xhoi  (Thermo Fisher)


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  • 99
    Name:
    XhoI 10 U µL
    Description:
    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 Note Also available as a FastDigest enzyme for rapid DNA digestion 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:
    er0691
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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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 Note Also available as a FastDigest enzyme for rapid DNA digestion 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 262 article reviews
    Price from $9.99 to $1999.99
    xhoi - by Bioz Stars, 2020-11
    99/100 stars

    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

    2) 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

    3) Product Images from "Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris"

    Article Title: Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2014.08.005

    pJ915-rhChymase vector map The P. pastoris ] using 5’ XhoI and 3’ NotI restriction sites. The GAP promoter provides constitutive expression of the fusion protein and the plasmid contains a Zeocin resistance selectable marker gene. The plasmid is linearized at the SwaI restriction site prior to electroporation.
    Figure Legend Snippet: pJ915-rhChymase vector map The P. pastoris ] using 5’ XhoI and 3’ NotI restriction sites. The GAP promoter provides constitutive expression of the fusion protein and the plasmid contains a Zeocin resistance selectable marker gene. The plasmid is linearized at the SwaI restriction site prior to electroporation.

    Techniques Used: Plasmid Preparation, Expressing, Marker, Electroporation

    4) Product Images from "Expression and characterization of UL16 gene from duck enteritis virus"

    Article Title: Expression and characterization of UL16 gene from duck enteritis virus

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-413

    Identification of recombination vector pET 32b (+)-UL16 by restriction enzymes digestion . Lane M1, DNA marker; Lane 1, PCR products from pET 32b(+)-UL16; Lane 2, recombination plasmid pET 32b(+)-UL16 was digested with two restriction enzymes HindIII and XhoI; Lane M2, DNA marker.
    Figure Legend Snippet: Identification of recombination vector pET 32b (+)-UL16 by restriction enzymes digestion . Lane M1, DNA marker; Lane 1, PCR products from pET 32b(+)-UL16; Lane 2, recombination plasmid pET 32b(+)-UL16 was digested with two restriction enzymes HindIII and XhoI; Lane M2, DNA marker.

    Techniques Used: Plasmid Preparation, Positron Emission Tomography, Marker, Polymerase Chain Reaction

    5) Product Images from "Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel"

    Article Title: Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.49.3.1150-1156.2005

    Southern blot analysis of plasmid DNA from Enterobacter sp. strains 1061 and 1434 and their transconjugants. Plasmid DNA was digested with the SmaI, XhoI, and BamHI endonucleases in lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12, respectively, and hybridized
    Figure Legend Snippet: Southern blot analysis of plasmid DNA from Enterobacter sp. strains 1061 and 1434 and their transconjugants. Plasmid DNA was digested with the SmaI, XhoI, and BamHI endonucleases in lanes 1 to 3, 4 to 6, 7 to 9, and 10 to 12, respectively, and hybridized

    Techniques Used: Southern Blot, Plasmid Preparation

    6) Product Images from "Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01170-z

    Reducing heteroduplex formation in half-shRNA library. Workflow showing steps to reduce heteroduplex formation in half-shRNA library. Step 1-8: same as in Fig. 3 . Step 8: PCR purification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Magnetic bead-based purification of the barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, the electropherogram from the Bioanalyzer, read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. For half-shRNA, library is 108 bp excluding A and P1 sequence.
    Figure Legend Snippet: Reducing heteroduplex formation in half-shRNA library. Workflow showing steps to reduce heteroduplex formation in half-shRNA library. Step 1-8: same as in Fig. 3 . Step 8: PCR purification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Magnetic bead-based purification of the barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, the electropherogram from the Bioanalyzer, read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. For half-shRNA, library is 108 bp excluding A and P1 sequence.

    Techniques Used: shRNA, Polymerase Chain Reaction, Purification, Amplification, Ligation, Sequencing, Agarose Gel Electrophoresis

    Elimination of the hairpin in shRNA library. Workflow showing steps to eliminate hairpin in the shRNA library. Step 1: XhoI digestion of purified PCR amplicon from gDNA. Step 2: PCR purification of XhoI- digested product. Step 3: Gel extraction of expected 316 bp XhoI- digested amplicon. Step 4: Preparation of the SalI adapter. Step 5: Adapter ligation to XhoI- digested amplicon. Step 6: PCR amplification of the ligated product. Step 7: PCR barcode labeling of the ligated product. Step 8: PCR amplification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Gel extraction of the 160 bp barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, electropherogram from the Bioanalyzer and read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. The half-shRNA library is 108 bp excluding A and P1 sequence.
    Figure Legend Snippet: Elimination of the hairpin in shRNA library. Workflow showing steps to eliminate hairpin in the shRNA library. Step 1: XhoI digestion of purified PCR amplicon from gDNA. Step 2: PCR purification of XhoI- digested product. Step 3: Gel extraction of expected 316 bp XhoI- digested amplicon. Step 4: Preparation of the SalI adapter. Step 5: Adapter ligation to XhoI- digested amplicon. Step 6: PCR amplification of the ligated product. Step 7: PCR barcode labeling of the ligated product. Step 8: PCR amplification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Gel extraction of the 160 bp barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, electropherogram from the Bioanalyzer and read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. The half-shRNA library is 108 bp excluding A and P1 sequence.

