ecori  (Thermo Fisher)


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  • 99
    Name:
    EcoRI
    Description:
    5'  G ↓A  A  T  T  C   3' 3'  C  T  T  A  A ↑G   5' Thermo Scientific EcoRI restriction enzyme recognizes G^AATTC sites and cuts best at 37°C in its own unique buffer. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes. Note: Also available as a FastDigest enzyme for rapid DNA digestion.Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that sent 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:
    ER0271
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Size:
    5 000 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, Restriction Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher ecori
    R17a replication in PFSK-1 cells. 2 µg low molecular weight <t>DNA</t> from PFSK-1 cells transfected with MCVSyn or R17a viral DNA was <t>EcoRI</t> and DpnI digested and separated on an agarose gel, followed by EtBr staining (left panel). The DNA was transferred via southern blotting and probed with a radioactively labelled LT-Ag PCR fragment (right panel). The blot was exposed for 24 h.
    5'  G ↓A  A  T  T  C   3' 3'  C  T  T  A  A ↑G   5' Thermo Scientific EcoRI restriction enzyme recognizes G^AATTC sites and cuts best at 37°C in its own unique buffer. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes. Note: Also available as a FastDigest enzyme for rapid DNA digestion.Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that sent 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/ecori/product/Thermo Fisher
    Average 99 stars, based on 209 article reviews
    Price from $9.99 to $1999.99
    ecori - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome"

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029112

    R17a replication in PFSK-1 cells. 2 µg low molecular weight DNA from PFSK-1 cells transfected with MCVSyn or R17a viral DNA was EcoRI and DpnI digested and separated on an agarose gel, followed by EtBr staining (left panel). The DNA was transferred via southern blotting and probed with a radioactively labelled LT-Ag PCR fragment (right panel). The blot was exposed for 24 h.
    Figure Legend Snippet: R17a replication in PFSK-1 cells. 2 µg low molecular weight DNA from PFSK-1 cells transfected with MCVSyn or R17a viral DNA was EcoRI and DpnI digested and separated on an agarose gel, followed by EtBr staining (left panel). The DNA was transferred via southern blotting and probed with a radioactively labelled LT-Ag PCR fragment (right panel). The blot was exposed for 24 h.

    Techniques Used: Molecular Weight, Transfection, Agarose Gel Electrophoresis, Staining, Southern Blot, Polymerase Chain Reaction

    SV40 replication and gene expression in CV-1 cells transfected with SV40 DNA. (A) 100 ng of intramolecular religated SV40 viral DNA was transfected in CV-1 cells and cells were lysed 12 h, 24 h, 36 h, 2d and 7d post transfection. Protein lysates were subsequently analyzed for SV40 LT-Ag (Pab419 antibody) and VP1 expression (α-VP1 polyclonal rabbit serum) by SDS-page and Western Blotting. Staining of actin was used to ensure that equal protein amounts were loaded per lane. (B) Low molecular weight DNA was isolated from SV40 DNA transfected CV-1 cells at the indicated time points by HIRT extraction, 1 µg DNA was DpnI and EcoRI digested; DNA was separated on an agarose gel and stained with EtBr (left panel), followed by southern blotting and detection of viral DNA using a 32 PdCTP-labeled SV40 LT-Ag PCR fragment as a probe. The blot was exposed for 30 min. Numbers below the lanes correspond to the quantification of newly replicated DNA using a Fuji phosphoimager FLA7000 and MultiGauge software.
    Figure Legend Snippet: SV40 replication and gene expression in CV-1 cells transfected with SV40 DNA. (A) 100 ng of intramolecular religated SV40 viral DNA was transfected in CV-1 cells and cells were lysed 12 h, 24 h, 36 h, 2d and 7d post transfection. Protein lysates were subsequently analyzed for SV40 LT-Ag (Pab419 antibody) and VP1 expression (α-VP1 polyclonal rabbit serum) by SDS-page and Western Blotting. Staining of actin was used to ensure that equal protein amounts were loaded per lane. (B) Low molecular weight DNA was isolated from SV40 DNA transfected CV-1 cells at the indicated time points by HIRT extraction, 1 µg DNA was DpnI and EcoRI digested; DNA was separated on an agarose gel and stained with EtBr (left panel), followed by southern blotting and detection of viral DNA using a 32 PdCTP-labeled SV40 LT-Ag PCR fragment as a probe. The blot was exposed for 30 min. Numbers below the lanes correspond to the quantification of newly replicated DNA using a Fuji phosphoimager FLA7000 and MultiGauge software.

    Techniques Used: Expressing, Transfection, SDS Page, Western Blot, Staining, Molecular Weight, Isolation, Agarose Gel Electrophoresis, Southern Blot, Labeling, Polymerase Chain Reaction, Software

    MCVSyn replication assays in H1299, PFSK-1 and 293 cells. 5 µg low molecular weight DNA isolated from cell cultures at the indicated time points post-transfection with MCVSyn DNA was digested with DpnI and EcoRI, separated on an agarose gel and stained with EtBr (left panels), then transferred via southern blot and probed with a radioactively labelled LT-Ag PCR fragment [13] . The blot was exposed for 24 h and scanned using a Fuji phosphoimager FLA7000; MultiGauge software was used for quantification.
    Figure Legend Snippet: MCVSyn replication assays in H1299, PFSK-1 and 293 cells. 5 µg low molecular weight DNA isolated from cell cultures at the indicated time points post-transfection with MCVSyn DNA was digested with DpnI and EcoRI, separated on an agarose gel and stained with EtBr (left panels), then transferred via southern blot and probed with a radioactively labelled LT-Ag PCR fragment [13] . The blot was exposed for 24 h and scanned using a Fuji phosphoimager FLA7000; MultiGauge software was used for quantification.

    Techniques Used: Molecular Weight, Isolation, Transfection, Agarose Gel Electrophoresis, Staining, Southern Blot, Polymerase Chain Reaction, Software

    2) Product Images from "Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences"

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences

    Journal:

    doi: 10.1101/gr.094953.109

    Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).
    Figure Legend Snippet: Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1 ) A 0.4–10-kb DNA ladder. (Lane 2 ) Total (unfractionated) DNA (NF). (Lane 3 ) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5 ) The corresponding insoluble pellet (SRDI). (Lane 4 ) DNA released from sperm after MNase digestion (MNDS). (Lane 6 ) The corresponding insoluble pellet (MNDI).

    Techniques Used:

    3) Product Images from "The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells"

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12378

    (A) Characterization of Pdx1‐ pCDNA 3.1(+) vector. Lane 1: the recombinant plasmid before digestion. Lane 2: the 850‐bp Pdx1 gene separated from the recombinant Pdx1‐ pCDNA 3.1(+) digested using EcoRI and Hind III enzymes. Lane 3: 900‐bp PCR product of recombinant Pdx1‐ pCDNA 3.1(+) vector. Lane 4: 1‐kb DNA ladder. (B) Western blot analysis results. The CHO cells transfected with Pdx1‐ pCDNA 3.1(+) (lane 1) showed a significantly higher expression of Pdx1 compared with the CHO cells transfected with pCDNA 3.1(+) alone (Lane 2).
    Figure Legend Snippet: (A) Characterization of Pdx1‐ pCDNA 3.1(+) vector. Lane 1: the recombinant plasmid before digestion. Lane 2: the 850‐bp Pdx1 gene separated from the recombinant Pdx1‐ pCDNA 3.1(+) digested using EcoRI and Hind III enzymes. Lane 3: 900‐bp PCR product of recombinant Pdx1‐ pCDNA 3.1(+) vector. Lane 4: 1‐kb DNA ladder. (B) Western blot analysis results. The CHO cells transfected with Pdx1‐ pCDNA 3.1(+) (lane 1) showed a significantly higher expression of Pdx1 compared with the CHO cells transfected with pCDNA 3.1(+) alone (Lane 2).

    Techniques Used: Plasmid Preparation, Recombinant, Polymerase Chain Reaction, Western Blot, Transfection, Expressing

    4) Product Images from "Virus found in a boreal lake links ssDNA and dsDNA viruses"

    Article Title: Virus found in a boreal lake links ssDNA and dsDNA viruses

    Journal:

    doi: 10.1073/pnas.1703834114

    Analysis of the ssDNA genome of FLiP. Agarose gel electrophoresis (AGE, 1% agarose) analysis of FLiP (lanes 2–9) and φX174 genomes (lanes 10–16). Lanes 2 and 10: untreated samples; lanes 3 and 11: samples treated with DNase I; lanes 4 and 12, samples treated with RNase A; lanes 5 and 13: samples treated with RNase I; lanes 6 and 14, samples treated with S1 nuclease; lanes 7 and 15: samples treated with Mung bean nuclease; lanes 8 and 16: lanes treated with EcoRI. The DNA ladder (Fermentas #R0491) is in lane 1.
    Figure Legend Snippet: Analysis of the ssDNA genome of FLiP. Agarose gel electrophoresis (AGE, 1% agarose) analysis of FLiP (lanes 2–9) and φX174 genomes (lanes 10–16). Lanes 2 and 10: untreated samples; lanes 3 and 11: samples treated with DNase I; lanes 4 and 12, samples treated with RNase A; lanes 5 and 13: samples treated with RNase I; lanes 6 and 14, samples treated with S1 nuclease; lanes 7 and 15: samples treated with Mung bean nuclease; lanes 8 and 16: lanes treated with EcoRI. The DNA ladder (Fermentas #R0491) is in lane 1.

    Techniques Used: Agarose Gel Electrophoresis

    5) Product Images from "Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System"

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158889

    Restriction patterns of DNA extracted from purified virions cleaved with the selected REases. Panel A: HindIII (H), Eco32I (E32), SalI (S) and EcoRI (EI). ND, undigested DNA. M, GeneRuler 100- to 10,000-bp size marker. λ/H, λ DNA cleaved with HindIII. ~14 kb restriction fragment of EcoR32I digested virion DNA is marked with a triangle. Panel B: Arrows indicate restriction fragments assigned to ФAH14a (left) and ФAH14b (right) genomic DNA, obtained by SalI digestion.
    Figure Legend Snippet: Restriction patterns of DNA extracted from purified virions cleaved with the selected REases. Panel A: HindIII (H), Eco32I (E32), SalI (S) and EcoRI (EI). ND, undigested DNA. M, GeneRuler 100- to 10,000-bp size marker. λ/H, λ DNA cleaved with HindIII. ~14 kb restriction fragment of EcoR32I digested virion DNA is marked with a triangle. Panel B: Arrows indicate restriction fragments assigned to ФAH14a (left) and ФAH14b (right) genomic DNA, obtained by SalI digestion.

    Techniques Used: Purification, Marker

    6) Product Images from "Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex"

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-599

    Isolation of the KS14 plasmid prophage . DNA was isolated using a QIAprep Spin Miniprep plasmid isolation kit (Qiagen) and digested with EcoRI (Invitrogen). Lane 1: 1 Kb Plus DNA ladder (Invitrogen), lane 2: B. cenocepacia C6433, lane 3: B. multivorans ATCC 17616, lane 4: B. cenocepacia CEP511 lane 5: B. cenocepacia K56-2, lane 6: blank, lane 7: 1 Kb Plus DNA ladder, lane 8: KS14-resistant C6433 isolate I, lane 9: KS14-resistant C6433 isolate II, lane 10: KS14-resistant C6433 isolate III, lane 11: KS14-resistant C6433 isolate IV, lane 12: KS14-resistant C6433 isolate V. The size of the markers (in kbp) is shown on the left. A KS14 EcoRI DNA digest and the size of the bands predicted for this digest ( > 1 kbp in size) are shown on the far right.
    Figure Legend Snippet: Isolation of the KS14 plasmid prophage . DNA was isolated using a QIAprep Spin Miniprep plasmid isolation kit (Qiagen) and digested with EcoRI (Invitrogen). Lane 1: 1 Kb Plus DNA ladder (Invitrogen), lane 2: B. cenocepacia C6433, lane 3: B. multivorans ATCC 17616, lane 4: B. cenocepacia CEP511 lane 5: B. cenocepacia K56-2, lane 6: blank, lane 7: 1 Kb Plus DNA ladder, lane 8: KS14-resistant C6433 isolate I, lane 9: KS14-resistant C6433 isolate II, lane 10: KS14-resistant C6433 isolate III, lane 11: KS14-resistant C6433 isolate IV, lane 12: KS14-resistant C6433 isolate V. The size of the markers (in kbp) is shown on the left. A KS14 EcoRI DNA digest and the size of the bands predicted for this digest ( > 1 kbp in size) are shown on the far right.

