pcr2 1 vector  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pcr2 1 vector
    SDS-PAGE analysis of LPSs expressed by gmhA -deficient E. coli χ711 and related E. coli strains. LPS present in proteinase K-treated whole-cell lysates was resolved by Tricine-SDS-PAGE and stained with silver. Lanes: A, E. coli χ705 with a wild-type E. coli gmhA gene; B, E. coli χ711; C, E. coli χ711 containing the <t>pCR2.1</t> vector; D, E. coli χ711 containing pCR253 with the wild-type H. ducreyi gmhA gene.
    Pcr2 1 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 38 article reviews
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    pcr2 1 vector - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence"

    Article Title: Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence

    Journal: Infection and Immunity

    doi:

    SDS-PAGE analysis of LPSs expressed by gmhA -deficient E. coli χ711 and related E. coli strains. LPS present in proteinase K-treated whole-cell lysates was resolved by Tricine-SDS-PAGE and stained with silver. Lanes: A, E. coli χ705 with a wild-type E. coli gmhA gene; B, E. coli χ711; C, E. coli χ711 containing the pCR2.1 vector; D, E. coli χ711 containing pCR253 with the wild-type H. ducreyi gmhA gene.
    Figure Legend Snippet: SDS-PAGE analysis of LPSs expressed by gmhA -deficient E. coli χ711 and related E. coli strains. LPS present in proteinase K-treated whole-cell lysates was resolved by Tricine-SDS-PAGE and stained with silver. Lanes: A, E. coli χ705 with a wild-type E. coli gmhA gene; B, E. coli χ711; C, E. coli χ711 containing the pCR2.1 vector; D, E. coli χ711 containing pCR253 with the wild-type H. ducreyi gmhA gene.

    Techniques Used: SDS Page, Staining, Plasmid Preparation

    2) Product Images from "Characterization of a WaaF (RfaF) Homolog Expressed by Haemophilus ducreyi"

    Article Title: Characterization of a WaaF (RfaF) Homolog Expressed by Haemophilus ducreyi

    Journal: Infection and Immunity

    doi:

    SDS-PAGE analysis of LPS expressed by the S. typhimurium waaF mutant strain 3789 and related S. typhimurium strains. LPS present in proteinase K-treated whole-cell lysates was resolved by Tricine-SDS-PAGE and stained with silver. Lanes: 1, S. typhimurium 3770, with a wild-type waaF gene; 2, S. typhimurium 3789; 3, S. typhimurium 3789 containing the pCR2.1 vector; 4, S. typhimurium 3789 containing pBB12 with the wild-type H. ducreyi waaF gene.
    Figure Legend Snippet: SDS-PAGE analysis of LPS expressed by the S. typhimurium waaF mutant strain 3789 and related S. typhimurium strains. LPS present in proteinase K-treated whole-cell lysates was resolved by Tricine-SDS-PAGE and stained with silver. Lanes: 1, S. typhimurium 3770, with a wild-type waaF gene; 2, S. typhimurium 3789; 3, S. typhimurium 3789 containing the pCR2.1 vector; 4, S. typhimurium 3789 containing pBB12 with the wild-type H. ducreyi waaF gene.

    Techniques Used: SDS Page, Mutagenesis, Staining, Plasmid Preparation

    3) Product Images from "The N-Terminal Part of the Enzyme Component (C2I) of the Binary Clostridium botulinum C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin"

    Article Title: The N-Terminal Part of the Enzyme Component (C2I) of the Binary Clostridium botulinum C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin

    Journal: Infection and Immunity

    doi:

    Construction of plasmid pGEX-C2IN. The C2I gene (1,293 bp) from C. botulinum KZZ 1577 was amplified from chromosomal DNA by PCR using primers C2IC, containing a Bgl II site, and C2IN, containing a Bam HI site, and cloned into pCR2.1 vector (A). For expression experiments the C2I gene was excised with Bgl II/ Bsa BI and cloned into Bam HI/ Sma I-digested pGEX2T, resulting in plasmid pGEX2T-C2I (B). pGEX2T-C2IN was constructed by Bam HI digestion of pGEX2T-C2I and religation of the pGEX2T-C2IN fragment. The construct was identified by DNA sequencing. To construct the fusion toxin C2IN-C3 (D), the C. limosum C3 gene was excised from the pCR2.1 vector harboring C3 (C) by restriction with Bgl II and Bam HI and ligated with Bam HI-digested pGEX-C2IN. The construct was confirmed by DNA sequencing.
    Figure Legend Snippet: Construction of plasmid pGEX-C2IN. The C2I gene (1,293 bp) from C. botulinum KZZ 1577 was amplified from chromosomal DNA by PCR using primers C2IC, containing a Bgl II site, and C2IN, containing a Bam HI site, and cloned into pCR2.1 vector (A). For expression experiments the C2I gene was excised with Bgl II/ Bsa BI and cloned into Bam HI/ Sma I-digested pGEX2T, resulting in plasmid pGEX2T-C2I (B). pGEX2T-C2IN was constructed by Bam HI digestion of pGEX2T-C2I and religation of the pGEX2T-C2IN fragment. The construct was identified by DNA sequencing. To construct the fusion toxin C2IN-C3 (D), the C. limosum C3 gene was excised from the pCR2.1 vector harboring C3 (C) by restriction with Bgl II and Bam HI and ligated with Bam HI-digested pGEX-C2IN. The construct was confirmed by DNA sequencing.