    Techniques Used: shRNA, Purification, Polymerase Chain Reaction, Amplification, Gel Extraction, Ligation, Labeling, Sequencing, Agarose Gel Electrophoresis

    7) Product Images from "Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli"

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

    Journal: BioMed Research International

    doi: 10.1155/2019/9308593

    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).
    Figure Legend 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).

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Software

    8) 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

    9) Product Images from "The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function"

    Article Title: The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04212-8

    CBX3 subfragments and lentiviral vectors. ( A ) Schematic representation of the CBX3 element with its CpG-sites (black bars) and deleted splice sites (red bars) as well as the CBX3 subfragments obtained by PCR (CBX3(1-339) - CBX3(340-508)) and the CBX3(503-679) subfragment generated by restriction digestion with XhoI and ApaI. ( B ) Third-generation self-inactivating (SIN) lentiviral constructs expressing an eGFP cDNA from the spleen focus forming virus (SFFV) promoter in the absence or presence of either the 1.5 kb A2UCOE, the 679 bp CBX3 or any of the CBX3 subfragments. Abbreviations: ΔLTR: Long terminal repeat harboring the SIN mutation in the U3 region of the LTR; ψ: extended encapsidation signal; RRE: Rev-response element; cPPT: central polypurine tract; GFP: (enhanced) green fluorescent protein; wPRE: woodchuck hepatitis virus post-transcriptional element.
    Figure Legend Snippet: CBX3 subfragments and lentiviral vectors. ( A ) Schematic representation of the CBX3 element with its CpG-sites (black bars) and deleted splice sites (red bars) as well as the CBX3 subfragments obtained by PCR (CBX3(1-339) - CBX3(340-508)) and the CBX3(503-679) subfragment generated by restriction digestion with XhoI and ApaI. ( B ) Third-generation self-inactivating (SIN) lentiviral constructs expressing an eGFP cDNA from the spleen focus forming virus (SFFV) promoter in the absence or presence of either the 1.5 kb A2UCOE, the 679 bp CBX3 or any of the CBX3 subfragments. Abbreviations: ΔLTR: Long terminal repeat harboring the SIN mutation in the U3 region of the LTR; ψ: extended encapsidation signal; RRE: Rev-response element; cPPT: central polypurine tract; GFP: (enhanced) green fluorescent protein; wPRE: woodchuck hepatitis virus post-transcriptional element.

    Techniques Used: Polymerase Chain Reaction, Generated, Construct, Expressing, Mutagenesis

    10) Product Images from "Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi"

    Article Title: Construction of a Novel DNA Vaccine Candidate encoding LmSTI1-PpSP42 Fusion Protein from Leishmania major and Phlebotomus papatasi Against Cutaneous Leishmaniasi

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).
    Figure Legend Snippet: A: Schematic diagram of pCLmSTI1Pp42 construct composed of pcDNA3.1(+) eukaryotic expression vector and in-frame L. major LMSTI1 and Ph. Papatasi SP42 genes. B: pCLmSTI1 double-digested with KpnI and EcoRI (expected digested fragments: ∼5400 bp and ∼1600 bp). C: pCLmSTI1Pp42 double-digested with KpnI and XhoI (expected digested fragments: ∼5400 bp and ∼2500 bp).

    Techniques Used: Construct, Expressing, Plasmid Preparation

    11) Product Images from "Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01170-z

    Reducing heteroduplex formation in half-shRNA library. Workflow showing steps to reduce heteroduplex formation in half-shRNA library. Step 1-8: same as in Fig. 3 . Step 8: PCR purification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Magnetic bead-based purification of the barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, the electropherogram from the Bioanalyzer, read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. For half-shRNA, library is 108 bp excluding A and P1 sequence.
    Figure Legend Snippet: Reducing heteroduplex formation in half-shRNA library. Workflow showing steps to reduce heteroduplex formation in half-shRNA library. Step 1-8: same as in Fig. 3 . Step 8: PCR purification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Magnetic bead-based purification of the barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, the electropherogram from the Bioanalyzer, read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. For half-shRNA, library is 108 bp excluding A and P1 sequence.

    Techniques Used: shRNA, Polymerase Chain Reaction, Purification, Amplification, Ligation, Sequencing, Agarose Gel Electrophoresis

    Elimination of the hairpin in shRNA library. Workflow showing steps to eliminate hairpin in the shRNA library. Step 1: XhoI digestion of purified PCR amplicon from gDNA. Step 2: PCR purification of XhoI- digested product. Step 3: Gel extraction of expected 316 bp XhoI- digested amplicon. Step 4: Preparation of the SalI adapter. Step 5: Adapter ligation to XhoI- digested amplicon. Step 6: PCR amplification of the ligated product. Step 7: PCR barcode labeling of the ligated product. Step 8: PCR amplification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Gel extraction of the 160 bp barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, electropherogram from the Bioanalyzer and read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. The half-shRNA library is 108 bp excluding A and P1 sequence.
    Figure Legend Snippet: Elimination of the hairpin in shRNA library. Workflow showing steps to eliminate hairpin in the shRNA library. Step 1: XhoI digestion of purified PCR amplicon from gDNA. Step 2: PCR purification of XhoI- digested product. Step 3: Gel extraction of expected 316 bp XhoI- digested amplicon. Step 4: Preparation of the SalI adapter. Step 5: Adapter ligation to XhoI- digested amplicon. Step 6: PCR amplification of the ligated product. Step 7: PCR barcode labeling of the ligated product. Step 8: PCR amplification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Gel extraction of the 160 bp barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, electropherogram from the Bioanalyzer and read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. The half-shRNA library is 108 bp excluding A and P1 sequence.