    Techniques Used: Isolation, Plasmid Preparation

    7) Product Images from "Transcription Factor Binding and Induced Transcription Alter Chromosomal c-myc Replicator Activity"

    Article Title: Transcription Factor Binding and Induced Transcription Alter Chromosomal c-myc Replicator Activity

    Journal:

    doi: 10.1128/MCB.24.23.10193-10207.2004

    Maps of DNA sequences used in this study. (A) HindIII-EcoRV fragment of the human c- myc locus. The first c- myc exon and the 5′ part of the second exon are indicated by open boxes. The HindIII-XhoI fragment contains the wild-type c- myc replicator core. (A, ApaI; Ev, EcoRv; H, HindIII; S, SpeI; N, NotI; Xh, XhoI). Downward-pointing arrowheads represent replication initiation sites previously mapped in vivo ( , , , , ). The in vivo DNA unwinding element (DUE) ( , , ) is indicated. P0 , P1 , P2 , P3 , c- myc promoters. A TATA box is located 28 bp 5′ to the P1 promoter. (B) Left, the clonal acceptor cell line HeLa/406 was generated through single-copy insertion of the plasmid pHyg.FRT.TK, containing an FLP recombinase target site (FRT), into the HeLa genome. Hyg, hygromycin resistance gene; TK, herpes simplex virus thymidine kinase gene (shaded rectangles). STS, sequence-tagged sites (Q-PCR primers); numbered arrows, analytical PCR primers (cf. Fig. ). Unfilled rectangles are vector sequences. The acceptor cell line is hygromycin resistant and ganciclovir sensitive. Right, the donor plasmid pFRT.myc containing a promoterless neomycin resistance gene (Neo) and the 2.4-kb HindIII-XhoI fragment of the c- myc replicator (solid black box) was integrated at the acceptor site by the FLP recombinase expressed from pOG44. (C) The resulting pFRT.myc cell line is resistant to hygromycin, neomycin, and ganciclovir. PCR primers 1, 2, and 3 (horizontal arrowheads) gave diagnostic PCR products for the empty acceptor site (primers 1 and 2) or after FLP-mediated integration of the donor plasmids (primers 1 and 3). Sequence-tagged sites for Q-PCR are indicated as STS-Hyg, STS-UV, STS-DV, and STS-TK. The positions of restriction sites (E, EcoRI; H, HindIII) and probes relevant to the Southern analysis are shown. Bars below each map correspond to diagnostic restriction fragments detected by Southern blotting. (D) The pFRT.myc.TET-GFP cell lines were generated by deleting the 3′ ApaI-ApaI c- myc sequence (pFRT.myc.3′Δ820, dashed lines) and replacing it with a GFP transcription unit under control of the tetracycline-inducible TRE promoter. The GFP genes were cloned in both orientations (indicated by thick arrows) relative to the c- myc sequences. All c- myc sequences except the 5′ HindIII-PinI fragment (79 bp) had been deleted from the pFRT.TRE-GFP cells. (E) The 3′ 1,420 bp of the c- myc core origin sequence was deleted from the donor plasmid pFRT.myc to construct the cell line pFRT.myc.3′Δ1420. (F) pFRT.myc.3′Δ1420-GAL4 was constructed by fusing four GAL4p binding sites to the 3′ end of the c- myc sequences in pFRT.myc.3′Δ1420.
    Figure Legend Snippet: Maps of DNA sequences used in this study. (A) HindIII-EcoRV fragment of the human c- myc locus. The first c- myc exon and the 5′ part of the second exon are indicated by open boxes. The HindIII-XhoI fragment contains the wild-type c- myc replicator core. (A, ApaI; Ev, EcoRv; H, HindIII; S, SpeI; N, NotI; Xh, XhoI). Downward-pointing arrowheads represent replication initiation sites previously mapped in vivo ( , , , , ). The in vivo DNA unwinding element (DUE) ( , , ) is indicated. P0 , P1 , P2 , P3 , c- myc promoters. A TATA box is located 28 bp 5′ to the P1 promoter. (B) Left, the clonal acceptor cell line HeLa/406 was generated through single-copy insertion of the plasmid pHyg.FRT.TK, containing an FLP recombinase target site (FRT), into the HeLa genome. Hyg, hygromycin resistance gene; TK, herpes simplex virus thymidine kinase gene (shaded rectangles). STS, sequence-tagged sites (Q-PCR primers); numbered arrows, analytical PCR primers (cf. Fig. ). Unfilled rectangles are vector sequences. The acceptor cell line is hygromycin resistant and ganciclovir sensitive. Right, the donor plasmid pFRT.myc containing a promoterless neomycin resistance gene (Neo) and the 2.4-kb HindIII-XhoI fragment of the c- myc replicator (solid black box) was integrated at the acceptor site by the FLP recombinase expressed from pOG44. (C) The resulting pFRT.myc cell line is resistant to hygromycin, neomycin, and ganciclovir. PCR primers 1, 2, and 3 (horizontal arrowheads) gave diagnostic PCR products for the empty acceptor site (primers 1 and 2) or after FLP-mediated integration of the donor plasmids (primers 1 and 3). Sequence-tagged sites for Q-PCR are indicated as STS-Hyg, STS-UV, STS-DV, and STS-TK. The positions of restriction sites (E, EcoRI; H, HindIII) and probes relevant to the Southern analysis are shown. Bars below each map correspond to diagnostic restriction fragments detected by Southern blotting. (D) The pFRT.myc.TET-GFP cell lines were generated by deleting the 3′ ApaI-ApaI c- myc sequence (pFRT.myc.3′Δ820, dashed lines) and replacing it with a GFP transcription unit under control of the tetracycline-inducible TRE promoter. The GFP genes were cloned in both orientations (indicated by thick arrows) relative to the c- myc sequences. All c- myc sequences except the 5′ HindIII-PinI fragment (79 bp) had been deleted from the pFRT.TRE-GFP cells. (E) The 3′ 1,420 bp of the c- myc core origin sequence was deleted from the donor plasmid pFRT.myc to construct the cell line pFRT.myc.3′Δ1420. (F) pFRT.myc.3′Δ1420-GAL4 was constructed by fusing four GAL4p binding sites to the 3′ end of the c- myc sequences in pFRT.myc.3′Δ1420.

    Techniques Used: In Vivo, Generated, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Diagnostic Assay, Southern Blot, Clone Assay, Construct, Binding Assay

    Structural confirmation of ectopic c- myc cell lines. (A) Genomic DNA was isolated from the cell lines whose results are shown in Fig. and amplified by PCR with primers yielding products diagnostic for the unoccupied acceptor site (primers 1 and 2) or the occupied acceptor site (primers 1 and 3). A, acceptor HeLa/406 cells; WT, Δ, F, and R, TRE-GFP cells in forward orientation (F), in reverse orientation (R), with pFRT.myc integrated (WT), and with pFRT.myc.3′Δ1420 integrated (Δ). (B) Hybridization to DNA from acceptor cells or cells transfected with pFRT constructs. Left, hybridization of the Neo probe internal to the pFRT donor plasmids to HindIII junction fragments between the acceptor site and chromosomal DNA. Right, hybridization of the acceptor site Hyg probe to EcoRI junction fragments. (C) Hybridization of the Neo probe to EcoRI-digested junction fragments (left) or HindIII junction fragments (right) from cells with integrated with pFRT.TRE-GFP. (D) Hybridization of the TK probe to HindIII-digested junction fragments from cells with integrated pFRT.myc or pFRT.myc.3′Δ820. (E) Hybridization of the Hyg probe (left) or the Neo probe (right) to EcoRI junction fragments from cells with integrated pFRT.myc.3′Δ1420-GAL4. Size markers are in kilobase pairs. The pFRT.myc.3′Δ1420 integrant has previously been characterized .
    Figure Legend Snippet: Structural confirmation of ectopic c- myc cell lines. (A) Genomic DNA was isolated from the cell lines whose results are shown in Fig. and amplified by PCR with primers yielding products diagnostic for the unoccupied acceptor site (primers 1 and 2) or the occupied acceptor site (primers 1 and 3). A, acceptor HeLa/406 cells; WT, Δ, F, and R, TRE-GFP cells in forward orientation (F), in reverse orientation (R), with pFRT.myc integrated (WT), and with pFRT.myc.3′Δ1420 integrated (Δ). (B) Hybridization to DNA from acceptor cells or cells transfected with pFRT constructs. Left, hybridization of the Neo probe internal to the pFRT donor plasmids to HindIII junction fragments between the acceptor site and chromosomal DNA. Right, hybridization of the acceptor site Hyg probe to EcoRI junction fragments. (C) Hybridization of the Neo probe to EcoRI-digested junction fragments (left) or HindIII junction fragments (right) from cells with integrated with pFRT.TRE-GFP. (D) Hybridization of the TK probe to HindIII-digested junction fragments from cells with integrated pFRT.myc or pFRT.myc.3′Δ820. (E) Hybridization of the Hyg probe (left) or the Neo probe (right) to EcoRI junction fragments from cells with integrated pFRT.myc.3′Δ1420-GAL4. Size markers are in kilobase pairs. The pFRT.myc.3′Δ1420 integrant has previously been characterized .

    Techniques Used: Isolation, Amplification, Polymerase Chain Reaction, Diagnostic Assay, Hybridization, Transfection, Construct

    8) Product Images from "A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis"