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay, Expressing, Construct, DNA Sequencing

    4) Product Images from "Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA"

    Article Title: Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA

    Journal: Journal of Virology

    doi:

    Construction of the full-length cDNA clone of IPNV segment A for the generation of plus-sense RNA transcript with T7 RNA polymerase. The gene structure of IPNV segment A and its encoded proteins are shown at the top. Overlapping cDNA segments of IPNV were generated by RT-PCR and cloned into a pCR2.1 vector to obtain various pCR clones, as indicated. These plasmids were digested with appropriate restriction enzymes, and the resulting segments were cloned into a pUC19 vector to obtain plasmid pUC19WBA. This plasmid contains a T7 RNA polymerase promoter sequence at its 5′ end. Restriction enzymes used for the construction or linearization of the full-length clone are indicated. Abbreviations: A, Apa I; E, Eco RI; K, Asp 718; S, Sal I.
    Figure Legend Snippet: Construction of the full-length cDNA clone of IPNV segment A for the generation of plus-sense RNA transcript with T7 RNA polymerase. The gene structure of IPNV segment A and its encoded proteins are shown at the top. Overlapping cDNA segments of IPNV were generated by RT-PCR and cloned into a pCR2.1 vector to obtain various pCR clones, as indicated. These plasmids were digested with appropriate restriction enzymes, and the resulting segments were cloned into a pUC19 vector to obtain plasmid pUC19WBA. This plasmid contains a T7 RNA polymerase promoter sequence at its 5′ end. Restriction enzymes used for the construction or linearization of the full-length clone are indicated. Abbreviations: A, Apa I; E, Eco RI; K, Asp 718; S, Sal I.

    Techniques Used: Generated, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Sequencing

    Construction of the full-length cDNA clone of IPNV segment B for the synthesis of plus-sense RNA transcript with T7 RNA polymerase. The genome segment B of IPNV encodes the RNA-dependent RNA polymerase, VP1, which is shown at the top. Overlapping cDNA segments of IPNV were cloned into the pCR2.1 vector to obtain various pCR clones, as shown. These plasmids were digested with appropriate restriction enzymes, and the resulting segments were cloned into the pUC19 vector to obtain plasmids pUC19B5′#2 and pUC19B3′#5. Finally, a full-length plasmid, pUC19WBB, which contains a T7 RNA polymerase promoter sequence at its 5′ end, was obtained from these two clones. Restriction enzymes used for the construction of the above plasmids or for linearization of the full-length clone are indicated. Abbreviations: B, Bam HI; E, Eco RI; H, Hin dIII; K, Asp 718; P, Pst I.
    Figure Legend Snippet: Construction of the full-length cDNA clone of IPNV segment B for the synthesis of plus-sense RNA transcript with T7 RNA polymerase. The genome segment B of IPNV encodes the RNA-dependent RNA polymerase, VP1, which is shown at the top. Overlapping cDNA segments of IPNV were cloned into the pCR2.1 vector to obtain various pCR clones, as shown. These plasmids were digested with appropriate restriction enzymes, and the resulting segments were cloned into the pUC19 vector to obtain plasmids pUC19B5′#2 and pUC19B3′#5. Finally, a full-length plasmid, pUC19WBB, which contains a T7 RNA polymerase promoter sequence at its 5′ end, was obtained from these two clones. Restriction enzymes used for the construction of the above plasmids or for linearization of the full-length clone are indicated. Abbreviations: B, Bam HI; E, Eco RI; H, Hin dIII; K, Asp 718; P, Pst I.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Sequencing

    5) Product Images from "Penicillin-Binding Proteins in Leptospira interrogans"

