII and XhoI digestion of pCR-2.1 vector. C/T transition, responsible of fluorescence switching off, is showed. B) Sequencing analysis showing wild type (WT; top panel) and mutated (Mut; bottom panel) pCEP4-eGFP in C1 and D1 cell clones, respectively. Arrows indicate the modified base (C→T). C) FACS density plot of C1 (WT; top) and D1 (Mut; bottom) respectively. D) pCEP4-eGFP copy number determination for each cell clone.

    Article Snippet: The mutated and the wild-type gene were extracted from pCR-2.1 vector by restriction with XhoI and HindIII and cloned inside the pCEP4 vector (Invitrogen, CA, USA), between pCMV promoter and SV40-pA, using the same restriction enzymes.

    Techniques: Sequencing, Polymerase Chain Reaction, Generated, Amplification, Plasmid Preparation, Fluorescence, Clone Assay, Modification, FACS

    Graphical representation of pET28a-bglA. Expression vector was created by insertion of bglA gene between NcoI and XhoI sites. Inserted sequence was adjacent to lac operator and terminates at 6xHis-tag included in the frame. Kan R : kanamycin resistance gene, Ori: origin of replication. The sketch was created using SnapGene® software (GSL Biotech).

    Journal: BioMed Research International

    Article Title: Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli

    doi: 10.1155/2019/9308593

    Figure Lengend Snippet: Graphical representation of pET28a-bglA. Expression vector was created by insertion of bglA gene between NcoI and XhoI sites. Inserted sequence was adjacent to lac operator and terminates at 6xHis-tag included in the frame. Kan R : kanamycin resistance gene, Ori: origin of replication. The sketch was created using SnapGene® software (GSL Biotech).

    Article Snippet: NcoI and XhoI (Fermentas Inc., Ontario, Canada) restriction enzymes were used for restriction of cloning and expression vectors.

    Techniques: Expressing, Plasmid Preparation, Sequencing, Software

    (A) Digestion of pTY-α-amylase with Nhe I and Xho I restriction enzymes. Lane 1: pTYB2 digested with Nhe I and Xho I; lane2: pTY-α-amylase digested with Nhe I and Xho I; lane 3: PCR product of α-amylase. (B) SDS-PAGE analysis of α-amylase expression. Lane 1: before IPTG induction; lane 2: after IPTG induction; lane 3: 16 hours after IPTG induction; lane 4: 20 hours after IPTG induction. (C) Western blot analysis of α-amylase with anti-intein polyclonal antibody. Lane1: α-amylase; lane 2: lysate of untransformed bacteria. IPTG = isopropyl- β-d-thiogalactopyranoside; M = molecular marker; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Journal: Osong Public Health and Research Perspectives

    Article Title: Cloning, Expression, and Purification of Hyperthermophile α-Amylase from Pyrococcus woesei

    doi: 10.1016/j.phrp.2015.10.003

    Figure Lengend Snippet: (A) Digestion of pTY-α-amylase with Nhe I and Xho I restriction enzymes. Lane 1: pTYB2 digested with Nhe I and Xho I; lane2: pTY-α-amylase digested with Nhe I and Xho I; lane 3: PCR product of α-amylase. (B) SDS-PAGE analysis of α-amylase expression. Lane 1: before IPTG induction; lane 2: after IPTG induction; lane 3: 16 hours after IPTG induction; lane 4: 20 hours after IPTG induction. (C) Western blot analysis of α-amylase with anti-intein polyclonal antibody. Lane1: α-amylase; lane 2: lysate of untransformed bacteria. IPTG = isopropyl- β-d-thiogalactopyranoside; M = molecular marker; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Article Snippet: P. woesei chromosomal DNA was used as a source of the α-amylase gene and polymerase chain reaction (PCR) amplification was done with the following primers: 5′- GCTAGC TTGGAGCTTGAAGAGGGAG-3′ and 5′- GAGACC ACAATAACTCCATACGGAG-3′ containing recognition sites for restriction endonucleases Nhe I and Xho I (Fermentas; Vilnius, Lithuania).