    Article Title: A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0804583105

    Truncated stau1 lacking dsRBD3 leads to reduced Stau1 protein levels and an impairment of RNA-binding capacity. ( A ) Targeting strategy for disrupting the Stau1 gene in mouse. WT genomic locus ( Top ) shows the location of the dsRBD3, the sites for homologous recombination with the targeting vector (IRESβgeopA, Middle ) and the final targeted, mutated Stau1 locus ( Bottom ). Arrowheads indicate the primers used for genotyping. 5′ probe, indicates the probe used for Southern blotting. ( B ) 3′ PCR ( Top ) and 5′ Southern blot ( Bottom ) analysis of WT (+/+), heterozygous (+/-), and mutant (−/−) Stau1 mice. For Southern blotting, genomic DNA was EcoRI-digested and probed with a PCR fragment (nt 325–568 of the Stau1 locus, 5′ probe). The DNA from stau1tm1Apa homozygous mice (−/−) shows a 1.5 kb deletion (from 10 to 8.5 kb) due to the presence of an additional EcoRI site in the recombinant locus. For verification of 3′ targeting ( Top , 3′ PCR), primers stauR5 and neo236 were used, generating a 2.9kbp PCR product in the correctly targeted clones. m, marker. ( C ) PCR genotyping indicating the two resulting PCR fragments for either WT (+/+) or stau1tm1Apa homozygous mice (−/−). M, marker. ( D ) Western blot of WT (+/+) and stau1tm1Apa (−/−) forebrain extracts probed with an immunopurified polyclonal anti-Stau1 antibody ( Upper ). In mutant brain, a faint band of lower molecular weight is visible (asterisk). The lower panel shows a blot probed with the preimmune serum (PIS) of the Stau1 rabbit. ( E ) Western blot of WT (+/+) and stau1tm1Apa (−/−) forebrain extracts probed with an immunopurified polyclonal anti-Stau2 antibody or a monoclonal anti-tubulin antibody as internal loading control. Expression levels of the three Stau2 isoforms (62, 59, and 52 kDa, respectively) are unchanged. ( F ) Detection of full-length Stau1 and Stau2 mRNAs isolated from brains of WT (+/+), heterozygous (+/-), or stau1tm1Apa homozygous mice (−/−) by RT-PCR. In contrast to WT (arrowhead), a shortened Stau1 transcript is found in stau1tm1Apa homozygous mice caused by an in-frame deletion of 207 nts in RBD3 (arrow). As internal control, the dsRBD5 of Stau2 was amplified in parallel. The scheme below shows skipping of exon 5 in stau1tm1Apa homozygous mice (−/−) leading to a deletion of 69 aa within the dsRBD3 (amino acids in italics, LQ is added in ΔStau1 due to alternative splicing). ( G ) ΔStau1 lacking RBD3 does not bind RNA in vitro . A Northwestern assay was performed on HeLa cell extracts transfected with WT (+/+) or ΔStau1 (−/−) and probed with an immunopurified polyclonal anti-Stau1 antibody (WB). The asterisk marks endogenous Stau1 protein in HeLa. Proteins were renatured on the filter and exposed to a synthetic 32 P-labeled poly rI/rC probe. Arrowhead indicates unspecific RNA binding to a protein present in all extracts. UT, untransfected control.
    Figure Legend Snippet: Truncated stau1 lacking dsRBD3 leads to reduced Stau1 protein levels and an impairment of RNA-binding capacity. ( A ) Targeting strategy for disrupting the Stau1 gene in mouse. WT genomic locus ( Top ) shows the location of the dsRBD3, the sites for homologous recombination with the targeting vector (IRESβgeopA, Middle ) and the final targeted, mutated Stau1 locus ( Bottom ). Arrowheads indicate the primers used for genotyping. 5′ probe, indicates the probe used for Southern blotting. ( B ) 3′ PCR ( Top ) and 5′ Southern blot ( Bottom ) analysis of WT (+/+), heterozygous (+/-), and mutant (−/−) Stau1 mice. For Southern blotting, genomic DNA was EcoRI-digested and probed with a PCR fragment (nt 325–568 of the Stau1 locus, 5′ probe). The DNA from stau1tm1Apa homozygous mice (−/−) shows a 1.5 kb deletion (from 10 to 8.5 kb) due to the presence of an additional EcoRI site in the recombinant locus. For verification of 3′ targeting ( Top , 3′ PCR), primers stauR5 and neo236 were used, generating a 2.9kbp PCR product in the correctly targeted clones. m, marker. ( C ) PCR genotyping indicating the two resulting PCR fragments for either WT (+/+) or stau1tm1Apa homozygous mice (−/−). M, marker. ( D ) Western blot of WT (+/+) and stau1tm1Apa (−/−) forebrain extracts probed with an immunopurified polyclonal anti-Stau1 antibody ( Upper ). In mutant brain, a faint band of lower molecular weight is visible (asterisk). The lower panel shows a blot probed with the preimmune serum (PIS) of the Stau1 rabbit. ( E ) Western blot of WT (+/+) and stau1tm1Apa (−/−) forebrain extracts probed with an immunopurified polyclonal anti-Stau2 antibody or a monoclonal anti-tubulin antibody as internal loading control. Expression levels of the three Stau2 isoforms (62, 59, and 52 kDa, respectively) are unchanged. ( F ) Detection of full-length Stau1 and Stau2 mRNAs isolated from brains of WT (+/+), heterozygous (+/-), or stau1tm1Apa homozygous mice (−/−) by RT-PCR. In contrast to WT (arrowhead), a shortened Stau1 transcript is found in stau1tm1Apa homozygous mice caused by an in-frame deletion of 207 nts in RBD3 (arrow). As internal control, the dsRBD5 of Stau2 was amplified in parallel. The scheme below shows skipping of exon 5 in stau1tm1Apa homozygous mice (−/−) leading to a deletion of 69 aa within the dsRBD3 (amino acids in italics, LQ is added in ΔStau1 due to alternative splicing). ( G ) ΔStau1 lacking RBD3 does not bind RNA in vitro . A Northwestern assay was performed on HeLa cell extracts transfected with WT (+/+) or ΔStau1 (−/−) and probed with an immunopurified polyclonal anti-Stau1 antibody (WB). The asterisk marks endogenous Stau1 protein in HeLa. Proteins were renatured on the filter and exposed to a synthetic 32 P-labeled poly rI/rC probe. Arrowhead indicates unspecific RNA binding to a protein present in all extracts. UT, untransfected control.

    Techniques Used: RNA Binding Assay, Homologous Recombination, Plasmid Preparation, Southern Blot, Polymerase Chain Reaction, Mutagenesis, Mouse Assay, Recombinant, Clone Assay, Marker, Western Blot, Molecular Weight, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, In Vitro, Transfection, Labeling

    9) Product Images from "Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site"

    Article Title: Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site

    Journal: Retrovirology

    doi: 10.1186/1742-4690-2-3

    RT-PCR analysis of virion RNAs. A. Plasmid structures of retroviral leader regions. L1 and L2, primers used for PCR amplification; sizes of DNA fragments and positions of EcoRI sites are indicated. B. Leader sequences in virion RNAs were PCR amplified and analyzed by restriction digestion. PCR products obtained from virion RNAs of ST2-1 and ST2-2 packaging cell clones (lines 1 and 3); PCR products obtained from virion RNAs of CT1 and CT2 cell clones (lines 2 and 4); M, molecular weight markers. The ratios of puro/neo retroviral RNAs for ST2-1, ST2-2, CT1, and CT2 cell clones were 1.8, 2.0, 1.6, and 2.5, respectively.
    Figure Legend Snippet: RT-PCR analysis of virion RNAs. A. Plasmid structures of retroviral leader regions. L1 and L2, primers used for PCR amplification; sizes of DNA fragments and positions of EcoRI sites are indicated. B. Leader sequences in virion RNAs were PCR amplified and analyzed by restriction digestion. PCR products obtained from virion RNAs of ST2-1 and ST2-2 packaging cell clones (lines 1 and 3); PCR products obtained from virion RNAs of CT1 and CT2 cell clones (lines 2 and 4); M, molecular weight markers. The ratios of puro/neo retroviral RNAs for ST2-1, ST2-2, CT1, and CT2 cell clones were 1.8, 2.0, 1.6, and 2.5, respectively.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Molecular Weight

    10) Product Images from "Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages"

    Article Title: Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-223

    RFLP analysis of KL1 and AH2 genomic DNA. 5 μg of genomic DNA were digested overnight with EcoRI and separated on a 0.8% agarose gel. The DNA in the ambient gel (left) was not heated, while the DNA in the 80°C gel (right) was incubated 20 min at 80°C and chilled on ice prior to loading. Arrows indicate bands containing cos site DNA. L: 1 Kb Plus DNA Ladder (Invitrogen).
    Figure Legend Snippet: RFLP analysis of KL1 and AH2 genomic DNA. 5 μg of genomic DNA were digested overnight with EcoRI and separated on a 0.8% agarose gel. The DNA in the ambient gel (left) was not heated, while the DNA in the 80°C gel (right) was incubated 20 min at 80°C and chilled on ice prior to loading. Arrows indicate bands containing cos site DNA. L: 1 Kb Plus DNA Ladder (Invitrogen).

    Techniques Used: Agarose Gel Electrophoresis, Incubation

    11) Product Images from "Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish"

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish

    Journal: Antibiotics

    doi: 10.3390/antibiotics7010016

    Restriction enzyme-digested fragments of the genomic DNA of A. hydrophila -phage 2. Footnote: Lane M: 1kb Plus Opti-DNA Marker (ABM, Canada); Lane L1: genomic DNA of Φ2; Lanes L2–L8: genomic DNA of Φ2 digested with EcoRV; EcoRI; Ncol; SalI; MspI; XmnI; KpnI, restriction enzymes respectively.
    Figure Legend Snippet: Restriction enzyme-digested fragments of the genomic DNA of A. hydrophila -phage 2. Footnote: Lane M: 1kb Plus Opti-DNA Marker (ABM, Canada); Lane L1: genomic DNA of Φ2; Lanes L2–L8: genomic DNA of Φ2 digested with EcoRV; EcoRI; Ncol; SalI; MspI; XmnI; KpnI, restriction enzymes respectively.

    Techniques Used: Marker

    12) Product Images from "Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function"

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function

    Journal: DARU : Journal of Faculty of Pharmacy, Tehran University of Medical Sciences

    doi:

    Digestion of pcDNA3 plasmid containing wild type V2R by XbaI-EcoRI enzymes. Products of digestion were electrophoresed on 0.7% agarose gel. The band of V2R (1200 bp) was digested out of the pcDNA3 plasmid. M: molecular weight marker, 250 bp unit.
    Figure Legend Snippet: Digestion of pcDNA3 plasmid containing wild type V2R by XbaI-EcoRI enzymes. Products of digestion were electrophoresed on 0.7% agarose gel. The band of V2R (1200 bp) was digested out of the pcDNA3 plasmid. M: molecular weight marker, 250 bp unit.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Molecular Weight, Marker

    13) Product Images from "Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates"

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

    Journal: The Korean Journal of Parasitology

    doi: 10.3347/kjp.2013.51.4.413

    Expression patterns of TDFs extracted from cDNA-AFLP PAGE. Sensitive amplification of cDNA-AFLP on a PAGE from 3 different primer combinations of S3 MboI /S2 EcoRI , S3 MboI /S1 EcoRI , and S4 MboI /S4 MboI . Arrows point to differentially expressed TDFs which were isolated with code numbers of 1, 2, and 3 (associated genes which derived from these TDFs mentioned in Table 2 ). M, 50 bp molecular weight marker; S, sensitve; R, resistant.
    Figure Legend Snippet: Expression patterns of TDFs extracted from cDNA-AFLP PAGE. Sensitive amplification of cDNA-AFLP on a PAGE from 3 different primer combinations of S3 MboI /S2 EcoRI , S3 MboI /S1 EcoRI , and S4 MboI /S4 MboI . Arrows point to differentially expressed TDFs which were isolated with code numbers of 1, 2, and 3 (associated genes which derived from these TDFs mentioned in Table 2 ). M, 50 bp molecular weight marker; S, sensitve; R, resistant.

    Techniques Used: Expressing, cDNA-AFLP Assay, Polyacrylamide Gel Electrophoresis, Amplification, Isolation, Derivative Assay, Molecular Weight, Marker

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202294

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

    Techniques Used: Plasmid Preparation, Clone Assay, Expressing

    15) Product Images from "The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes"

    Article Title: The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr789

    Observation of the pUC19 plasmid DNA states during cleavage by Spd1. Aliquots were taken from the reaction mixture at the indicated time points, and digested products were analysed by electrophoresis in 1% agarose gel, staining with SYBR® Safe (Invitrogen). Control lanes 1: Hyperladder I (Bioline); Lane 2: Untreated pUC19; Lane 3: EcoRI linearized pUC19; Lane 4: Reaction mixture prior to the addition of Spd1. The digestion of plasmid DNA proceeds via single-strand nick mediated relaxation of supercoiled DNA, this can be seen 30 s to 1 min into the reaction; within the same time frame a linear form can also be seen emerging.
    Figure Legend Snippet: Observation of the pUC19 plasmid DNA states during cleavage by Spd1. Aliquots were taken from the reaction mixture at the indicated time points, and digested products were analysed by electrophoresis in 1% agarose gel, staining with SYBR® Safe (Invitrogen). Control lanes 1: Hyperladder I (Bioline); Lane 2: Untreated pUC19; Lane 3: EcoRI linearized pUC19; Lane 4: Reaction mixture prior to the addition of Spd1. The digestion of plasmid DNA proceeds via single-strand nick mediated relaxation of supercoiled DNA, this can be seen 30 s to 1 min into the reaction; within the same time frame a linear form can also be seen emerging.

    Techniques Used: Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Staining

    16) Product Images from "Detection of Molecular Diversity in Bacillus atrophaeus by Amplified Fragment Length Polymorphism Analysis"

    Article Title: Detection of Molecular Diversity in Bacillus atrophaeus by Amplified Fragment Length Polymorphism Analysis

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.70.5.2786-2790.2004

    Digitized AFLP patterns of Bacillus taxa generated using primer sets EcoRI plus C/MseI plus CA (A) and EcoRI plus C/MseI plus CC (B). Across the top of each image is the fragment size scale (in bases). The Bacillus species and strain designations for
    Figure Legend Snippet: Digitized AFLP patterns of Bacillus taxa generated using primer sets EcoRI plus C/MseI plus CA (A) and EcoRI plus C/MseI plus CC (B). Across the top of each image is the fragment size scale (in bases). The Bacillus species and strain designations for

    Techniques Used: Generated

    17) Product Images from "Producing Recombinant mTEX101; a Murine Testis Specific Protein"

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein

    Journal: Journal of Reproduction & Infertility

    doi:

    Double digestion of pET-28a (+) expression vector with restriction enzymes. 1: digested pET-28a (+) with EcoRI and NotI, 2: uncut pET-28a (+), 3: 1 Kb DNA ladder.
    Figure Legend Snippet: Double digestion of pET-28a (+) expression vector with restriction enzymes. 1: digested pET-28a (+) with EcoRI and NotI, 2: uncut pET-28a (+), 3: 1 Kb DNA ladder.