    Article Title: Penicillin-Binding Proteins in Leptospira interrogans

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.45.3.870-877.2001

    Binding of DIG-AMP to L. interrogans PBP 1 and PBP 3. Lysates of E. coli cells harboring recombinant or vector plasmids were incubated with DIG-AMP, separated by SDS-PAGE, and transferred to a membrane, and the modified proteins were detected as described in Materials and Methods. (A) Production of strain Verdun PBP 3 from pET-PBP3, a recombinant plasmid in E. coli . pET-26b(+) is the expression vector (lane 1). pET-PBP3 is the recombinant plasmid carrying pbpB from strain Verdun (lanes 2 and 3). Binding of DIG-AMP was also assayed in the presence of free AMP (lane 3). The positions of E. coli PBP 5, PBP 6, and PBP 7 and L. interrogans strain Verdun PBP 3 are indicated on the left. The migration of size standards is indicated on the right (in kilodaltons). (B) Production of strain RZ11 PBP 1 from p513-3, a recombinant plasmid in E. coli . pCR2.1 is the expression vector (lane 1). p513-3 is the recombinant plasmid carrying ponA from strain RZ11 (lane 2). The migration of size standards is indicated on the right (in kilodaltons). L. interrogans strain RZ11 PBP 1 is labeled.
    Figure Legend Snippet: Binding of DIG-AMP to L. interrogans PBP 1 and PBP 3. Lysates of E. coli cells harboring recombinant or vector plasmids were incubated with DIG-AMP, separated by SDS-PAGE, and transferred to a membrane, and the modified proteins were detected as described in Materials and Methods. (A) Production of strain Verdun PBP 3 from pET-PBP3, a recombinant plasmid in E. coli . pET-26b(+) is the expression vector (lane 1). pET-PBP3 is the recombinant plasmid carrying pbpB from strain Verdun (lanes 2 and 3). Binding of DIG-AMP was also assayed in the presence of free AMP (lane 3). The positions of E. coli PBP 5, PBP 6, and PBP 7 and L. interrogans strain Verdun PBP 3 are indicated on the left. The migration of size standards is indicated on the right (in kilodaltons). (B) Production of strain RZ11 PBP 1 from p513-3, a recombinant plasmid in E. coli . pCR2.1 is the expression vector (lane 1). p513-3 is the recombinant plasmid carrying ponA from strain RZ11 (lane 2). The migration of size standards is indicated on the right (in kilodaltons). L. interrogans strain RZ11 PBP 1 is labeled.

    Techniques Used: Binding Assay, Recombinant, Plasmid Preparation, Incubation, SDS Page, Modification, Positron Emission Tomography, Expressing, Migration, Labeling

    6) Product Images from "Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity"

    Article Title: Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity

    Journal: Journal of Virology

    doi:

    Expression of HVS-Bcl-2 and Bcl-x L in transfected cell lines. (A) Transcripts of HVS-Bcl-2 were detected by RNase protection. Different HVS- bcl-2 -transfected clones derived from Jurkat cells (Ju) or DO cells were analyzed. Control cells contained the expression vector pCEP4 without insert. In cell lines transfected with HVS- bcl-2 , the riboprobe of 700 nucleotides (nt) specifically protected a fragment of 321 nucleotides. This protected fragment of 321 nucleotides consists of 250 nucleotides of HVS- bcl-2 , 48 nucleotides of pCR2.1, and 23 nucleotides of pCEP4. As a positive control, we used T-cell cultures from Callithrix jacchus that release infectious virus and transcribe HVS- bcl-2 ). When RNA from these cells is used, the applied riboprobe protects a fragment of about 250 nucleotides, representing the calculated length of the viral transcript that is mirrored in the riboprobe. (B) The expression of Bcl-x L in transfected Jurkat cells was detected by Western blotting.
    Figure Legend Snippet: Expression of HVS-Bcl-2 and Bcl-x L in transfected cell lines. (A) Transcripts of HVS-Bcl-2 were detected by RNase protection. Different HVS- bcl-2 -transfected clones derived from Jurkat cells (Ju) or DO cells were analyzed. Control cells contained the expression vector pCEP4 without insert. In cell lines transfected with HVS- bcl-2 , the riboprobe of 700 nucleotides (nt) specifically protected a fragment of 321 nucleotides. This protected fragment of 321 nucleotides consists of 250 nucleotides of HVS- bcl-2 , 48 nucleotides of pCR2.1, and 23 nucleotides of pCEP4. As a positive control, we used T-cell cultures from Callithrix jacchus that release infectious virus and transcribe HVS- bcl-2 ). When RNA from these cells is used, the applied riboprobe protects a fragment of about 250 nucleotides, representing the calculated length of the viral transcript that is mirrored in the riboprobe. (B) The expression of Bcl-x L in transfected Jurkat cells was detected by Western blotting.