    Techniques Used: shRNA, Purification, Polymerase Chain Reaction, Amplification, Gel Extraction, Ligation, Labeling, Sequencing, Agarose Gel Electrophoresis

    12) Product Images from "Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin"

    Article Title: Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin

    Journal: Chemistry & Biology

    doi: 10.1016/j.chembiol.2015.05.010

    One-Step Cloning and Heterologous Expression of the Conglobatin Gene Cluster (A) A 41-kbp XhoI-EcoRI DNA fragment (black) containing the five genes congA–E is generated by XhoI and EcoRI digestion of total genomic DNA. A 5.3-kbp pSET152 fragment was obtained by PCR amplification using as template the pSET152-derived plasmid pIB139. The resulting linear vector fragment had 39- and 41-bp flanking regions, respectively, identical to the termini of the target DNA (see Experimental Procedures ). (B) Gibson assembly leads to specific cloning of the target fragment, to give the bifunctional E. coli - Streptomyces plasmid pYJ24. The deduced open reading frame functions in the fragment are given in Table S1 . (C) Heterologous expression in S. coelicolor M1154 is confirmed by HPLC-MS and comparison with authentic compound produced by S. conglobatus (see also Figure S1 ). The mass extraction of m / z 499–500 is used to display the data. The y axis scale of S. conglobatus is 20 times larger than that of pYJ24/M1154 or M1154.
    Figure Legend Snippet: One-Step Cloning and Heterologous Expression of the Conglobatin Gene Cluster (A) A 41-kbp XhoI-EcoRI DNA fragment (black) containing the five genes congA–E is generated by XhoI and EcoRI digestion of total genomic DNA. A 5.3-kbp pSET152 fragment was obtained by PCR amplification using as template the pSET152-derived plasmid pIB139. The resulting linear vector fragment had 39- and 41-bp flanking regions, respectively, identical to the termini of the target DNA (see Experimental Procedures ). (B) Gibson assembly leads to specific cloning of the target fragment, to give the bifunctional E. coli - Streptomyces plasmid pYJ24. The deduced open reading frame functions in the fragment are given in Table S1 . (C) Heterologous expression in S. coelicolor M1154 is confirmed by HPLC-MS and comparison with authentic compound produced by S. conglobatus (see also Figure S1 ). The mass extraction of m / z 499–500 is used to display the data. The y axis scale of S. conglobatus is 20 times larger than that of pYJ24/M1154 or M1154.

    Techniques Used: Clone Assay, Expressing, Generated, Polymerase Chain Reaction, Amplification, Derivative Assay, Plasmid Preparation, High Performance Liquid Chromatography, Mass Spectrometry, Produced

    13) Product Images from "SVEP1 as a Genetic Modifier of TEK-Related Primary Congenital Glaucoma"

    Article Title: SVEP1 as a Genetic Modifier of TEK-Related Primary Congenital Glaucoma

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.61.12.6

    Exon trapping of the TEK c.1624+5G > A donor splice site variant leads to skipping of exon 11. ( a ) Reference and variant mini-genes were generated by incorporating genomic regions of TEK gene exon 11 into the pSPL3 exon trapping vector via XhoI and NdeI restriction sites. Vector exons VE1 and VE2 are depicted as black boxes and TEK exon 11 is in gray. Reference (R) and variant (V) splicing products are indicated by dashed lines above and below the construct, respectively. The location of the splice site variant is shown (*). ( b ) Gel electrophoresis of RT-PCR products from transfected Cos-7 cells, using vector exon-specific primers (indicated by half-arrows in a). pSPL3, cDNA template from cells transfected with empty pSPL3 vector only; No TF, cDNA template from cells transfected without plasmid DNA. TEK mRNA reference sequence: NM_000459.4.
    Figure Legend Snippet: Exon trapping of the TEK c.1624+5G > A donor splice site variant leads to skipping of exon 11. ( a ) Reference and variant mini-genes were generated by incorporating genomic regions of TEK gene exon 11 into the pSPL3 exon trapping vector via XhoI and NdeI restriction sites. Vector exons VE1 and VE2 are depicted as black boxes and TEK exon 11 is in gray. Reference (R) and variant (V) splicing products are indicated by dashed lines above and below the construct, respectively. The location of the splice site variant is shown (*). ( b ) Gel electrophoresis of RT-PCR products from transfected Cos-7 cells, using vector exon-specific primers (indicated by half-arrows in a). pSPL3, cDNA template from cells transfected with empty pSPL3 vector only; No TF, cDNA template from cells transfected without plasmid DNA. TEK mRNA reference sequence: NM_000459.4.