    Techniques Used: Positron Emission Tomography, Expressing, Plasmid Preparation

    Double digestion of pGEM-T easy vector containing mTEX101 fragment with EcoRI and NotI restriction enzymes. 1: digested vector with mTEX101 750 bp insert cut out of the vector, 2: DNA ladder VIII.
    Figure Legend Snippet: Double digestion of pGEM-T easy vector containing mTEX101 fragment with EcoRI and NotI restriction enzymes. 1: digested vector with mTEX101 750 bp insert cut out of the vector, 2: DNA ladder VIII.

    Techniques Used: Plasmid Preparation

    18) Product Images from "Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs"

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs

    Journal:

    doi: 10.1128/JVI.00236-10

    Mapping of the RNA sequence(s) in HBV pregenomic RNA responsible for HBV RNA decay. (A) Schematic diagram of the truncation and deletion mutants of HBV pregenomic RNA. The numbers indicate the HBV DNA sequence with 1 at the unique EcoRI site in the HBV genome. (B) Huh7 cells were transfected with 0.1 μg of luciferase fusion constructs containing the truncation mutants of HBV DNA and 0.2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h, and the luciferase activity was then assessed. For each transfection, 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P < 0.05. (C) Huh7 cells were transfected with 1 μg of pCDNA3.1-Luc, Luc-HBV(1804-2454), or Luc-HBV(1151-1684) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (D) Huh7 cells were transfected with 1 μg of Luc-ΔHBV(1151-1684) or Luc-ΔHBV(1804-2454) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (E) Huh7 cells were transfected with 1 μg of pCMV-HBV, pCMV-HBVΔ1804-2454, or pCMV-HBVΔ1151-1684 (also named pCMV-HBVΔPRE in this study) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control.
    Figure Legend Snippet: Mapping of the RNA sequence(s) in HBV pregenomic RNA responsible for HBV RNA decay. (A) Schematic diagram of the truncation and deletion mutants of HBV pregenomic RNA. The numbers indicate the HBV DNA sequence with 1 at the unique EcoRI site in the HBV genome. (B) Huh7 cells were transfected with 0.1 μg of luciferase fusion constructs containing the truncation mutants of HBV DNA and 0.2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h, and the luciferase activity was then assessed. For each transfection, 0.01 μg of pRL-tk was included as an internal control of transfection efficiency. Results represent the means of data from three independent experiments performed in duplicate. *, P < 0.05. (C) Huh7 cells were transfected with 1 μg of pCDNA3.1-Luc, Luc-HBV(1804-2454), or Luc-HBV(1151-1684) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (D) Huh7 cells were transfected with 1 μg of Luc-ΔHBV(1151-1684) or Luc-ΔHBV(1804-2454) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control. (E) Huh7 cells were transfected with 1 μg of pCMV-HBV, pCMV-HBVΔ1804-2454, or pCMV-HBVΔ1151-1684 (also named pCMV-HBVΔPRE in this study) together with 2 μg of pCMV/Myc or pCMV/Myc-MyD88 for 48 h. Levels of luciferase mRNA were determined by Northern blot analysis. GAPDH was analyzed as a loading control.

    Techniques Used: Sequencing, Transfection, Luciferase, Construct, Activity Assay, Northern Blot

    19) 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

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    Article Snippet: The amplified DNA fragments were analyzed by agarose gel electrophoresis and, if necessary, purified using a Gel Out kit (Thermo Fisher Scientific, Waltham, MA, USA). .. The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates
    Article Snippet: Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr. .. Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr.

    Synthesized:

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli
    Article Snippet: The coding regions for the following HSL synthases were synthesized as double-stranded oligos (IDT) with an EcoRI binding site upstream and a XbaI cut site downstream: AubI, BjaI, BraI, CerI, EsaI, LasI, LuxI, RhlI, RpaI, and SinI. .. The Modular Sender Vector and each HSL synthase oligo were cut with EcoRI and XbaI (Thermo Fisher Scientific) and ligated using T4 ligase (New England Biolabs).

    Article Title: Virus found in a boreal lake links ssDNA and dsDNA viruses
    Article Snippet: The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions. .. The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions.

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates
    Article Snippet: Single strand cDNA (sscDNA) was synthesized using 10 µg RNA, 20 pmol/µl oligo-dT (Fermentas, Burlington, Canada), 20 pmol/µl random hexamer (Fermentas), 10 mM of dNTP mix (Fermentas) incubated at 65℃ for 10 min, followed by addition of 20 U RNase inhibitor (Fermentas), 4 µL of 5× reverse transcriptase (RT) buffer containing Tris-HCl (pH 8.3) (Fermentas), and 200 U RevertAid premium RT (Fermentas). .. Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr.

    Construct:

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System
    Article Snippet: The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA). .. The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs
    Article Snippet: The pRSV138PRE-CAT and pRSV-CAT constructs were described previously ( ). .. To construct Myc-tagged MyD88, polypyrimidine tract-binding protein 1 (PTB1), and nuclear export signal (NES)(−)RanBP plasmids, the complete cDNAs of human MyD88 carried on pCDNA3-MyD88 , PTB1 carried on His-PTB1 , and NES(−)RanBP carried on green fluorescent protein (GFP)-NES(−)RanBP1 ( ) were inserted into the EcoRI and NotI sites of pCMV/Myc vectors (Invitrogen). cDNA fragments of HBV pregenomic RNA from pCMV-HBV and the complete HBV cDNA from pCMV-HBVΔPRE were inserted into pcDNA3.1/Luc ( ) at the 3′ end of the luciferase coding sequence before the bovine growth hormone (BGH) polyadenylation signal (see Fig. ). .. The sequence of HBV(1804-2454) was deleted from pCMV-HBV by inverse PCR using a KOD-Plus mutagenesis kit (Toyobo) according to the manufacturer's instructions, and the resultant plasmid was named pCMV-HBVΔ1804-2454.

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: Paragraph title: Plasmids and constructs ... The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas).

    Article Title:
    Article Snippet: The resultant clone was called mNectarine in pBAD/His B. .. Mammalian expression construct mNectarine (pDEJ6) was generated by digestion of mNectarine in pBAD/His B with XhoI and EcoRI and ligation into pcDNA3.1(−) (Invitrogen). hCNT3 in the mammalian expression vector pcDNA3.1(+) was generated by digestion of hCNT3 in yeast expression vector pYPGE15 ( ) with KpnI and EcoRI and ligation into pcDNA3.1(+) (Invitrogen). mNectarine.hCNT3 (pDEJ13) was constructed by two steps of PCR and cloning. .. The forward primer (5′-GCGGTACCGGTGGTGAGCTGAGGAGTACAGCAGC-3′) included a KpnI site, two glycine codons, and the first 20 bp of hCNT3.

    Electrophoresis:

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: After electrophoresis on 0.7% agarose gel (Roche, Germany) and determination of the size of the obtained DNA, the products of the first PCR were used as the template for the second (nested) PCR with sense and anti-sense of inner primers in V2R ( ). .. The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas).

    Incubation:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The recombinant plasmids were isolated from confirmed colonies by miniperp kit (QIAGEN) and then digested by EcoRI and NotI restriction enzymes. .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C . .. The mTEX101 fragment in pGEM-T Easy was extracted form agarose gel and then subcloned into a pET-28a (+) expression vector (Merck, Darmstadt, Germany) and was digested by the same restriction enzymes above.

    Article Title: Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages
    Article Snippet: To eliminate residual DNase I activity, the phage suspension was incubated at 37°C 10 min with 40 μl 20 mg/ml proteinase K. Following extraction of the phages with an equal volume of chloroform and the addition of EDTA to 100 mM, ½ volume of 6 M guanidine thiocyanate was added to disrupt the capsids and release the phage DNA. .. RFLP analysis was performed using 5 μg of phage DNA digested overnight at 37°C with EcoRI (Invitrogen, Carlsbad, CA).

    Article Title: Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin
    Article Snippet: S. conglobatus genomic DNA was digested with XhoI and EcoRI (FastDigest, Thermo Scientific) at 37°C for 3.5 hr. .. S. conglobatus genomic DNA was digested with XhoI and EcoRI (FastDigest, Thermo Scientific) at 37°C for 3.5 hr.

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates
    Article Snippet: Single strand cDNA (sscDNA) was synthesized using 10 µg RNA, 20 pmol/µl oligo-dT (Fermentas, Burlington, Canada), 20 pmol/µl random hexamer (Fermentas), 10 mM of dNTP mix (Fermentas) incubated at 65℃ for 10 min, followed by addition of 20 U RNase inhibitor (Fermentas), 4 µL of 5× reverse transcriptase (RT) buffer containing Tris-HCl (pH 8.3) (Fermentas), and 200 U RevertAid premium RT (Fermentas). .. Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr.

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences). .. The purified linear vector and insert were subjected to ligation reaction using T4 DNA ligase (Fermentas).

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences
    Article Snippet: Pellets were subsequently processed as described previously with sequential extraction in 10% (w/v) mixed CTAB (alklytrimethylammonium bromide), followed by sequential incubation in 1% (w/v) CTAB in 10 mM Tris-HCl (pH 8.0) containing 0.05% (w/v) digitonin; Tris-buffered saline (TBS) containing 0.05% (w/v) digitonin, 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA, 0.05% (w/v) digitonin, EDTA-free protease inhibitor cocktail (Roche), and finally extraction in 0.65 M NaCl. .. The pellets containing ∼5 × 108 nuclei were then resuspended in BamHI buffer and digested with 2000 U each of EcoRI and BamHI (Invitrogen) for 90 min at 37°C with gentle agitation, allowing digested DNA to leach out of the nuclei.

    Luciferase:

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs
    Article Snippet: The pRSV138PRE-CAT and pRSV-CAT constructs were described previously ( ). .. To construct Myc-tagged MyD88, polypyrimidine tract-binding protein 1 (PTB1), and nuclear export signal (NES)(−)RanBP plasmids, the complete cDNAs of human MyD88 carried on pCDNA3-MyD88 , PTB1 carried on His-PTB1 , and NES(−)RanBP carried on green fluorescent protein (GFP)-NES(−)RanBP1 ( ) were inserted into the EcoRI and NotI sites of pCMV/Myc vectors (Invitrogen). cDNA fragments of HBV pregenomic RNA from pCMV-HBV and the complete HBV cDNA from pCMV-HBVΔPRE were inserted into pcDNA3.1/Luc ( ) at the 3′ end of the luciferase coding sequence before the bovine growth hormone (BGH) polyadenylation signal (see Fig. ). .. The sequence of HBV(1804-2454) was deleted from pCMV-HBV by inverse PCR using a KOD-Plus mutagenesis kit (Toyobo) according to the manufacturer's instructions, and the resultant plasmid was named pCMV-HBVΔ1804-2454.

    Activity Assay:

    Article Title: Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages
    Article Snippet: To eliminate residual DNase I activity, the phage suspension was incubated at 37°C 10 min with 40 μl 20 mg/ml proteinase K. Following extraction of the phages with an equal volume of chloroform and the addition of EDTA to 100 mM, ½ volume of 6 M guanidine thiocyanate was added to disrupt the capsids and release the phage DNA. .. RFLP analysis was performed using 5 μg of phage DNA digested overnight at 37°C with EcoRI (Invitrogen, Carlsbad, CA).

    Expressing:

    Article Title:
    Article Snippet: The resultant clone was called mNectarine in pBAD/His B. .. Mammalian expression construct mNectarine (pDEJ6) was generated by digestion of mNectarine in pBAD/His B with XhoI and EcoRI and ligation into pcDNA3.1(−) (Invitrogen). hCNT3 in the mammalian expression vector pcDNA3.1(+) was generated by digestion of hCNT3 in yeast expression vector pYPGE15 ( ) with KpnI and EcoRI and ligation into pcDNA3.1(+) (Invitrogen). mNectarine.hCNT3 (pDEJ13) was constructed by two steps of PCR and cloning. .. The forward primer (5′-GCGGTACCGGTGGTGAGCTGAGGAGTACAGCAGC-3′) included a KpnI site, two glycine codons, and the first 20 bp of hCNT3.