    Techniques: Polymerase Chain Reaction, SDS Page, Expressing, Western Blot, Marker, Polyacrylamide Gel Electrophoresis

    Assessment of def1 Δ in Meiosis and Mitosis (A) Immunocytology of nuclear spreads of SK1 wild-type and def1Δ strains after 8 h of sporulation. The meiosis-specific subunit of cohesin, Rec8, was tagged with multiple Haemagglutinin (HA) epitopes. Using antibodies for HA and Zip1 allowed analysis of sister chormatid cohesion and synaptonemal complex formation, respectively. It can be seen in the wild-type example that all 16 chromosomes have long cohesin axes and close to full chromosome synapsis except for the rDNA region on Chromosome XII. Whereas from the first panel for def1Δ it can be seen that axes are aligned but synapsis is minimal. The second and third panels for def1Δ again show aligned axes, but homologues are only partially synapsed. However, as shown in the final panel, synapsis was observed in some meiotic nuclei of the def1Δ strain. Polycomplexes (PCs) of Zip1 were observed in 20% of the nuclei counted for def1Δ at this time point whereas less than 1% PCs were observed for the wild type. (B) Time course of the meiotic nuclei counted using immunocytology for both wild type and def1Δ during meiosis. The def1Δ mutant synapsis phenotype represented in (A) was counted as “aligned” axes in the Rec8 analysis graph. At least 200 nuclei were counted per time point. (C) Ectopic URA3 - ARG4 interval on Chromosome III described in Figure 3 . XhoI and EcoRI restriction sites are indicated by “X” and “E,” respectively. To detect NCOs, COs, and DSBs, DNA is digested with XhoI and EcoRI then probed with HIS4 sequences (hisU; [ 44 ]). For graphs (D–F), wild-type and def1Δ are represented by blue squares and pink diamonds, respectively. The corresponding XhoI and EcoRI double digest Southern blots, the XhoI single digest Southern blots, together with the molecular analyses, are presented in Figure S5 . (D) Molecular analysis for DSB (DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (E) Molecular analysis for NCO (NCO1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO (CO1') signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Southern blot of DNA isolated from wild-type and def1Δ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. DNA was digested with XhoI and EcoRI then probed to detect NCOs, COs, and DSBs; mw represents the 1-kb molecular weight marker (Fermentas).

    Journal: PLoS Genetics

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing

    doi: 10.1371/journal.pgen.0030222

    Figure Lengend Snippet: Assessment of def1 Δ in Meiosis and Mitosis (A) Immunocytology of nuclear spreads of SK1 wild-type and def1Δ strains after 8 h of sporulation. The meiosis-specific subunit of cohesin, Rec8, was tagged with multiple Haemagglutinin (HA) epitopes. Using antibodies for HA and Zip1 allowed analysis of sister chormatid cohesion and synaptonemal complex formation, respectively. It can be seen in the wild-type example that all 16 chromosomes have long cohesin axes and close to full chromosome synapsis except for the rDNA region on Chromosome XII. Whereas from the first panel for def1Δ it can be seen that axes are aligned but synapsis is minimal. The second and third panels for def1Δ again show aligned axes, but homologues are only partially synapsed. However, as shown in the final panel, synapsis was observed in some meiotic nuclei of the def1Δ strain. Polycomplexes (PCs) of Zip1 were observed in 20% of the nuclei counted for def1Δ at this time point whereas less than 1% PCs were observed for the wild type. (B) Time course of the meiotic nuclei counted using immunocytology for both wild type and def1Δ during meiosis. The def1Δ mutant synapsis phenotype represented in (A) was counted as “aligned” axes in the Rec8 analysis graph. At least 200 nuclei were counted per time point. (C) Ectopic URA3 - ARG4 interval on Chromosome III described in Figure 3 . XhoI and EcoRI restriction sites are indicated by “X” and “E,” respectively. To detect NCOs, COs, and DSBs, DNA is digested with XhoI and EcoRI then probed with HIS4 sequences (hisU; [ 44 ]). For graphs (D–F), wild-type and def1Δ are represented by blue squares and pink diamonds, respectively. The corresponding XhoI and EcoRI double digest Southern blots, the XhoI single digest Southern blots, together with the molecular analyses, are presented in Figure S5 . (D) Molecular analysis for DSB (DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (E) Molecular analysis for NCO (NCO1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO (CO1') signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Southern blot of DNA isolated from wild-type and def1Δ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. DNA was digested with XhoI and EcoRI then probed to detect NCOs, COs, and DSBs; mw represents the 1-kb molecular weight marker (Fermentas).