    Techniques Used: Variant Assay, Generated, Plasmid Preparation, Construct, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Transfection, Sequencing

    14) Product Images from "Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli"

    Article Title: Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli

    Journal: Reports of Biochemistry & Molecular Biology

    doi:

    A) Schematic view of the hspX/esxS construct. HindIII and XhoI restriction sites were added at the 5’ and 3´ ends, respectively, the hspX and esxS genes are 435 and 294 bps in length, respectively, and the 15-amino acid flexible linker was 2x GGGGS. B) HspX/EsxS fusion protein profile. Amino acid composition; length, molecular weight, and tertiary structure are shown. The fusion protein tertiary structure was predicted by I-TASSER; the blue band (LYS1-THR145) is HspX, the green band (GLY159-GLU260) is EsxS, and the yellow band (THR145-GLY159) is the linker.
    Figure Legend Snippet: A) Schematic view of the hspX/esxS construct. HindIII and XhoI restriction sites were added at the 5’ and 3´ ends, respectively, the hspX and esxS genes are 435 and 294 bps in length, respectively, and the 15-amino acid flexible linker was 2x GGGGS. B) HspX/EsxS fusion protein profile. Amino acid composition; length, molecular weight, and tertiary structure are shown. The fusion protein tertiary structure was predicted by I-TASSER; the blue band (LYS1-THR145) is HspX, the green band (GLY159-GLU260) is EsxS, and the yellow band (THR145-GLY159) is the linker.

    Techniques Used: Construct, Molecular Weight

    15) Product Images from "Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01170-z

    Reducing heteroduplex formation in half-shRNA library. Workflow showing steps to reduce heteroduplex formation in half-shRNA library. Step 1-8: same as in Fig. 3 . Step 8: PCR purification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Magnetic bead-based purification of the barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, the electropherogram from the Bioanalyzer, read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. For half-shRNA, library is 108 bp excluding A and P1 sequence.
    Figure Legend Snippet: Reducing heteroduplex formation in half-shRNA library. Workflow showing steps to reduce heteroduplex formation in half-shRNA library. Step 1-8: same as in Fig. 3 . Step 8: PCR purification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Magnetic bead-based purification of the barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, the electropherogram from the Bioanalyzer, read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. For half-shRNA, library is 108 bp excluding A and P1 sequence.

    Techniques Used: shRNA, Polymerase Chain Reaction, Purification, Amplification, Ligation, Sequencing, Agarose Gel Electrophoresis

    Elimination of the hairpin in shRNA library. Workflow showing steps to eliminate hairpin in the shRNA library. Step 1: XhoI digestion of purified PCR amplicon from gDNA. Step 2: PCR purification of XhoI- digested product. Step 3: Gel extraction of expected 316 bp XhoI- digested amplicon. Step 4: Preparation of the SalI adapter. Step 5: Adapter ligation to XhoI- digested amplicon. Step 6: PCR amplification of the ligated product. Step 7: PCR barcode labeling of the ligated product. Step 8: PCR amplification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Gel extraction of the 160 bp barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, electropherogram from the Bioanalyzer and read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. The half-shRNA library is 108 bp excluding A and P1 sequence.
    Figure Legend Snippet: Elimination of the hairpin in shRNA library. Workflow showing steps to eliminate hairpin in the shRNA library. Step 1: XhoI digestion of purified PCR amplicon from gDNA. Step 2: PCR purification of XhoI- digested product. Step 3: Gel extraction of expected 316 bp XhoI- digested amplicon. Step 4: Preparation of the SalI adapter. Step 5: Adapter ligation to XhoI- digested amplicon. Step 6: PCR amplification of the ligated product. Step 7: PCR barcode labeling of the ligated product. Step 8: PCR amplification of the barcoded PCR product. Step 9: XhoI digestion of the amplicon from XhoI self-ligated product. Step 10: PCR amplification after digestion of amplicon from XhoI self-ligation. Step 11: Gel extraction of the 160 bp barcoded product. Step 12: Quality assessment of the barcoded product on the Bioanalyzer. Step 13: Half-shRNA library sequencing on Ion Torrent platform. Representative agarose gel, electropherogram from the Bioanalyzer and read-length histogram from Ion Torrent are shown. The sequence length is plotted in the X-axis and the frequency is plotted in the Y-axis. The half-shRNA library is 108 bp excluding A and P1 sequence.

    Techniques Used: shRNA, Purification, Polymerase Chain Reaction, Amplification, Gel Extraction, Ligation, Labeling, Sequencing, Agarose Gel Electrophoresis

    16) Product Images from "Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030851

    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.
    Figure Legend 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.

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

    17) 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

    18) Product Images from "Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris"

    Article Title: Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2014.08.005

    pJ915-rhChymase vector map The P. pastoris ] using 5’ XhoI and 3’ NotI restriction sites. The GAP promoter provides constitutive expression of the fusion protein and the plasmid contains a Zeocin resistance selectable marker gene. The plasmid is linearized at the SwaI restriction site prior to electroporation.
    Figure Legend Snippet: pJ915-rhChymase vector map The P. pastoris ] using 5’ XhoI and 3’ NotI restriction sites. The GAP promoter provides constitutive expression of the fusion protein and the plasmid contains a Zeocin resistance selectable marker gene. The plasmid is linearized at the SwaI restriction site prior to electroporation.