    Transformation Assay:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The transformed bacteria were left for 1 hr in LB broth at 37°C for recovery, and later 100µl of the transformation culture was plated onto an ampicillin (100mg/ml ) (Sigma, Louis, MO, USA), IPTG (Sigma) (0.5mM ) and X-gal (Sigma), (80µg/ml ) containing LB agar plate and was cultured for 16hrs at 37°C . .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C .

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: Preliminary sequence analysis was performed using a shotgun cloning protocol. .. Phage DNA was digested using EcoRI (Invitrogen), separated on 0.8% (wt/vol) agarose gels, purified using the GeneClean II kit (Qbiogene, Irvine, CA), ligated into pUC19 or pGEM-7Z and transformed into DH5α (Invitrogen). .. Following blue-white selection on LB solid medium containing 100 μg/ml ampicillin, constructs with phage DNA inserts were isolated using a QIAprep Spin Miniprep kit (Qiagen), digested using EcoRI and viewed using gel electrophoresis.

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences). .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences).

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas). .. The DRI insert was ligated to pcDNA3 vector with a molar ratio of 3/1 (insert to vector).

    Hybridization:

    Article Title: Transcription Factor Binding and Induced Transcription Alter Chromosomal c-myc Replicator Activity
    Article Snippet: Paragraph title: Southern blot hybridization and diagnostic PCR. ... A total of 10 μg of genomic DNA was digested with either HindIII or EcoRI (Gibco-BRL) overnight, electrophoresed in 0.8% agarose gels in 1× Tris-acetate-EDTA, and blot transferred to Hybond N+ membranes (Amersham) following standard protocols ( ).

    Article Title: A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis
    Article Snippet: In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas). .. In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas).

    Electron Microscopy:

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish
    Article Snippet: For transmission electron microscopy (TEM): A 200 mesh copper grid was immersed in 40 µL of phage solution for five min before fixing the phage with glutaraldehyde solution (1%) for five min. Then, the phage samples were negatively stained with 5% (w/v ) uranyl acetate and observed by TEM (JEOL JEM-1010) operating at a voltage of 80 kV at the Vietnam National Institute of Hygiene and Epidemiology. .. The genomic DNA phages were digested using the restriction enzymes: EcoRV, EcoRI, Ncol, SalI, MspI, XmnI, and KpnI, as per the manufacturer’s instruction (ThermoFisher Scientific).

    Transfection:

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome
    Article Snippet: At 2, 4, 8 and 12d post transfection, low molecular weight DNA was purified by HIRT extraction as described before . .. 1 µg of the isolated DNA was digested with DpnI and EcoRI for 30 min at 37°C (FASTdigest, Fermentas) and separated on a 0.8% DNA Agarose gel.

    Article Title: Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site
    Article Snippet: Virion RNA was purified from filtered culture medium from transfected cells and used in RT-PCR assays [ ]. .. PCR products were digested with 10 units of EcoRI (Fermentas) according to the manufacturer's recommendations and analyzed by 2 % agarose gel.

    Sequencing:

    Article Title: Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages
    Article Snippet: Paragraph title: DNA isolation, RFLP analysis, and sequencing ... RFLP analysis was performed using 5 μg of phage DNA digested overnight at 37°C with EcoRI (Invitrogen, Carlsbad, CA).

    Article Title: Virus found in a boreal lake links ssDNA and dsDNA viruses
    Article Snippet: Paragraph title: Isolation, Sequencing, and Bioinformatics of the Phage Genome. ... The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions.

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs
    Article Snippet: The pRSV138PRE-CAT and pRSV-CAT constructs were described previously ( ). .. To construct Myc-tagged MyD88, polypyrimidine tract-binding protein 1 (PTB1), and nuclear export signal (NES)(−)RanBP plasmids, the complete cDNAs of human MyD88 carried on pCDNA3-MyD88 , PTB1 carried on His-PTB1 , and NES(−)RanBP carried on green fluorescent protein (GFP)-NES(−)RanBP1 ( ) were inserted into the EcoRI and NotI sites of pCMV/Myc vectors (Invitrogen). cDNA fragments of HBV pregenomic RNA from pCMV-HBV and the complete HBV cDNA from pCMV-HBVΔPRE were inserted into pcDNA3.1/Luc ( ) at the 3′ end of the luciferase coding sequence before the bovine growth hormone (BGH) polyadenylation signal (see Fig. ). .. The sequence of HBV(1804-2454) was deleted from pCMV-HBV by inverse PCR using a KOD-Plus mutagenesis kit (Toyobo) according to the manufacturer's instructions, and the resultant plasmid was named pCMV-HBVΔ1804-2454.

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: Paragraph title: Sequencing and bioinformatics analysis ... Phage DNA was digested using EcoRI (Invitrogen), separated on 0.8% (wt/vol) agarose gels, purified using the GeneClean II kit (Qbiogene, Irvine, CA), ligated into pUC19 or pGEM-7Z and transformed into DH5α (Invitrogen).

    Southern Blot:

    Article Title: Transcription Factor Binding and Induced Transcription Alter Chromosomal c-myc Replicator Activity
    Article Snippet: Paragraph title: Southern blot hybridization and diagnostic PCR. ... A total of 10 μg of genomic DNA was digested with either HindIII or EcoRI (Gibco-BRL) overnight, electrophoresed in 0.8% agarose gels in 1× Tris-acetate-EDTA, and blot transferred to Hybond N+ membranes (Amersham) following standard protocols ( ).

    Article Title: A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis
    Article Snippet: In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas). .. In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas).

    Ligation:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega). .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C .

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences). .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences).

    Article Title:
    Article Snippet: The resultant clone was called mNectarine in pBAD/His B. .. Mammalian expression construct mNectarine (pDEJ6) was generated by digestion of mNectarine in pBAD/His B with XhoI and EcoRI and ligation into pcDNA3.1(−) (Invitrogen). hCNT3 in the mammalian expression vector pcDNA3.1(+) was generated by digestion of hCNT3 in yeast expression vector pYPGE15 ( ) with KpnI and EcoRI and ligation into pcDNA3.1(+) (Invitrogen). mNectarine.hCNT3 (pDEJ13) was constructed by two steps of PCR and cloning. .. The forward primer (5′-GCGGTACCGGTGGTGAGCTGAGGAGTACAGCAGC-3′) included a KpnI site, two glycine codons, and the first 20 bp of hCNT3.

    Protease Inhibitor:

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences
    Article Snippet: Pellets were subsequently processed as described previously with sequential extraction in 10% (w/v) mixed CTAB (alklytrimethylammonium bromide), followed by sequential incubation in 1% (w/v) CTAB in 10 mM Tris-HCl (pH 8.0) containing 0.05% (w/v) digitonin; Tris-buffered saline (TBS) containing 0.05% (w/v) digitonin, 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA, 0.05% (w/v) digitonin, EDTA-free protease inhibitor cocktail (Roche), and finally extraction in 0.65 M NaCl. .. The pellets containing ∼5 × 108 nuclei were then resuspended in BamHI buffer and digested with 2000 U each of EcoRI and BamHI (Invitrogen) for 90 min at 37°C with gentle agitation, allowing digested DNA to leach out of the nuclei.

    Northern Blot:

    Article Title: Transcription Factor Binding and Induced Transcription Alter Chromosomal c-myc Replicator Activity
    Article Snippet: A total of 10 μg of genomic DNA was digested with either HindIII or EcoRI (Gibco-BRL) overnight, electrophoresed in 0.8% agarose gels in 1× Tris-acetate-EDTA, and blot transferred to Hybond N+ membranes (Amersham) following standard protocols ( ). .. Hybridization was performed with the prehybridization solution without salmon sperm DNA overnight at 42°C by use of the 407-bp NcoI-SmaI fragment from pFRT.myc (Neo probe), the 1.5-kb EcoRI-XbaI fragment from pHyg.FRT.TK (thymidine kinase [TK] probe), or the 756-bp EcoRI-XbaI fragment from pHyg.FRT.TK (hygromycin resistance gene [Hyg] probe).

    Cell Culture:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The transformed bacteria were left for 1 hr in LB broth at 37°C for recovery, and later 100µl of the transformation culture was plated onto an ampicillin (100mg/ml ) (Sigma, Louis, MO, USA), IPTG (Sigma) (0.5mM ) and X-gal (Sigma), (80µg/ml ) containing LB agar plate and was cultured for 16hrs at 37°C . .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C .

    Generated:

    Article Title:
    Article Snippet: The resultant clone was called mNectarine in pBAD/His B. .. Mammalian expression construct mNectarine (pDEJ6) was generated by digestion of mNectarine in pBAD/His B with XhoI and EcoRI and ligation into pcDNA3.1(−) (Invitrogen). hCNT3 in the mammalian expression vector pcDNA3.1(+) was generated by digestion of hCNT3 in yeast expression vector pYPGE15 ( ) with KpnI and EcoRI and ligation into pcDNA3.1(+) (Invitrogen). mNectarine.hCNT3 (pDEJ13) was constructed by two steps of PCR and cloning. .. The forward primer (5′-GCGGTACCGGTGGTGAGCTGAGGAGTACAGCAGC-3′) included a KpnI site, two glycine codons, and the first 20 bp of hCNT3.

    cDNA-AFLP Assay:

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates
    Article Snippet: Paragraph title: cDNA-AFLP ... Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr.

    DNA Sequencing:

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System
    Article Snippet: The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA). .. The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs
    Article Snippet: To construct Myc-tagged MyD88, polypyrimidine tract-binding protein 1 (PTB1), and nuclear export signal (NES)(−)RanBP plasmids, the complete cDNAs of human MyD88 carried on pCDNA3-MyD88 , PTB1 carried on His-PTB1 , and NES(−)RanBP carried on green fluorescent protein (GFP)-NES(−)RanBP1 ( ) were inserted into the EcoRI and NotI sites of pCMV/Myc vectors (Invitrogen). cDNA fragments of HBV pregenomic RNA from pCMV-HBV and the complete HBV cDNA from pCMV-HBVΔPRE were inserted into pcDNA3.1/Luc ( ) at the 3′ end of the luciferase coding sequence before the bovine growth hormone (BGH) polyadenylation signal (see Fig. ). .. To construct Myc-tagged MyD88, polypyrimidine tract-binding protein 1 (PTB1), and nuclear export signal (NES)(−)RanBP plasmids, the complete cDNAs of human MyD88 carried on pCDNA3-MyD88 , PTB1 carried on His-PTB1 , and NES(−)RanBP carried on green fluorescent protein (GFP)-NES(−)RanBP1 ( ) were inserted into the EcoRI and NotI sites of pCMV/Myc vectors (Invitrogen). cDNA fragments of HBV pregenomic RNA from pCMV-HBV and the complete HBV cDNA from pCMV-HBVΔPRE were inserted into pcDNA3.1/Luc ( ) at the 3′ end of the luciferase coding sequence before the bovine growth hormone (BGH) polyadenylation signal (see Fig. ).

    Transmission Assay:

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish
    Article Snippet: For transmission electron microscopy (TEM): A 200 mesh copper grid was immersed in 40 µL of phage solution for five min before fixing the phage with glutaraldehyde solution (1%) for five min. Then, the phage samples were negatively stained with 5% (w/v ) uranyl acetate and observed by TEM (JEOL JEM-1010) operating at a voltage of 80 kV at the Vietnam National Institute of Hygiene and Epidemiology. .. The genomic DNA phages were digested using the restriction enzymes: EcoRV, EcoRI, Ncol, SalI, MspI, XmnI, and KpnI, as per the manufacturer’s instruction (ThermoFisher Scientific).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site
    Article Snippet: Paragraph title: RT-PCR analysis ... PCR products were digested with 10 units of EcoRI (Fermentas) according to the manufacturer's recommendations and analyzed by 2 % agarose gel.

    Binding Assay:

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli
    Article Snippet: The coding regions for the following HSL synthases were synthesized as double-stranded oligos (IDT) with an EcoRI binding site upstream and a XbaI cut site downstream: AubI, BjaI, BraI, CerI, EsaI, LasI, LuxI, RhlI, RpaI, and SinI. .. The Modular Sender Vector and each HSL synthase oligo were cut with EcoRI and XbaI (Thermo Fisher Scientific) and ligated using T4 ligase (New England Biolabs).