    Techniques Used: Expressing, Transfection, Clone Assay, Derivative Assay, Plasmid Preparation, Positive Control, Western Blot

    7) Product Images from "SipA Is Required for Pilus Formation in Streptococcus pyogenes Serotype M3 ▿"

    Article Title: SipA Is Required for Pilus Formation in Streptococcus pyogenes Serotype M3 ▿

    Journal:

    doi: 10.1128/JB.01520-07

    CpaHA-T3 linkage in E. coli . (A) Western immunoblot with cell lysates of E. coli TOP10 strains containing vector pCR2.1 (lane 1), plasmid pJRS1325 (lane 2), and pJRS1326 (lane 3) reacted with HA-7 monoclonal antibody. The positions of monomeric CpaHA
    Figure Legend Snippet: CpaHA-T3 linkage in E. coli . (A) Western immunoblot with cell lysates of E. coli TOP10 strains containing vector pCR2.1 (lane 1), plasmid pJRS1325 (lane 2), and pJRS1326 (lane 3) reacted with HA-7 monoclonal antibody. The positions of monomeric CpaHA

    Techniques Used: Western Blot, Plasmid Preparation

    FCT region of serotype M3 GAS strain AM3 and derived constructs. The positions of HA tags and mutations introduced by site-specific mutagenesis are indicated by arrowheads. Vectors pCR2.1 and pCR-XL are E. coli cloning vectors (Invitrogen), and pNZ276
    Figure Legend Snippet: FCT region of serotype M3 GAS strain AM3 and derived constructs. The positions of HA tags and mutations introduced by site-specific mutagenesis are indicated by arrowheads. Vectors pCR2.1 and pCR-XL are E. coli cloning vectors (Invitrogen), and pNZ276

    Techniques Used: Derivative Assay, Construct, Mutagenesis, Polymerase Chain Reaction, Clone Assay

    T3 polymerization in E. coli : Western immunoblot analysis of cell lysates of E. coli TOP10 strains containing the vector pCR2.1 (lane 1), plasmid pEU7655 (lane 2), pEU7664 (lane 3), pEU7665 (lane 4), and pEU7657 (lane 5) reacted with polyclonal anti-T3
    Figure Legend Snippet: T3 polymerization in E. coli : Western immunoblot analysis of cell lysates of E. coli TOP10 strains containing the vector pCR2.1 (lane 1), plasmid pEU7655 (lane 2), pEU7664 (lane 3), pEU7665 (lane 4), and pEU7657 (lane 5) reacted with polyclonal anti-T3

    Techniques Used: Western Blot, Plasmid Preparation

    Effect of sipA2 deletion on CpaHA-T3 polymerization: Western immunoblots with cell lysates of E. coli TOP10 strains resuspended in RIPA buffer and disrupted by sonication. The lanes contained cell lysates from cells with vector pCR2.1 (lane 1), pJRS1326
    Figure Legend Snippet: Effect of sipA2 deletion on CpaHA-T3 polymerization: Western immunoblots with cell lysates of E. coli TOP10 strains resuspended in RIPA buffer and disrupted by sonication. The lanes contained cell lysates from cells with vector pCR2.1 (lane 1), pJRS1326

    Techniques Used: Western Blot, Sonication, Plasmid Preparation

    8) Product Images from "Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein"

    Article Title: Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein

    Journal: Journal of Virology

    doi:

    Characterization of products of integration reactions with lysed virions by digestion with restriction enzymes. Unintegrated HIV cDNA and solo LTR substrates have U3 sequences at one end and U5 sequences at the other end. (A) Autoradiogram of integration products analyzed by cleavage with restriction enzymes. Lane 1 displays the isolated integration product. Lane 2 presents the partially purified tagged circle form. Lane 3 presents the isolated two-LTR coupled form. Lanes 4 and 5 show these two forms cleaved with Ppu MI. Lanes 6 to 8 contain the indicated markers. (B) Expected structure of the predominant tagged circle form. (C) Major coupled two-LTR products. The target DNA used was circular pCR2.1 (3.9 kb); the LTR DNA was 636 bp in length. The major products detected in panel A are as expected from the diagrams in panels B and C. The expected tagged circle form cleaved with Ppu M1 runs between the uncleaved form and the relaxed circle form of the target DNA. Cleavage of the two-LTR coupled-joining product yielded two bands that migrated between the 4,822- and 4,361-bp markers; the expected sizes are 4,700 and 4,530 bp. Due to preferential joining of the U5 end, coupled products contained either two U5 ends or one U5 end and one U3 end joined to the target (C).
    Figure Legend Snippet: Characterization of products of integration reactions with lysed virions by digestion with restriction enzymes. Unintegrated HIV cDNA and solo LTR substrates have U3 sequences at one end and U5 sequences at the other end. (A) Autoradiogram of integration products analyzed by cleavage with restriction enzymes. Lane 1 displays the isolated integration product. Lane 2 presents the partially purified tagged circle form. Lane 3 presents the isolated two-LTR coupled form. Lanes 4 and 5 show these two forms cleaved with Ppu MI. Lanes 6 to 8 contain the indicated markers. (B) Expected structure of the predominant tagged circle form. (C) Major coupled two-LTR products. The target DNA used was circular pCR2.1 (3.9 kb); the LTR DNA was 636 bp in length. The major products detected in panel A are as expected from the diagrams in panels B and C. The expected tagged circle form cleaved with Ppu M1 runs between the uncleaved form and the relaxed circle form of the target DNA. Cleavage of the two-LTR coupled-joining product yielded two bands that migrated between the 4,822- and 4,361-bp markers; the expected sizes are 4,700 and 4,530 bp. Due to preferential joining of the U5 end, coupled products contained either two U5 ends or one U5 end and one U3 end joined to the target (C).