    Techniques Used: Plasmid Preparation, Expressing, Marker, Electroporation

    19) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155 ▿Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155 ▿ †"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155 ▿Kaposi's Sarcoma-Associated Herpesvirus Encodes an Ortholog of miR-155 ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01804-07

    Seed match site 2 is essential for combinatorial regulation of BACH-1 by miR-155 and miR-K12-11. (A) Schematic of the BACH-1 3′UTR with potential miRNA seed match binding sites (sites 1 to 4) and restriction enzyme sites. “A” to “D” are truncation mutants generated by restriction enzyme digestion. (B) Mapping of miRNA binding sites. BACH-1 fragments A to D were inserted into pGL3-Promoter, and 40 ng of each reporter was cotransfected with 10 ng pEF-RL and 800 ng either pcDNA3.1, pmiR-155, or pmiR-K12-11 into 293 cells. (C) Site-directed mutagenesis to validate mapping data. Individual or double seed match sites within pGL3-BACH-1 were mutated to an XhoI site. Forty nanograms of each reporter was cotransfected with 10 ng pEF-RL and 800 ng miRNA expression vectors as described above (B). For B and C, lysates were harvested at 72 h, and relative light units (RLU) were normalized to Renilla ). The binding position within the 3′UTR and the minimum free hybridization energy (mfe) for each miRNA are shown.
    Figure Legend Snippet: Seed match site 2 is essential for combinatorial regulation of BACH-1 by miR-155 and miR-K12-11. (A) Schematic of the BACH-1 3′UTR with potential miRNA seed match binding sites (sites 1 to 4) and restriction enzyme sites. “A” to “D” are truncation mutants generated by restriction enzyme digestion. (B) Mapping of miRNA binding sites. BACH-1 fragments A to D were inserted into pGL3-Promoter, and 40 ng of each reporter was cotransfected with 10 ng pEF-RL and 800 ng either pcDNA3.1, pmiR-155, or pmiR-K12-11 into 293 cells. (C) Site-directed mutagenesis to validate mapping data. Individual or double seed match sites within pGL3-BACH-1 were mutated to an XhoI site. Forty nanograms of each reporter was cotransfected with 10 ng pEF-RL and 800 ng miRNA expression vectors as described above (B). For B and C, lysates were harvested at 72 h, and relative light units (RLU) were normalized to Renilla ). The binding position within the 3′UTR and the minimum free hybridization energy (mfe) for each miRNA are shown.

    Techniques Used: Binding Assay, Generated, Mutagenesis, Expressing, Hybridization

    20) Product Images from "Introducing cloned genes into cultured neurons providing novel in vitro models for neuropathology and neurotoxicity studies"

    Article Title: Introducing cloned genes into cultured neurons providing novel in vitro models for neuropathology and neurotoxicity studies

    Journal: Neuromethods

    doi: 10.1007/978-1-61779-077-5_9

    Insert-vector ligation and strategies to avoid recircularization. A) Ligation reaction of an EcoRI cleaved DNA fragment (red) in an EcoRI cleaved vector (black) without further treatment. Self-circularization of vector is highly favored. B) Second cleavage of vector and insert with XhoI make incompatible both ends of the vector, avoiding self-circularization. C) Vector dephosphorylation with alkaline phosphatase eliminates 5’phosphate groups at the ends of the vector, necessary for recircularization. Note that in B) , as well as in C) , the only possible ligation reaction is between vector and insert.
    Figure Legend Snippet: Insert-vector ligation and strategies to avoid recircularization. A) Ligation reaction of an EcoRI cleaved DNA fragment (red) in an EcoRI cleaved vector (black) without further treatment. Self-circularization of vector is highly favored. B) Second cleavage of vector and insert with XhoI make incompatible both ends of the vector, avoiding self-circularization. C) Vector dephosphorylation with alkaline phosphatase eliminates 5’phosphate groups at the ends of the vector, necessary for recircularization. Note that in B) , as well as in C) , the only possible ligation reaction is between vector and insert.

    Techniques Used: Plasmid Preparation, Ligation, De-Phosphorylation Assay

    21) Product Images from "Induction of a robust immune response against avian influenza virus following transdermal inoculation with H5-DNA vaccine formulated in modified dendrimer-based delivery system in mouse model"

    Article Title: Induction of a robust immune response against avian influenza virus following transdermal inoculation with H5-DNA vaccine formulated in modified dendrimer-based delivery system in mouse model

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S139126

    Analysis of the inserted gene (H5-GFP) by using restriction enzyme digestion and polymerase chain reaction. Notes: M: GeneRulerTM DNA ladder Kit, 1 Kbp (Fermentas, Burlington, Canada); lane 1, 2, 3: single digestion with XhoI; lane 4, 5: double digestion with NotI and XhoI. Single digestion of pBud-H5-GFP with XhoI produced a single fragment of about 7,672 bp, which correlate with the expected size of the recombinant DNA plasmid. Double digestion of the plasmid with NotI and XhoI, resulted in two fragments, 4,546 and 3,126 bp related to pBud CE4.1 and the H5-GFP gene, respectively. Abbreviations: DNA, deoxyribonucleic acid; GFP, green fluorescent protein.
    Figure Legend Snippet: Analysis of the inserted gene (H5-GFP) by using restriction enzyme digestion and polymerase chain reaction. Notes: M: GeneRulerTM DNA ladder Kit, 1 Kbp (Fermentas, Burlington, Canada); lane 1, 2, 3: single digestion with XhoI; lane 4, 5: double digestion with NotI and XhoI. Single digestion of pBud-H5-GFP with XhoI produced a single fragment of about 7,672 bp, which correlate with the expected size of the recombinant DNA plasmid. Double digestion of the plasmid with NotI and XhoI, resulted in two fragments, 4,546 and 3,126 bp related to pBud CE4.1 and the H5-GFP gene, respectively. Abbreviations: DNA, deoxyribonucleic acid; GFP, green fluorescent protein.