    Molecular Weight:

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome
    Article Snippet: At 2, 4, 8 and 12d post transfection, low molecular weight DNA was purified by HIRT extraction as described before . .. 1 µg of the isolated DNA was digested with DpnI and EcoRI for 30 min at 37°C (FASTdigest, Fermentas) and separated on a 0.8% DNA Agarose gel.

    DNA Extraction:

    Article Title: A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis
    Article Snippet: In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas). .. In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas).

    Article Title: Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages
    Article Snippet: Paragraph title: DNA isolation, RFLP analysis, and sequencing ... RFLP analysis was performed using 5 μg of phage DNA digested overnight at 37°C with EcoRI (Invitrogen, Carlsbad, CA).

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish
    Article Snippet: Phage genomic DNA extraction and restriction analyses: Phage genomic DNA was extracted using the Phage DNA Isolation Kit (Norgen Biotek Corp, Thorold, Canada). .. The genomic DNA phages were digested using the restriction enzymes: EcoRV, EcoRI, Ncol, SalI, MspI, XmnI, and KpnI, as per the manufacturer’s instruction (ThermoFisher Scientific).

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences
    Article Snippet: The pellets containing ∼5 × 108 nuclei were then resuspended in BamHI buffer and digested with 2000 U each of EcoRI and BamHI (Invitrogen) for 90 min at 37°C with gentle agitation, allowing digested DNA to leach out of the nuclei. .. Following centrifugation, supernatants containing solubilized DNA were removed and treated with proteinase K (200 μg/mL) and SDS (0.5% [w/v]) for 10 h at 55°C.

    Plaque Assay:

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish
    Article Snippet: Phage titres were determined using both surface spread [ , ] and double-layer [ ] agar plaque assay techniques where agar plates were previously seeded with the Aeromonas sp. (×106 CFU/mL). .. The genomic DNA phages were digested using the restriction enzymes: EcoRV, EcoRI, Ncol, SalI, MspI, XmnI, and KpnI, as per the manufacturer’s instruction (ThermoFisher Scientific).

    Mutagenesis:

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs
    Article Snippet: For these plasmids, the synthesis of the pregenomic RNA is driven by the cytomegalovirus (CMV) promoter and the Tet promoter, respectively. pCMV-HBV M2 is a derivative of pCMV-HBV in which the La protein-binding sites have been mutated ( ). pCMV-HBVΔPRE is a PRE deletion mutant of pCMV-HBV ( ). .. To construct Myc-tagged MyD88, polypyrimidine tract-binding protein 1 (PTB1), and nuclear export signal (NES)(−)RanBP plasmids, the complete cDNAs of human MyD88 carried on pCDNA3-MyD88 , PTB1 carried on His-PTB1 , and NES(−)RanBP carried on green fluorescent protein (GFP)-NES(−)RanBP1 ( ) were inserted into the EcoRI and NotI sites of pCMV/Myc vectors (Invitrogen). cDNA fragments of HBV pregenomic RNA from pCMV-HBV and the complete HBV cDNA from pCMV-HBVΔPRE were inserted into pcDNA3.1/Luc ( ) at the 3′ end of the luciferase coding sequence before the bovine growth hormone (BGH) polyadenylation signal (see Fig. ).

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: The PCR products were confirmed by staining of the agarose gel with ethidium bromide using a UV transilluminator, and the obtained DNA bands were detected. .. The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas). .. DNA was extracted from the agarose gel using QIAquic kit (Qiagen, Valencia, CA, USA).

    Isolation:

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome
    Article Snippet: At 2, 4, 8 and 12d post transfection, low molecular weight DNA was purified by HIRT extraction as described before . .. 1 µg of the isolated DNA was digested with DpnI and EcoRI for 30 min at 37°C (FASTdigest, Fermentas) and separated on a 0.8% DNA Agarose gel. .. DNA was transferred to Hybond N+ membrane (GE Healthcare) and analyzed by Southern blot analysis using a 32 P dCTP labelled PCR amplified LT-fragment (rediPrime, GE Healthcare) resuspended in ULTRAhyb Solution (Ambion) as a probe.

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The recombinant plasmids were isolated from confirmed colonies by miniperp kit (QIAGEN) and then digested by EcoRI and NotI restriction enzymes. .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C .

    Article Title: Virus found in a boreal lake links ssDNA and dsDNA viruses
    Article Snippet: Paragraph title: Isolation, Sequencing, and Bioinformatics of the Phage Genome. ... The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions.

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish
    Article Snippet: Paragraph title: 4.4. Isolation and Characterization of Bacteriophages ... The genomic DNA phages were digested using the restriction enzymes: EcoRV, EcoRI, Ncol, SalI, MspI, XmnI, and KpnI, as per the manufacturer’s instruction (ThermoFisher Scientific).

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences
    Article Snippet: The pellets containing ∼5 × 108 nuclei were then resuspended in BamHI buffer and digested with 2000 U each of EcoRI and BamHI (Invitrogen) for 90 min at 37°C with gentle agitation, allowing digested DNA to leach out of the nuclei. .. Prior to DNA extraction, RNAse A was added to the digest (10 mg/mL) and incubated for 1 h at 37°C.

    Size-exclusion Chromatography:

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates
    Article Snippet: The PCR condition was an initial denaturing step of 94℃ for 5 min and 30 repetitions of denaturation at 94℃ for 30 sec, annealing at 60℃ for 30 sec, and extension at 72℃ for 45 sec with a final extension of 72℃ for 7 min. .. Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr.

    Mouse Assay:

    Article Title: A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis
    Article Snippet: Paragraph title: Generation of stau1tm1Apa Homozygous Mice. ... In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas).

    Polymerase Chain Reaction:

    Article Title: Transcription Factor Binding and Induced Transcription Alter Chromosomal c-myc Replicator Activity
    Article Snippet: Paragraph title: Southern blot hybridization and diagnostic PCR. ... A total of 10 μg of genomic DNA was digested with either HindIII or EcoRI (Gibco-BRL) overnight, electrophoresed in 0.8% agarose gels in 1× Tris-acetate-EDTA, and blot transferred to Hybond N+ membranes (Amersham) following standard protocols ( ).

    Article Title: A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis
    Article Snippet: Clones were screened for the presence of correct targeting by Southern blotting with a 5′ probe and 3′ PCR as shown in B . .. In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas).

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: White colonies were screened by 25 cycles of colony PCR under the aforementioned conditions. .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C .

    Article Title: Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin
    Article Snippet: S. conglobatus genomic DNA was digested with XhoI and EcoRI (FastDigest, Thermo Scientific) at 37°C for 3.5 hr. .. The mixture was incubated at −20°C for 10 min before precipitating the DNA by centrifugation for 10 min. After washing with 80% ethanol, the DNA was dissolved in 20 μl of water, giving a concentration of 67 ng/μl.

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System
    Article Snippet: The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA). .. The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates
    Article Snippet: Double strand cDNA (dscDNA) synthesis was performed using 40 U DNA Polymerase I (Fermentas), 1.5 U Ribonuclease H (Roche, Mannheim, Germany), and 10 mM of dNTP mix at 16℃ for 2 hr, followed by purification with High Pure PCR Clean up Micro Kit (Roche). .. Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr.

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The purification of the Pdx1 PCR product from agarose gel was performed using the Gel DNA Recovery Kit (SinaClon BioSciences, Tehran, Iran) as per the manufacturer's recommendations. .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences). .. The digested fragments were purified using the Gel DNA Recovery Kit (SinaClon BioSciences) based on the manufacturer's instructions.

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: The PCR products were confirmed by staining of the agarose gel with ethidium bromide using a UV transilluminator, and the obtained DNA bands were detected. .. The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas).

    Article Title: Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site
    Article Snippet: A third of the resultant cDNA was subjected to PCR (94°C for 1 min, 50°C for 1 min, 72°C for 1 min, for 30 cycles) with AmpliTaq DNA polymerase (Perkin-Elmer), using the same primer that was used in the RT reaction and paired with a sense R-specific primer (L1) beginning at nt 1 (5'-GCGCCAGTCCTCCGA-3'). .. PCR products were digested with 10 units of EcoRI (Fermentas) according to the manufacturer's recommendations and analyzed by 2 % agarose gel. .. A GelDoc™ EQ system (Biorad) with SigmaGel v.1.0 software (Jandel Scientific) was used to quantitate the ethidium bromide fluorescence intensity of each band.

    Article Title:
    Article Snippet: The resultant clone was called mNectarine in pBAD/His B. .. Mammalian expression construct mNectarine (pDEJ6) was generated by digestion of mNectarine in pBAD/His B with XhoI and EcoRI and ligation into pcDNA3.1(−) (Invitrogen). hCNT3 in the mammalian expression vector pcDNA3.1(+) was generated by digestion of hCNT3 in yeast expression vector pYPGE15 ( ) with KpnI and EcoRI and ligation into pcDNA3.1(+) (Invitrogen). mNectarine.hCNT3 (pDEJ13) was constructed by two steps of PCR and cloning. .. The forward primer (5′-GCGGTACCGGTGGTGAGCTGAGGAGTACAGCAGC-3′) included a KpnI site, two glycine codons, and the first 20 bp of hCNT3.

    Positron Emission Tomography:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C . .. The mTEX101 fragment in pGEM-T Easy was extracted form agarose gel and then subcloned into a pET-28a (+) expression vector (Merck, Darmstadt, Germany) and was digested by the same restriction enzymes above.

    Nested PCR:

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: After electrophoresis on 0.7% agarose gel (Roche, Germany) and determination of the size of the obtained DNA, the products of the first PCR were used as the template for the second (nested) PCR with sense and anti-sense of inner primers in V2R ( ). .. The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas).

    Purification:

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome
    Article Snippet: At 2, 4, 8 and 12d post transfection, low molecular weight DNA was purified by HIRT extraction as described before . .. 1 µg of the isolated DNA was digested with DpnI and EcoRI for 30 min at 37°C (FASTdigest, Fermentas) and separated on a 0.8% DNA Agarose gel.

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: Ligation reac tion was set up by 50ng of pGEM-T Easy Vector (Promega), three units of T4 DNA ligase, 75ng of mTEX101 purified fragment, and 6µl of Rapid 2X Ligation Buffer (Promega). .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C .

    Article Title: Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages
    Article Snippet: DNA was then purified using the GENECLEAN Turbo Kit (Qbiogene, Irvine, CA). .. RFLP analysis was performed using 5 μg of phage DNA digested overnight at 37°C with EcoRI (Invitrogen, Carlsbad, CA).

    Article Title: Virus found in a boreal lake links ssDNA and dsDNA viruses
    Article Snippet: To isolate phage DNA, purified phage particles were disrupted by treatment with 2% SDS and 1.2 µg/mL proteinase K (37 °C, 45 min), followed by phenol-ether extraction and precipitation with sodium acetate and ethanol. .. The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions. .. The same treatments were performed for the single-stranded DNA genome of φX174 (Thermo Scientific) as control.

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System
    Article Snippet: The amplified DNA fragments were analyzed by agarose gel electrophoresis and, if necessary, purified using a Gel Out kit (Thermo Fisher Scientific, Waltham, MA, USA). .. The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates
    Article Snippet: Double strand cDNA (dscDNA) synthesis was performed using 40 U DNA Polymerase I (Fermentas), 1.5 U Ribonuclease H (Roche, Mannheim, Germany), and 10 mM of dNTP mix at 16℃ for 2 hr, followed by purification with High Pure PCR Clean up Micro Kit (Roche). .. Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr. .. Digested dscDNA fragments were ligated to AFLP adaptors , 8 µg AD Mbo1, AD EcoR1, 4 µg ad Mbo1, and ad EcoR1 in a final volume of 60 µl.

    Article Title: Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex
    Article Snippet: Preliminary sequence analysis was performed using a shotgun cloning protocol. .. Phage DNA was digested using EcoRI (Invitrogen), separated on 0.8% (wt/vol) agarose gels, purified using the GeneClean II kit (Qbiogene, Irvine, CA), ligated into pUC19 or pGEM-7Z and transformed into DH5α (Invitrogen). .. Following blue-white selection on LB solid medium containing 100 μg/ml ampicillin, constructs with phage DNA inserts were isolated using a QIAprep Spin Miniprep kit (Qiagen), digested using EcoRI and viewed using gel electrophoresis.