    Techniques Used: Isolation, Purification

    9) Product Images from "Identification of the source of elevated hepatocyte growth factor levels in multiple myeloma patients"

    Article Title: Identification of the source of elevated hepatocyte growth factor levels in multiple myeloma patients

    Journal: Biomarker Research

    doi: 10.1186/2050-7771-2-8

    Characterization of DATE in the HGF gene promoter region. (A) and (B) Representative DNA sequencing traces of patients MM 14 and MM 22 with DATEs of 29 and 15 nucleotides, respectively. DATEs of individual patients were amplified by nested PCR, cloned into TA cloning vector pCR2.1 and sequenced using M13 standard primers. (C) HGF mRNA levels in CD138 + cells are plotted against the number of nucleotides present in DATE of the corresponding samples. CD138 + cells purified from bone marrow of myeloma patients were used to quantify HGF mRNA levels by real-time PCR. Corresponding samples were used to isolate genomic DNA for sequencing of DATE in the HGF promoter region. Data shown are HGF mRNA mean fold change ± standard deviation and the number of nucleotides quantified by sequencing.
    Figure Legend Snippet: Characterization of DATE in the HGF gene promoter region. (A) and (B) Representative DNA sequencing traces of patients MM 14 and MM 22 with DATEs of 29 and 15 nucleotides, respectively. DATEs of individual patients were amplified by nested PCR, cloned into TA cloning vector pCR2.1 and sequenced using M13 standard primers. (C) HGF mRNA levels in CD138 + cells are plotted against the number of nucleotides present in DATE of the corresponding samples. CD138 + cells purified from bone marrow of myeloma patients were used to quantify HGF mRNA levels by real-time PCR. Corresponding samples were used to isolate genomic DNA for sequencing of DATE in the HGF promoter region. Data shown are HGF mRNA mean fold change ± standard deviation and the number of nucleotides quantified by sequencing.

    Techniques Used: DNA Sequencing, Amplification, Nested PCR, Clone Assay, TA Cloning, Plasmid Preparation, Purification, Real-time Polymerase Chain Reaction, Sequencing, Standard Deviation

    10) Product Images from "Conservation and Heterogeneity of vlsE among Human and Tick Isolates of Borrelia burgdorferi"

    Article Title: Conservation and Heterogeneity of vlsE among Human and Tick Isolates of Borrelia burgdorferi

    Journal: Infection and Immunity

    doi:

    Comparison of sequences from representative clinical isolate and tick vlsE PCR products with the B. burgdorferi B31-5A3 vlsE sequence. PCR products were cloned into the plasmid pCR2.1 and sequenced as described in the text. Deduced amino acid sequences were aligned with the B31-5A3 vlsE sequence by using ClustalW and formatted by using Boxshade. Dashes indicate identity with the B31-5A3 sequence, lowercase letters identify similar residues, and capital letters represent dissimilar amino acids. Variable regions (VR) I through VI previously identified by alignment of B31-5A3 vls silent cassette sequences with vlsE ) are shown. (A) Alignment of sequences from Lyme disease clinical isolates B14, B294, B296, and B247. Deduced sequences from two different PCR product clones (e.g., B14-2 and B14-3) are shown for each of the isolates except B247, where only one PCR product clone was sequenced. (B) Sequences obtained from PCR product clones obtained from tick W2F (clones W2F-2 and W2F-3) and tick W12M (clone W12M-1).
    Figure Legend Snippet: Comparison of sequences from representative clinical isolate and tick vlsE PCR products with the B. burgdorferi B31-5A3 vlsE sequence. PCR products were cloned into the plasmid pCR2.1 and sequenced as described in the text. Deduced amino acid sequences were aligned with the B31-5A3 vlsE sequence by using ClustalW and formatted by using Boxshade. Dashes indicate identity with the B31-5A3 sequence, lowercase letters identify similar residues, and capital letters represent dissimilar amino acids. Variable regions (VR) I through VI previously identified by alignment of B31-5A3 vls silent cassette sequences with vlsE ) are shown. (A) Alignment of sequences from Lyme disease clinical isolates B14, B294, B296, and B247. Deduced sequences from two different PCR product clones (e.g., B14-2 and B14-3) are shown for each of the isolates except B247, where only one PCR product clone was sequenced. (B) Sequences obtained from PCR product clones obtained from tick W2F (clones W2F-2 and W2F-3) and tick W12M (clone W12M-1).