    Techniques Used: Polymerase Chain Reaction, Produced, Recombinant, Plasmid Preparation

    Related Articles

    Clone Assay:

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

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: .. The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately. .. The digestion products were electro-phoresed and purified from agarose gel by Bioneer DNA extraction kit (South Korea).

    Agarose Gel Electrophoresis:

    Article Title: Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel
    Article Snippet: .. Plasmid DNA was digested with SmaI, XhoI, and BamHI endonucleases (MBI Fermentas); and the resulting restriction pattern was visualized in a 1% agarose gel by ethidium bromide staining. .. DNA was transferred from the agarose gel to a positively charged Hybond N+ membrane (Amersham Biosciences, Little Chalfont, United Kingdom) and cross-linked with UV light.

    Southern Blot:

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing
    Article Snippet: .. Southern Blot of DNA Isolated from (A) Wild Type, (B) soh1Δ , and (C) ygl250wΔ SK1 Strains Containing the Ectopic URA3 -ARG4 Interval on Chromosome III (D) 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). (465 KB DOC) Click here for additional data file. .. FACS Analysis of Pre-Meiotic DNA Replication for VID21, BRE1, LGE1, RMD11, SGF73, and DEF1 Mutants Raw output from FACS analysis of each SK1 strain synchronized for entry into the meiotic cell cycle.

    Ligation:

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

    Isolation:

    Article Title: Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris
    Article Snippet: .. The DNA encoding human Chymase (hChymase) with an added N-terminal Kex2 protease cleavage site was isolated from the previously described plasmid pPICzα-rhChymase [ ] using simultaneous digestion with XhoI and NotI restriction endonucleases (Thermo Scientific, FastDigest). .. P. pastoris expression plasmid pJ915 (DNA 2.0), which provides secretion directed by α-mating factor and the GAP promoter for constitutive expression, was separately treated in the same manner.

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing
    Article Snippet: .. Southern Blot of DNA Isolated from (A) Wild Type, (B) soh1Δ , and (C) ygl250wΔ SK1 Strains Containing the Ectopic URA3 -ARG4 Interval on Chromosome III (D) 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). (465 KB DOC) Click here for additional data file. .. FACS Analysis of Pre-Meiotic DNA Replication for VID21, BRE1, LGE1, RMD11, SGF73, and DEF1 Mutants Raw output from FACS analysis of each SK1 strain synchronized for entry into the meiotic cell cycle.

    Marker:

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

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing
    Article Snippet: .. Southern Blot of DNA Isolated from (A) Wild Type, (B) soh1Δ , and (C) ygl250wΔ SK1 Strains Containing the Ectopic URA3 -ARG4 Interval on Chromosome III (D) 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). (465 KB DOC) Click here for additional data file. .. FACS Analysis of Pre-Meiotic DNA Replication for VID21, BRE1, LGE1, RMD11, SGF73, and DEF1 Mutants Raw output from FACS analysis of each SK1 strain synchronized for entry into the meiotic cell cycle.

    Polymerase Chain Reaction:

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

    Molecular Weight:

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

    Article Title: Novel Roles for Selected Genes in Meiotic DNA Processing
    Article Snippet: .. Southern Blot of DNA Isolated from (A) Wild Type, (B) soh1Δ , and (C) ygl250wΔ SK1 Strains Containing the Ectopic URA3 -ARG4 Interval on Chromosome III (D) 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). (465 KB DOC) Click here for additional data file. .. FACS Analysis of Pre-Meiotic DNA Replication for VID21, BRE1, LGE1, RMD11, SGF73, and DEF1 Mutants Raw output from FACS analysis of each SK1 strain synchronized for entry into the meiotic cell cycle.

    Expressing:

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

    Sequencing:

    Article Title: Expression and characterization of UL16 gene from duck enteritis virus
    Article Snippet: .. The T-clone plasmid, pMD18-T- UL16, was digested with the endonucleases HindIII and XhoI, and the UL16 target sequence was subcloned into the same multicloning sites of pET32b (+) (Invitrogen). .. The recombinant plasmid pET32b-UL16 was transformed into Escherichia coli Rossetta (DE3).

    Staining:

    Article Title: Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel
    Article Snippet: .. Plasmid DNA was digested with SmaI, XhoI, and BamHI endonucleases (MBI Fermentas); and the resulting restriction pattern was visualized in a 1% agarose gel by ethidium bromide staining. .. DNA was transferred from the agarose gel to a positively charged Hybond N+ membrane (Amersham Biosciences, Little Chalfont, United Kingdom) and cross-linked with UV light.

    Plasmid Preparation:

    Article Title: Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel
    Article Snippet: .. Plasmid DNA was digested with SmaI, XhoI, and BamHI endonucleases (MBI Fermentas); and the resulting restriction pattern was visualized in a 1% agarose gel by ethidium bromide staining. .. DNA was transferred from the agarose gel to a positively charged Hybond N+ membrane (Amersham Biosciences, Little Chalfont, United Kingdom) and cross-linked with UV light.