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The purification of the Pdx1 PCR product from agarose gel was performed using the Gel DNA Recovery Kit (SinaClon BioSciences, Tehran, Iran) as per the manufacturer's recommendations. .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences). .. The digested fragments were purified using the Gel DNA Recovery Kit (SinaClon BioSciences) based on the manufacturer's instructions.

    Article Title: Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site
    Article Snippet: Virion RNA was purified from filtered culture medium from transfected cells and used in RT-PCR assays [ ]. .. PCR products were digested with 10 units of EcoRI (Fermentas) according to the manufacturer's recommendations and analyzed by 2 % agarose gel.

    Plasmid Preparation:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The recombinant plasmids were isolated from confirmed colonies by miniperp kit (QIAGEN) and then digested by EcoRI and NotI restriction enzymes. .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C . .. The mTEX101 fragment in pGEM-T Easy was extracted form agarose gel and then subcloned into a pET-28a (+) expression vector (Merck, Darmstadt, Germany) and was digested by the same restriction enzymes above.

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli
    Article Snippet: The coding regions for the following HSL synthases were synthesized as double-stranded oligos (IDT) with an EcoRI binding site upstream and a XbaI cut site downstream: AubI, BjaI, BraI, CerI, EsaI, LasI, LuxI, RhlI, RpaI, and SinI. .. The Modular Sender Vector and each HSL synthase oligo were cut with EcoRI and XbaI (Thermo Fisher Scientific) and ligated using T4 ligase (New England Biolabs). .. Plasmids containing synthases not already in the iGEM registry were submitted with the following identification keys: AubI BBa_K2033000, BjaI BBa_K2033002, BraI BBa_K2033004, CerI BBa_K2033006, SinI BBa_K2033008.

    Article Title: The structural characterization of a prophage-encoded extracellular DNase from Streptococcus pyogenes
    Article Snippet: DNase activity was assayed by a continuous method based on the differential fluorescence output of a DNA dye called PicoGreen® (Invitrogen) as described by Tolun et al. ( ) .. The plasmid vector pUC19 was linearized with EcoRI (Fermentas) and used as a homogeneous substrate for the continuous assay. .. The linearization was verified by agarose gel electrophoresis, and the restriction endonuclease inactivated in accordance with the manufacturer's instructions (heat denaturation at 65°C for 20 min).

    Article Title: Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin
    Article Snippet: S. conglobatus genomic DNA was digested with XhoI and EcoRI (FastDigest, Thermo Scientific) at 37°C for 3.5 hr. .. The mixture was incubated at −20°C for 10 min before precipitating the DNA by centrifugation for 10 min. After washing with 80% ethanol, the DNA was dissolved in 20 μl of water, giving a concentration of 67 ng/μl.

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs
    Article Snippet: To construct HBV plasmid pHBV1.3, a terminally redundant (1.3× copy), replication-competent HBV genome (subtype adw; nucleotides 957 to 1952; GenBank accession number ) was inserted into pUC19 (Promega). pCIdA-HBV expresses all the HBV proteins, but it lacks the 5′ ɛ RNA signal, which eliminates pregenomic RNA packaging and DNA replication ( ). .. To construct Myc-tagged MyD88, polypyrimidine tract-binding protein 1 (PTB1), and nuclear export signal (NES)(−)RanBP plasmids, the complete cDNAs of human MyD88 carried on pCDNA3-MyD88 , PTB1 carried on His-PTB1 , and NES(−)RanBP carried on green fluorescent protein (GFP)-NES(−)RanBP1 ( ) were inserted into the EcoRI and NotI sites of pCMV/Myc vectors (Invitrogen). cDNA fragments of HBV pregenomic RNA from pCMV-HBV and the complete HBV cDNA from pCMV-HBVΔPRE were inserted into pcDNA3.1/Luc ( ) at the 3′ end of the luciferase coding sequence before the bovine growth hormone (BGH) polyadenylation signal (see Fig. ).

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The purification of the Pdx1 PCR product from agarose gel was performed using the Gel DNA Recovery Kit (SinaClon BioSciences, Tehran, Iran) as per the manufacturer's recommendations. .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences). .. The digested fragments were purified using the Gel DNA Recovery Kit (SinaClon BioSciences) based on the manufacturer's instructions.

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas). .. The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas).

    Article Title:
    Article Snippet: The resultant clone was called mNectarine in pBAD/His B. .. Mammalian expression construct mNectarine (pDEJ6) was generated by digestion of mNectarine in pBAD/His B with XhoI and EcoRI and ligation into pcDNA3.1(−) (Invitrogen). hCNT3 in the mammalian expression vector pcDNA3.1(+) was generated by digestion of hCNT3 in yeast expression vector pYPGE15 ( ) with KpnI and EcoRI and ligation into pcDNA3.1(+) (Invitrogen). mNectarine.hCNT3 (pDEJ13) was constructed by two steps of PCR and cloning. .. The forward primer (5′-GCGGTACCGGTGGTGAGCTGAGGAGTACAGCAGC-3′) included a KpnI site, two glycine codons, and the first 20 bp of hCNT3.

    Software:

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome
    Article Snippet: 1 µg of the isolated DNA was digested with DpnI and EcoRI for 30 min at 37°C (FASTdigest, Fermentas) and separated on a 0.8% DNA Agarose gel. .. 1 µg of the isolated DNA was digested with DpnI and EcoRI for 30 min at 37°C (FASTdigest, Fermentas) and separated on a 0.8% DNA Agarose gel.

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The primers were designed using primer premier 5.0 software (Premier Biosoft, Palo alto, CA, USA) with restriction sites at the 5′ (HindIII) and 3′ (EcoRI) ends. .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences).

    Recombinant:

    Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein
    Article Snippet: The recombinant plasmids were isolated from confirmed colonies by miniperp kit (QIAGEN) and then digested by EcoRI and NotI restriction enzymes. .. Plasmid DNA (800ng ) was used in 25µl of the total volume, including NotI (15units), (Invitrogen, Carlsbad, CA, USA), EcoRI (15 units), (Invitrogen), and 2.5µl of 10 X Reaction buffer 3 (Invitrogen) and incubated for 1.5 hr at 37°C .

    Agarose Gel Electrophoresis:

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome
    Article Snippet: At 2, 4, 8 and 12d post transfection, low molecular weight DNA was purified by HIRT extraction as described before . .. 1 µg of the isolated DNA was digested with DpnI and EcoRI for 30 min at 37°C (FASTdigest, Fermentas) and separated on a 0.8% DNA Agarose gel. .. DNA was transferred to Hybond N+ membrane (GE Healthcare) and analyzed by Southern blot analysis using a 32 P dCTP labelled PCR amplified LT-fragment (rediPrime, GE Healthcare) resuspended in ULTRAhyb Solution (Ambion) as a probe.

    Article Title: Virus found in a boreal lake links ssDNA and dsDNA viruses
    Article Snippet: The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions. .. The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions.

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System
    Article Snippet: The amplified DNA fragments were analyzed by agarose gel electrophoresis and, if necessary, purified using a Gel Out kit (Thermo Fisher Scientific, Waltham, MA, USA). .. The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The purification of the Pdx1 PCR product from agarose gel was performed using the Gel DNA Recovery Kit (SinaClon BioSciences, Tehran, Iran) as per the manufacturer's recommendations. .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences). .. The digested fragments were purified using the Gel DNA Recovery Kit (SinaClon BioSciences) based on the manufacturer's instructions.

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: The PCR products were confirmed by staining of the agarose gel with ethidium bromide using a UV transilluminator, and the obtained DNA bands were detected. .. The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas).

    Article Title: Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site
    Article Snippet: A third of the resultant cDNA was subjected to PCR (94°C for 1 min, 50°C for 1 min, 72°C for 1 min, for 30 cycles) with AmpliTaq DNA polymerase (Perkin-Elmer), using the same primer that was used in the RT reaction and paired with a sense R-specific primer (L1) beginning at nt 1 (5'-GCGCCAGTCCTCCGA-3'). .. PCR products were digested with 10 units of EcoRI (Fermentas) according to the manufacturer's recommendations and analyzed by 2 % agarose gel. .. A GelDoc™ EQ system (Biorad) with SigmaGel v.1.0 software (Jandel Scientific) was used to quantitate the ethidium bromide fluorescence intensity of each band.

    Protein Binding:

    Article Title: Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs
    Article Snippet: For these plasmids, the synthesis of the pregenomic RNA is driven by the cytomegalovirus (CMV) promoter and the Tet promoter, respectively. pCMV-HBV M2 is a derivative of pCMV-HBV in which the La protein-binding sites have been mutated ( ). pCMV-HBVΔPRE is a PRE deletion mutant of pCMV-HBV ( ). .. To construct Myc-tagged MyD88, polypyrimidine tract-binding protein 1 (PTB1), and nuclear export signal (NES)(−)RanBP plasmids, the complete cDNAs of human MyD88 carried on pCDNA3-MyD88 , PTB1 carried on His-PTB1 , and NES(−)RanBP carried on green fluorescent protein (GFP)-NES(−)RanBP1 ( ) were inserted into the EcoRI and NotI sites of pCMV/Myc vectors (Invitrogen). cDNA fragments of HBV pregenomic RNA from pCMV-HBV and the complete HBV cDNA from pCMV-HBVΔPRE were inserted into pcDNA3.1/Luc ( ) at the 3′ end of the luciferase coding sequence before the bovine growth hormone (BGH) polyadenylation signal (see Fig. ).

    Random Hexamer Labeling:

    Article Title: Virus found in a boreal lake links ssDNA and dsDNA viruses
    Article Snippet: The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions. .. The purified phage genome was digested with different nucleases [DNase I (Fermentas), RNase A (Sigma-Aldrich), RNase I (Thermo Scientific), S1 nuclease (Thermo Scientific), Mung bean nuclease (Promega), and EcoRI (Fermentas)] according to the manufacturers’ instructions.

    Article Title: Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates
    Article Snippet: Single strand cDNA (sscDNA) was synthesized using 10 µg RNA, 20 pmol/µl oligo-dT (Fermentas, Burlington, Canada), 20 pmol/µl random hexamer (Fermentas), 10 mM of dNTP mix (Fermentas) incubated at 65℃ for 10 min, followed by addition of 20 U RNase inhibitor (Fermentas), 4 µL of 5× reverse transcriptase (RT) buffer containing Tris-HCl (pH 8.3) (Fermentas), and 200 U RevertAid premium RT (Fermentas). .. Double restriction digestion was carried out with 5 U MboI and 5 U EcoRI (Fermentas) on 5 µg purified dscDNA at 37℃ for 2 hr.

    Spectrophotometry:

    Article Title: Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages
    Article Snippet: Phage DNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA). .. RFLP analysis was performed using 5 μg of phage DNA digested overnight at 37°C with EcoRI (Invitrogen, Carlsbad, CA).

    Concentration Assay:

    Article Title: Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin
    Article Snippet: S. conglobatus genomic DNA was digested with XhoI and EcoRI (FastDigest, Thermo Scientific) at 37°C for 3.5 hr. .. S. conglobatus genomic DNA was digested with XhoI and EcoRI (FastDigest, Thermo Scientific) at 37°C for 3.5 hr.

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The final MgCl2 concentration was adjusted to 1.5 mm . .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences).

    Alkaline Lysis:

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas). .. The DRI insert was ligated to pcDNA3 vector with a molar ratio of 3/1 (insert to vector).

    Transmission Electron Microscopy:

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish
    Article Snippet: For transmission electron microscopy (TEM): A 200 mesh copper grid was immersed in 40 µL of phage solution for five min before fixing the phage with glutaraldehyde solution (1%) for five min. Then, the phage samples were negatively stained with 5% (w/v ) uranyl acetate and observed by TEM (JEOL JEM-1010) operating at a voltage of 80 kV at the Vietnam National Institute of Hygiene and Epidemiology. .. The genomic DNA phages were digested using the restriction enzymes: EcoRV, EcoRI, Ncol, SalI, MspI, XmnI, and KpnI, as per the manufacturer’s instruction (ThermoFisher Scientific).