    Techniques Used: Polymerase Chain Reaction, Sequencing, Clone Assay, Plasmid Preparation

    11) Product Images from "Clostridium perfringens Delta Toxin Is Sequence Related to Beta Toxin, NetB, and Staphylococcus Pore-Forming Toxins, but Shows Functional Differences"

    Article Title: Clostridium perfringens Delta Toxin Is Sequence Related to Beta Toxin, NetB, and Staphylococcus Pore-Forming Toxins, but Shows Functional Differences

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0003764

    Delta gene organization in C. perfringens and cloning strategy of the Delta toxin gene. C. perfringens cloned DNA fragments into pCR2.1 yielding pMRP980 (insert of 1048 bp) and pMRP680 (insert of 1949 bp) are shown. P723 is an oligonucleotide deduced from an internal protein sequence ( Table 1 ), P1212 and P1283 are the oligonucleotides designed for cloning the upstream part of cpd by inverse PCR.
    Figure Legend Snippet: Delta gene organization in C. perfringens and cloning strategy of the Delta toxin gene. C. perfringens cloned DNA fragments into pCR2.1 yielding pMRP980 (insert of 1048 bp) and pMRP680 (insert of 1949 bp) are shown. P723 is an oligonucleotide deduced from an internal protein sequence ( Table 1 ), P1212 and P1283 are the oligonucleotides designed for cloning the upstream part of cpd by inverse PCR.

    Techniques Used: Clone Assay, Sequencing, Inverse PCR

    12) Product Images from "IFNγ induces DNA methylation-silenced GPR109A expression via pSTAT1/p300 and H3K18 acetylation in colon cancer"

    Article Title: IFNγ induces DNA methylation-silenced GPR109A expression via pSTAT1/p300 and H3K18 acetylation in colon cancer

    Journal: Cancer immunology research

    doi: 10.1158/2326-6066.CIR-14-0164

    The human GPR109A promoter is methylated and GPR109A expression is silenced in human colon carcinoma cells A . GPR109A expression level in matched pairs of human normal colon and colon carcinoma tissues. Colon carcinoma tissues and adjacent normal tissues were collected from 6 patients, and analyzed for GPR109A expression by RT-PCR. GAPDH was used as normalization control. Bottom panel: the GPR109A levels were quantified using the NIH J program. The ratio of GPR109A vs GAPDH in patient #1 was arbitrarily set at 1. The GPR109A expression levels of the remaining five specimens were then normalized based on patient #1. B . Methylation status of the GPR109A gene promoter in human colon carcinoma specimens. Genomic DNA was isolated from colon carcinoma specimens of 5 colon cancer patients and modified with bisulfate. The modified DNA was then analyzed by MS-PCR (U, unmethylated; M, methylated). Numbers above the figure are patient codes. C . Inhibition of DNA methylation increases GPR109A expression. SW116 and T84 cells were treated with Aza-dC for 3 days at the indicated doses and analyzed for GPR109A expression level by semi-quantitative RT-PCR (top panel) and real-time RT-PCR (bottom panel). The GPR109A expression levels of untreated cells were arbitrarily set at 1. Column : mean, bar : SD. D . Methylation level of the human GPR109A gene promoter in human colon carcinoma cell lines. The human GPR109A gene DNA sequence was exported from the human genome database and analyzed for CpG islands using MethyPrimer computer program. Top panel: the human GPR109A gene promoter structure. Vertical bars under the line indicate location of CpG dinucleotides, and the number under the line indicates nucleotide number relative to GPR109A transcription initiation site (+1). Bottom panel: methylation level of the GPR109A gene promoter in the indicated cell lines. Genomic DNA was modified with bisulfite. The indicated DNA fragment was amplified by PCR and cloned into pCR2.1 vector. Individual clones for each cell line were sequenced and the methylation level of the cytosine in the CpGs was analyzed using QUMA computer program (open circle, unmethylated CpG; closed circle, methylated CpG).
    Figure Legend Snippet: The human GPR109A promoter is methylated and GPR109A expression is silenced in human colon carcinoma cells A . GPR109A expression level in matched pairs of human normal colon and colon carcinoma tissues. Colon carcinoma tissues and adjacent normal tissues were collected from 6 patients, and analyzed for GPR109A expression by RT-PCR. GAPDH was used as normalization control. Bottom panel: the GPR109A levels were quantified using the NIH J program. The ratio of GPR109A vs GAPDH in patient #1 was arbitrarily set at 1. The GPR109A expression levels of the remaining five specimens were then normalized based on patient #1. B . Methylation status of the GPR109A gene promoter in human colon carcinoma specimens. Genomic DNA was isolated from colon carcinoma specimens of 5 colon cancer patients and modified with bisulfate. The modified DNA was then analyzed by MS-PCR (U, unmethylated; M, methylated). Numbers above the figure are patient codes. C . Inhibition of DNA methylation increases GPR109A expression. SW116 and T84 cells were treated with Aza-dC for 3 days at the indicated doses and analyzed for GPR109A expression level by semi-quantitative RT-PCR (top panel) and real-time RT-PCR (bottom panel). The GPR109A expression levels of untreated cells were arbitrarily set at 1. Column : mean, bar : SD. D . Methylation level of the human GPR109A gene promoter in human colon carcinoma cell lines. The human GPR109A gene DNA sequence was exported from the human genome database and analyzed for CpG islands using MethyPrimer computer program. Top panel: the human GPR109A gene promoter structure. Vertical bars under the line indicate location of CpG dinucleotides, and the number under the line indicates nucleotide number relative to GPR109A transcription initiation site (+1). Bottom panel: methylation level of the GPR109A gene promoter in the indicated cell lines. Genomic DNA was modified with bisulfite. The indicated DNA fragment was amplified by PCR and cloned into pCR2.1 vector. Individual clones for each cell line were sequenced and the methylation level of the cytosine in the CpGs was analyzed using QUMA computer program (open circle, unmethylated CpG; closed circle, methylated CpG).