    Article Title: Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris
    Article Snippet: .. The DNA encoding human Chymase (hChymase) with an added N-terminal Kex2 protease cleavage site was isolated from the previously described plasmid pPICzα-rhChymase [ ] using simultaneous digestion with XhoI and NotI restriction endonucleases (Thermo Scientific, FastDigest). .. P. pastoris expression plasmid pJ915 (DNA 2.0), which provides secretion directed by α-mating factor and the GAP promoter for constitutive expression, was separately treated in the same manner.

    Article Title: Expression and characterization of UL16 gene from duck enteritis virus
    Article Snippet: .. The T-clone plasmid, pMD18-T- UL16, was digested with the endonucleases HindIII and XhoI, and the UL16 target sequence was subcloned into the same multicloning sites of pET32b (+) (Invitrogen). .. The recombinant plasmid pET32b-UL16 was transformed into Escherichia coli Rossetta (DE3).

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression
    Article Snippet: .. The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately. .. The digestion products were electro-phoresed and purified from agarose gel by Bioneer DNA extraction kit (South Korea).

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    Thermo Fisher xhoi restriction enzymes
    (A) Digestion on extracted plasmid of one of positive clones with NheI and <t>XhoI</t> confirmed HBsAg fragment insertion in <t>pcDNA</t> vector. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested circular plasmid. Column 3: 696 bp and 5501 bp bands that confirmed cloning. (B) Digestion of recombinant plasmid with BglII enzyme. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested plasmid. Column 3: A 6197 bp linearized plasmid.
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    (A) Digestion on extracted plasmid of one of positive clones with NheI and XhoI confirmed HBsAg fragment insertion in pcDNA vector. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested circular plasmid. Column 3: 696 bp and 5501 bp bands that confirmed cloning. (B) Digestion of recombinant plasmid with BglII enzyme. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested plasmid. Column 3: A 6197 bp linearized plasmid.

    Journal: Research in Pharmaceutical Sciences

    Article Title: Exposition of hepatitis B surface antigen (HBsAg) on the surface of HEK293T cell and evaluation of its expression

    doi: 10.4103/1735-5362.192485

    Figure Lengend Snippet: (A) Digestion on extracted plasmid of one of positive clones with NheI and XhoI confirmed HBsAg fragment insertion in pcDNA vector. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested circular plasmid. Column 3: 696 bp and 5501 bp bands that confirmed cloning. (B) Digestion of recombinant plasmid with BglII enzyme. Column 1: Mix DNA ladder (Thermoscientific, USA). Column 2: Undigested plasmid. Column 3: A 6197 bp linearized plasmid.

    Article Snippet: The extracted plasmid of one of the resulted clones and pcDNA 3.1 Hygro (+) plasmid (Invitrogen, USA) were digested by NheI and XhoI restriction enzymes (Thermoscience, USA) separately.

    Techniques: Plasmid Preparation, Clone Assay, Recombinant

    pJ915-rhChymase vector map The P. pastoris ] using 5’ XhoI and 3’ NotI restriction sites. The GAP promoter provides constitutive expression of the fusion protein and the plasmid contains a Zeocin resistance selectable marker gene. The plasmid is linearized at the SwaI restriction site prior to electroporation.

    Journal: Protein expression and purification

    Article Title: Expression of Recombinant Human Mast Cell Chymase with Asn-linked Glycans in Glycoengineered Pichia pastoris

    doi: 10.1016/j.pep.2014.08.005

    Figure Lengend Snippet: pJ915-rhChymase vector map The P. pastoris ] using 5’ XhoI and 3’ NotI restriction sites. The GAP promoter provides constitutive expression of the fusion protein and the plasmid contains a Zeocin resistance selectable marker gene. The plasmid is linearized at the SwaI restriction site prior to electroporation.

    Article Snippet: The DNA encoding human Chymase (hChymase) with an added N-terminal Kex2 protease cleavage site was isolated from the previously described plasmid pPICzα-rhChymase [ ] using simultaneous digestion with XhoI and NotI restriction endonucleases (Thermo Scientific, FastDigest).

    Techniques: Plasmid Preparation, Expressing, Marker, Electroporation

    mtDNA replication is reduced in 16-cell cysts of homoplasmic mt:CoI T300I germaria at 29°C. ( a ) Mutated nucleotide and amino acid residue (red) in mt:CoI T300I , including XhoI site (yellow). ( b ) Eclosion rate (mean±s.d., from total > 50 animals in 5 groups) of wt, homoplasmic and heteroplasmic mt:CoI T300I animals at 18°C and 29°C. * indicates zero mt:CoI T300I eclosed at 29°C. ( c ) COX activities of wt and mt:CoI T300I flies were examined on consecutive days shifting to 29°C. Data represent 3 biological replicates. Values were normalized with average COX activities of wt at 29°C 0 day and shown as mean±s.d. ( d ) Quantification of EdU puncta (mean±s.d.) in posterior cyst of region 2B at 18°C (wt n= 7, mt:CoI T300I n= 7) or 29°C (wt n= 5, mt:CoI T300I n= 8). The number of EdU puncta in mt:CoI T300I at 29°C is significantly less than that at 18°C or wild type at 29°C ( P