    Ethanol Precipitation:

    Article Title: Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences
    Article Snippet: The pellets containing ∼5 × 108 nuclei were then resuspended in BamHI buffer and digested with 2000 U each of EcoRI and BamHI (Invitrogen) for 90 min at 37°C with gentle agitation, allowing digested DNA to leach out of the nuclei. .. Prior to DNA extraction, RNAse A was added to the digest (10 mg/mL) and incubated for 1 h at 37°C.

    Staining:

    Article Title: Protective Effects of Bacteriophages against Aeromonas hydrophila Causing Motile Aeromonas Septicemia (MAS) in Striped Catfish
    Article Snippet: For transmission electron microscopy (TEM): A 200 mesh copper grid was immersed in 40 µL of phage solution for five min before fixing the phage with glutaraldehyde solution (1%) for five min. Then, the phage samples were negatively stained with 5% (w/v ) uranyl acetate and observed by TEM (JEOL JEM-1010) operating at a voltage of 80 kV at the Vietnam National Institute of Hygiene and Epidemiology. .. The genomic DNA phages were digested using the restriction enzymes: EcoRV, EcoRI, Ncol, SalI, MspI, XmnI, and KpnI, as per the manufacturer’s instruction (ThermoFisher Scientific).

    Article Title: The combined effect of Pdx1 overexpression and Shh manipulation on the function of insulin‐producing cells derived from adipose‐tissue stem cells
    Article Snippet: The purification of the Pdx1 PCR product from agarose gel was performed using the Gel DNA Recovery Kit (SinaClon BioSciences, Tehran, Iran) as per the manufacturer's recommendations. .. The purified Pdx1 PCR product and pcDNA3.1+ vector (ThermoFisher Scientific) were double‐digested with EcoRI and HindIII restriction enzymes (Fermentas, Waltham, MA, USA) at 37 °C for 2 h. The digested fragments were electrophoresed on 1% agarose gel stained by Safe stain (SinaClon BioSciences). .. The digested fragments were purified using the Gel DNA Recovery Kit (SinaClon BioSciences) based on the manufacturer's instructions.

    Article Title: Mutation of the histidin residue of the DRH motif in vasopressin V2 receptor expression and function
    Article Snippet: The PCR products were confirmed by staining of the agarose gel with ethidium bromide using a UV transilluminator, and the obtained DNA bands were detected. .. The products containing the desired mutation were digested by BamHI, EcoRI and XbaI restriction enzymes (Fermentas).

    Homologous Recombination:

    Article Title: A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis
    Article Snippet: In brief, 10 μg of genomic DNA were digested with EcoRI (Fermentas). .. Genotyping of the mice was determined by using an [α-32 P]dCTP-radiolabeled Stau1 probe that has been generated with the Prime-It RmT random primer labeling kit (Stratagene) according to the manufacturer's protocol.

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    Thermo Fisher ecori restriction enzyme
    Integration and HDR Analysis of CRISPR/Cas9-Edited EBS hKc After co-transfection of the respective Cas9/sgRNA combinations (spCas9-sgRNA1, spCas9-sgRNA2, or both Cas9n-sgRNA1 and Cas9n-sgRNA2) and the MC donor plasmid into EBS hKc, we performed two rounds of blasticidin selection. The resulting blasticidin-resistant cells were then analyzed for site-specific integration of the donor template at the genomic level. (A) Integration-specific <t>PCR</t> using a forward primer binding the restriction sites <t>EcoRI</t> and NheI (white box) provided by the MC and a reverse primer binding downstream to the 3′ UTR of the KRT14 gene resulted in a PCR product of 893 bp. (B) EcoRI digestion to estimate the relative recombination efficiency in the treated cell pool. We performed a PCR using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 3′ UTR. The PCR resulted in a specific product of 1,189 bp. EcoRI digestion generated the expected fragments of 874 and 315 bp, only visible in the Cas9n 1- and 2-treated EBS hKc. (C) Schematic depiction of the primer binding sites used for amplification of the integration-specific PCR fragments. Gray arrows indicate a forward primer binding to the restriction sites EcoRI and NheI integrated via HDR. Black arrows indicate forward and reverse primers binding to endogenous KRT14 . Black star: missense mutation (c.1231G > A) in exon 6; gray stars: silent mutations.
    Ecori Restriction Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 216 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Integration and HDR Analysis of CRISPR/Cas9-Edited EBS hKc After co-transfection of the respective Cas9/sgRNA combinations (spCas9-sgRNA1, spCas9-sgRNA2, or both Cas9n-sgRNA1 and Cas9n-sgRNA2) and the MC donor plasmid into EBS hKc, we performed two rounds of blasticidin selection. The resulting blasticidin-resistant cells were then analyzed for site-specific integration of the donor template at the genomic level. (A) Integration-specific PCR using a forward primer binding the restriction sites EcoRI and NheI (white box) provided by the MC and a reverse primer binding downstream to the 3′ UTR of the KRT14 gene resulted in a PCR product of 893 bp. (B) EcoRI digestion to estimate the relative recombination efficiency in the treated cell pool. We performed a PCR using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 3′ UTR. The PCR resulted in a specific product of 1,189 bp. EcoRI digestion generated the expected fragments of 874 and 315 bp, only visible in the Cas9n 1- and 2-treated EBS hKc. (C) Schematic depiction of the primer binding sites used for amplification of the integration-specific PCR fragments. Gray arrows indicate a forward primer binding to the restriction sites EcoRI and NheI integrated via HDR. Black arrows indicate forward and reverse primers binding to endogenous KRT14 . Black star: missense mutation (c.1231G > A) in exon 6; gray stars: silent mutations.

    Journal: Molecular Therapy

    Article Title: Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases

    doi: 10.1016/j.ymthe.2017.08.015

    Figure Lengend Snippet: Integration and HDR Analysis of CRISPR/Cas9-Edited EBS hKc After co-transfection of the respective Cas9/sgRNA combinations (spCas9-sgRNA1, spCas9-sgRNA2, or both Cas9n-sgRNA1 and Cas9n-sgRNA2) and the MC donor plasmid into EBS hKc, we performed two rounds of blasticidin selection. The resulting blasticidin-resistant cells were then analyzed for site-specific integration of the donor template at the genomic level. (A) Integration-specific PCR using a forward primer binding the restriction sites EcoRI and NheI (white box) provided by the MC and a reverse primer binding downstream to the 3′ UTR of the KRT14 gene resulted in a PCR product of 893 bp. (B) EcoRI digestion to estimate the relative recombination efficiency in the treated cell pool. We performed a PCR using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 3′ UTR. The PCR resulted in a specific product of 1,189 bp. EcoRI digestion generated the expected fragments of 874 and 315 bp, only visible in the Cas9n 1- and 2-treated EBS hKc. (C) Schematic depiction of the primer binding sites used for amplification of the integration-specific PCR fragments. Gray arrows indicate a forward primer binding to the restriction sites EcoRI and NheI integrated via HDR. Black arrows indicate forward and reverse primers binding to endogenous KRT14 . Black star: missense mutation (c.1231G > A) in exon 6; gray stars: silent mutations.

    Article Snippet: For estimation of the HDR efficiency, we PCR-amplified the KRT14 target region using a forward primer binding to KRT14 exon 6 (5′-CAGGAGATGATTGGCAGCGTGG-3′) and again a reverse primer binding to the endogenous 3′ downstream sequence (5′-GATGCTTCTCCCACTTTCTCCCC-3′) of the KRT14 gene, resulting in a PCR product of 1,189 bp, which was subsequently digested with EcoRI restriction enzyme (Thermo Fisher).

    Techniques: CRISPR, Cotransfection, Plasmid Preparation, Selection, Polymerase Chain Reaction, Binding Assay, Sequencing, Generated, Amplification, Mutagenesis

    Analysis of HDR Efficiency (A) PCR analysis of genomic DNA from CRISPR/Cas9-treated EBS hKc using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 . To minimize the size of the PCR product and to facilitate subcloning, we performed a nested PCR using a forward primer binding to exon 6 and a reverse primer binding to intron 7, resulting in a specific product of 507 bp. An EcoRI restriction digest confirmed the presence of recombined KRT14 alleles in treated EBS cells. Upon bacterial transformation, single colonies were picked for colony PCR and amplified product digested with EcoRI to identify recombined alleles. (B) Sequence analysis of one representative single clone, containing a modified wild-type allele (green arrow: wild-type sequence at mutation site [position 178] within exon 6), is shown. The allele additionally contains the introduced restriction sites NheI/EcoRI and the silent mutations (blue stars). Sequence alignment was performed with the software “Multiple sequence alignment with hierarchical clustering.” 21

    Journal: Molecular Therapy

    Article Title: Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases

    doi: 10.1016/j.ymthe.2017.08.015

    Figure Lengend Snippet: Analysis of HDR Efficiency (A) PCR analysis of genomic DNA from CRISPR/Cas9-treated EBS hKc using a forward primer binding to exon 6 and a reverse primer binding a sequence downstream of KRT14 . To minimize the size of the PCR product and to facilitate subcloning, we performed a nested PCR using a forward primer binding to exon 6 and a reverse primer binding to intron 7, resulting in a specific product of 507 bp. An EcoRI restriction digest confirmed the presence of recombined KRT14 alleles in treated EBS cells. Upon bacterial transformation, single colonies were picked for colony PCR and amplified product digested with EcoRI to identify recombined alleles. (B) Sequence analysis of one representative single clone, containing a modified wild-type allele (green arrow: wild-type sequence at mutation site [position 178] within exon 6), is shown. The allele additionally contains the introduced restriction sites NheI/EcoRI and the silent mutations (blue stars). Sequence alignment was performed with the software “Multiple sequence alignment with hierarchical clustering.” 21

    Article Snippet: For estimation of the HDR efficiency, we PCR-amplified the KRT14 target region using a forward primer binding to KRT14 exon 6 (5′-CAGGAGATGATTGGCAGCGTGG-3′) and again a reverse primer binding to the endogenous 3′ downstream sequence (5′-GATGCTTCTCCCACTTTCTCCCC-3′) of the KRT14 gene, resulting in a PCR product of 1,189 bp, which was subsequently digested with EcoRI restriction enzyme (Thermo Fisher).

    Techniques: Polymerase Chain Reaction, CRISPR, Binding Assay, Sequencing, Subcloning, Nested PCR, Electroporation Bacterial Transformation, Amplification, Modification, Mutagenesis, Software

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0202294

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

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

    Techniques: Plasmid Preparation, Clone Assay, Expressing

    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.

    Journal: Chemistry & Biology

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

    doi: 10.1016/j.chembiol.2015.05.010

    Figure Lengend 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.

    Article Snippet: S. conglobatus genomic DNA was digested with XhoI and EcoRI (FastDigest, Thermo Scientific) at 37°C for 3.5 hr.

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

    Restriction patterns of DNA extracted from purified virions cleaved with the selected REases. Panel A: HindIII (H), Eco32I (E32), SalI (S) and EcoRI (EI). ND, undigested DNA. M, GeneRuler 100- to 10,000-bp size marker. λ/H, λ DNA cleaved with HindIII. ~14 kb restriction fragment of EcoR32I digested virion DNA is marked with a triangle. Panel B: Arrows indicate restriction fragments assigned to ФAH14a (left) and ФAH14b (right) genomic DNA, obtained by SalI digestion.

    Journal: PLoS ONE

    Article Title: Two Inducible Prophages of an Antarctic Pseudomonas sp. ANT_H14 Use the Same Capsid for Packaging Their Genomes – Characterization of a Novel Phage Helper-Satellite System

    doi: 10.1371/journal.pone.0158889

    Figure Lengend Snippet: Restriction patterns of DNA extracted from purified virions cleaved with the selected REases. Panel A: HindIII (H), Eco32I (E32), SalI (S) and EcoRI (EI). ND, undigested DNA. M, GeneRuler 100- to 10,000-bp size marker. λ/H, λ DNA cleaved with HindIII. ~14 kb restriction fragment of EcoR32I digested virion DNA is marked with a triangle. Panel B: Arrows indicate restriction fragments assigned to ФAH14a (left) and ФAH14b (right) genomic DNA, obtained by SalI digestion.

    Article Snippet: The test for the presence of cohesive ends of the phage genome was performed as previously described [ ], using the following REases: HindIII, SalI, EcoRI, Eco32I and PstI (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Purification, Marker