    Techniques Used: Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Modification, Mass Spectrometry, Polymerase Chain Reaction, Inhibition, DNA Methylation Assay, Quantitative RT-PCR, Sequencing, Amplification, Clone Assay, Plasmid Preparation

    13) Product Images from "The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa"

    Article Title: The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006032

    Interactions between H1 domains of the LadS hybrid HK, the GacS unorthodox HK and the RetS hybrid HK using pull-down and two-hybrid experiments. (A) Pull-down experiments. N-terminal FLAG or Strep versions of the H1 domain of GacS, LadS and RetS HKs were constructed in pBBRMCS3 and pCR2.1 vectors, respectively, and expressed in E . coli . Cell lysates were immunoprecipitated using anti-Strep antibody-coupled beads, and FLAG and Strep derivatives were further detected using StrepTactin Alkaline Phosphatase conjugate (upper panel) and anti-FLAG antibody detection (lower panel). (B) In two-hybrid experiments, the ladSH1 , retSH1 and gacSH1 DNA regions were cloned into the two-hybrid pUT18C or pKT25 vectors and corresponding vectors were co-transformed in BTH101 cells that were further streaked on LB plates containing X-gal. A blue color of colonies reflects interaction between chimeric proteins, while white color attests to the absence of interaction. The interactions were further quantified by measuring the corresponding ß-galactosidase levels expressed in Miller units (values and standard deviations of 3 independent clones below corresponding colonies).
    Figure Legend Snippet: Interactions between H1 domains of the LadS hybrid HK, the GacS unorthodox HK and the RetS hybrid HK using pull-down and two-hybrid experiments. (A) Pull-down experiments. N-terminal FLAG or Strep versions of the H1 domain of GacS, LadS and RetS HKs were constructed in pBBRMCS3 and pCR2.1 vectors, respectively, and expressed in E . coli . Cell lysates were immunoprecipitated using anti-Strep antibody-coupled beads, and FLAG and Strep derivatives were further detected using StrepTactin Alkaline Phosphatase conjugate (upper panel) and anti-FLAG antibody detection (lower panel). (B) In two-hybrid experiments, the ladSH1 , retSH1 and gacSH1 DNA regions were cloned into the two-hybrid pUT18C or pKT25 vectors and corresponding vectors were co-transformed in BTH101 cells that were further streaked on LB plates containing X-gal. A blue color of colonies reflects interaction between chimeric proteins, while white color attests to the absence of interaction. The interactions were further quantified by measuring the corresponding ß-galactosidase levels expressed in Miller units (values and standard deviations of 3 independent clones below corresponding colonies).

    Techniques Used: Construct, Immunoprecipitation, Clone Assay, Transformation Assay

    Related Articles

    Clone Assay:

    Article Title: Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA
    Article Snippet: .. Amplified fragments were cloned into the Eco RI site of the pCR2.1 vector (Invitrogen Corp.) to obtain plasmids pCR#8, pCR#11, and pCR#23 (Fig. ). .. The insert DNA in all these plasmids was sequenced by the dideoxy chain termination method with an Applied Biosystem automated DNA sequencer, and the sequence data were analyzed by using PC/GENE (Intelligenetics) software.

    Article Title: Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein
    Article Snippet: .. The PCR product was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.). .. Plasmid pTA-LTRsupF, which contains a supF gene within the LTR sequence, was constructed as follows.