    Journal: Nature genetics

    Article Title: Selective propagation of functional mtDNA during oogenesis restricts the transmission of a deleterious mitochondrial variant

    doi: 10.1038/ng.2920

    Figure Lengend Snippet: mtDNA replication is reduced in 16-cell cysts of homoplasmic mt:CoI T300I germaria at 29°C. ( a ) Mutated nucleotide and amino acid residue (red) in mt:CoI T300I , including XhoI site (yellow). ( b ) Eclosion rate (mean±s.d., from total > 50 animals in 5 groups) of wt, homoplasmic and heteroplasmic mt:CoI T300I animals at 18°C and 29°C. * indicates zero mt:CoI T300I eclosed at 29°C. ( c ) COX activities of wt and mt:CoI T300I flies were examined on consecutive days shifting to 29°C. Data represent 3 biological replicates. Values were normalized with average COX activities of wt at 29°C 0 day and shown as mean±s.d. ( d ) Quantification of EdU puncta (mean±s.d.) in posterior cyst of region 2B at 18°C (wt n= 7, mt:CoI T300I n= 7) or 29°C (wt n= 5, mt:CoI T300I n= 8). The number of EdU puncta in mt:CoI T300I at 29°C is significantly less than that at 18°C or wild type at 29°C ( P

    Article Snippet: Molecular confirmation and quantification of heteroplasmy A 4 kb mtDNA fragment spanning the XhoI site in mt:CoI was PCR amplified from total animal DNA as described previously , and purified using the Thermo Scientific gel purification kit.

    Techniques:

    Germline selection against mt:CoI T300I at the restrictive temperature. ( a ) Frequency of mt:CoI T300I mutation in heteroplasmic flies maintained at 29°C or 18°C over generations. ( b ) XhoI digestion of PCR fragment spanning mt:CoI , amplified from single larvae produced by the same heteroplasmic mother at 18°C or 29°C. ( c ) Proportion of mutant mtDNA in 10 single larvae at 18°C or 29°C, calculated by quantifying band intensity in b . Average level of mutant mtDNA, 18°C, 83±5%; 29°C, 60±9%, n=10, P

    Journal: Nature genetics

    Article Title: Selective propagation of functional mtDNA during oogenesis restricts the transmission of a deleterious mitochondrial variant

    doi: 10.1038/ng.2920

    Figure Lengend Snippet: Germline selection against mt:CoI T300I at the restrictive temperature. ( a ) Frequency of mt:CoI T300I mutation in heteroplasmic flies maintained at 29°C or 18°C over generations. ( b ) XhoI digestion of PCR fragment spanning mt:CoI , amplified from single larvae produced by the same heteroplasmic mother at 18°C or 29°C. ( c ) Proportion of mutant mtDNA in 10 single larvae at 18°C or 29°C, calculated by quantifying band intensity in b . Average level of mutant mtDNA, 18°C, 83±5%; 29°C, 60±9%, n=10, P

    Article Snippet: Molecular confirmation and quantification of heteroplasmy A 4 kb mtDNA fragment spanning the XhoI site in mt:CoI was PCR amplified from total animal DNA as described previously , and purified using the Thermo Scientific gel purification kit.

    Techniques: Selection, Mutagenesis, Polymerase Chain Reaction, Amplification, Produced

    CBX3 subfragments and lentiviral vectors. ( A ) Schematic representation of the CBX3 element with its CpG-sites (black bars) and deleted splice sites (red bars) as well as the CBX3 subfragments obtained by PCR (CBX3(1-339) - CBX3(340-508)) and the CBX3(503-679) subfragment generated by restriction digestion with XhoI and ApaI. ( B ) Third-generation self-inactivating (SIN) lentiviral constructs expressing an eGFP cDNA from the spleen focus forming virus (SFFV) promoter in the absence or presence of either the 1.5 kb A2UCOE, the 679 bp CBX3 or any of the CBX3 subfragments. Abbreviations: ΔLTR: Long terminal repeat harboring the SIN mutation in the U3 region of the LTR; ψ: extended encapsidation signal; RRE: Rev-response element; cPPT: central polypurine tract; GFP: (enhanced) green fluorescent protein; wPRE: woodchuck hepatitis virus post-transcriptional element.

    Journal: Scientific Reports

    Article Title: The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function

    doi: 10.1038/s41598-017-04212-8

    Figure Lengend Snippet: CBX3 subfragments and lentiviral vectors. ( A ) Schematic representation of the CBX3 element with its CpG-sites (black bars) and deleted splice sites (red bars) as well as the CBX3 subfragments obtained by PCR (CBX3(1-339) - CBX3(340-508)) and the CBX3(503-679) subfragment generated by restriction digestion with XhoI and ApaI. ( B ) Third-generation self-inactivating (SIN) lentiviral constructs expressing an eGFP cDNA from the spleen focus forming virus (SFFV) promoter in the absence or presence of either the 1.5 kb A2UCOE, the 679 bp CBX3 or any of the CBX3 subfragments. Abbreviations: ΔLTR: Long terminal repeat harboring the SIN mutation in the U3 region of the LTR; ψ: extended encapsidation signal; RRE: Rev-response element; cPPT: central polypurine tract; GFP: (enhanced) green fluorescent protein; wPRE: woodchuck hepatitis virus post-transcriptional element.

    Article Snippet: The CBX3(503-679) subfragment was generated by digesting the CBX3.SFFV.eGFP with XhoI and ApaI, filling the ends by using Klenow fragment (ThermoFisher Scientific) and subsequently self-ligating the vector to generate the construct CBX3(503-679).SFFV.eGFP.

    Techniques: Polymerase Chain Reaction, Generated, Construct, Expressing, Mutagenesis