    Article Snippet: The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). (E) Molecular analysis for DSB (DSB1 + DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO1 signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO2 signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (H) Molecular analysis for CO (CO1 + CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (676 KB DOC) Click here for additional data file.

    Techniques: Mutagenesis, Southern Blot, Isolation, Molecular Weight, Marker

    Assessment of YGL250W , SOH1 , and BRE5 (A) Schematic representation of Chromosome II from the diploid W303 background which consists of two LYS2 heteroalleles ( lys2–5′nde I − and lys2–3′nde I − ). These were used to measure meiotic gene conversion (see Materials and Methods ). (B) Spot test of wild type, ygl250wΔ , soh1Δ , and bre5Δ on haploid selection plates and haploid selection plates without lysine to measure meiotic gene conversion. The reduction in meiotic gene conversion of ygl250wΔ , soh1Δ , and bre5Δ was further assessed by random spore analysis ( Table 1 ). (C) Southern blot of DNA isolated from wild type, soh1Δ , and ygl250wΔ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). The full-sized Southern blots are presented in Figure S1 . For graphs (D–G), wild type, ygl250wΔ , and soh1Δ are represented by black diamonds, black circles, and white squares, respectively. The corresponding XhoI-digested Southern blots are presented in Figure 3 C. (D) Pre-meiotic DNA replication was assessed for synchronized meiotic cultures by fluorescence-activated cell sorting (FACS) and the change from 2c to 4c DNA content was plotted over time. (E) Nuclear divisions (MI and MII) of the synchronized meiotic cultures in (E) were assessed with fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) staining to visualize nuclear division. (F) Molecular analysis for DSB (DSB1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO (CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures.

    Journal: PLoS Genetics

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing

    doi: 10.1371/journal.pgen.0030222

    Figure Lengend Snippet: Assessment of YGL250W , SOH1 , and BRE5 (A) Schematic representation of Chromosome II from the diploid W303 background which consists of two LYS2 heteroalleles ( lys2–5′nde I − and lys2–3′nde I − ). These were used to measure meiotic gene conversion (see Materials and Methods ). (B) Spot test of wild type, ygl250wΔ , soh1Δ , and bre5Δ on haploid selection plates and haploid selection plates without lysine to measure meiotic gene conversion. The reduction in meiotic gene conversion of ygl250wΔ , soh1Δ , and bre5Δ was further assessed by random spore analysis ( Table 1 ). (C) Southern blot of DNA isolated from wild type, soh1Δ , and ygl250wΔ SK1 strains containing the ectopic URA3 - ARG4 interval on Chromosome III. The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). The full-sized Southern blots are presented in Figure S1 . For graphs (D–G), wild type, ygl250wΔ , and soh1Δ are represented by black diamonds, black circles, and white squares, respectively. The corresponding XhoI-digested Southern blots are presented in Figure 3 C. (D) Pre-meiotic DNA replication was assessed for synchronized meiotic cultures by fluorescence-activated cell sorting (FACS) and the change from 2c to 4c DNA content was plotted over time. (E) Nuclear divisions (MI and MII) of the synchronized meiotic cultures in (E) were assessed with fluorescence microscopy using 4′,6-diamidino-2-phenylindole (DAPI) staining to visualize nuclear division. (F) Molecular analysis for DSB (DSB1) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO (CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures.

    Article Snippet: The DNA from the indicated times after initiation of sporulation were digested with XhoI then probed to detect COs and DSBs; mw1 represents the λ-HindIII molecular weight marker (Fermentas) and mw2 represents the 1-kb molecular weight marker (Fermentas). (E) Molecular analysis for DSB (DSB1 + DSB2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (F) Molecular analysis for CO1 signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (G) Molecular analysis for CO2 signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (H) Molecular analysis for CO (CO1 + CO2) signal/total lane signal from Southern blots of DNA extracted from synchronized meiotic cultures. (676 KB DOC) Click here for additional data file.

    Techniques: Spot Test, Selection, Southern Blot, Isolation, Molecular Weight, Marker, Fluorescence, FACS, Microscopy, Staining