    Article Title: SipA Is Required for Pilus Formation in Streptococcus pyogenes Serotype M3 ▿
    Article Snippet: .. sipA2, tee3 , and srtC2 were amplified in different combinations (Fig. ) using primers located at the 5′ ends of sipA2 (sipA_F_BamHI), tee3 (Orf100_F_BamHI), and srtC2 (SrtC2_F_BamHI) and the 3′ ends of sipA2 (sipA_R_BamHI), tee3 (Orf100_R_BamHI), and srtC2 (SrtC2_R_BamHI) and were cloned into the pCR2.1 vector using the TOPO-TA cloning kit (Invitrogen). ..

    Article Title: Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity
    Article Snippet: .. The complete sequence of open reading frame 16 of HVS strain C488 , which codes for HVS-Bcl-2, was amplified by PCR, cloned with the pCR2.1 vector (Invitrogen, De Schelp, The Netherlands), and confirmed by sequencing by the Dye Dideoxy terminator method (ABI, Weiterstadt, Germany). .. HVS-Bcl-2 was excised with Asp 718 and Not I from pCR2.1 and then inserted into the eukaryotic expression vector pCEP4 (Invitrogen).

    Article Title: The N-Terminal Part of the Enzyme Component (C2I) of the Binary Clostridium botulinum C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin
    Article Snippet: .. The resulting PCR product (1 μl) was cloned into pCR2.1 vector (Invitrogen, NV Leek, The Netherlands) according to the manufacturer’s instructions (Fig. A). .. For expression experiments, the C2I gene was excised with Bgl II/ Bsa BI and cloned into Bam HI/ Sma I-digested pGEX2T, resulting in plasmid pGEX2T-C2I. pGEX2T-C2IN was constructed by Bam HI digestion of pGEX2T-C2I.

    Amplification:

    Article Title: Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA
    Article Snippet: .. Amplified fragments were cloned into the Eco RI site of the pCR2.1 vector (Invitrogen Corp.) to obtain plasmids pCR#8, pCR#11, and pCR#23 (Fig. ). .. The insert DNA in all these plasmids was sequenced by the dideoxy chain termination method with an Applied Biosystem automated DNA sequencer, and the sequence data were analyzed by using PC/GENE (Intelligenetics) software.

    Article Title: SipA Is Required for Pilus Formation in Streptococcus pyogenes Serotype M3 ▿
    Article Snippet: .. sipA2, tee3 , and srtC2 were amplified in different combinations (Fig. ) using primers located at the 5′ ends of sipA2 (sipA_F_BamHI), tee3 (Orf100_F_BamHI), and srtC2 (SrtC2_F_BamHI) and the 3′ ends of sipA2 (sipA_R_BamHI), tee3 (Orf100_R_BamHI), and srtC2 (SrtC2_R_BamHI) and were cloned into the pCR2.1 vector using the TOPO-TA cloning kit (Invitrogen). ..

    Article Title: Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity
    Article Snippet: .. The complete sequence of open reading frame 16 of HVS strain C488 , which codes for HVS-Bcl-2, was amplified by PCR, cloned with the pCR2.1 vector (Invitrogen, De Schelp, The Netherlands), and confirmed by sequencing by the Dye Dideoxy terminator method (ABI, Weiterstadt, Germany). .. HVS-Bcl-2 was excised with Asp 718 and Not I from pCR2.1 and then inserted into the eukaryotic expression vector pCEP4 (Invitrogen).

    TA Cloning:

    Article Title: Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence
    Article Snippet: .. This 1-kb fragment was ligated into the pCR2.1 vector from a TA cloning kit (Invitrogen, Carlsbad, Calif.). .. The ligation reaction mixture was used to transform E. coli DH5α; transformants were selected on LB agar supplemented with ampicillin.

    Polymerase Chain Reaction:

    Article Title: Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA
    Article Snippet: .. Amplified fragments were cloned into the Eco RI site of the pCR2.1 vector (Invitrogen Corp.) to obtain plasmids pCR#8, pCR#11, and pCR#23 (Fig. ). .. The insert DNA in all these plasmids was sequenced by the dideoxy chain termination method with an Applied Biosystem automated DNA sequencer, and the sequence data were analyzed by using PC/GENE (Intelligenetics) software.

    Article Title: Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein
    Article Snippet: .. The PCR product was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.). .. Plasmid pTA-LTRsupF, which contains a supF gene within the LTR sequence, was constructed as follows.

    Article Title: Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity
    Article Snippet: .. The complete sequence of open reading frame 16 of HVS strain C488 , which codes for HVS-Bcl-2, was amplified by PCR, cloned with the pCR2.1 vector (Invitrogen, De Schelp, The Netherlands), and confirmed by sequencing by the Dye Dideoxy terminator method (ABI, Weiterstadt, Germany). .. HVS-Bcl-2 was excised with Asp 718 and Not I from pCR2.1 and then inserted into the eukaryotic expression vector pCEP4 (Invitrogen).

    Article Title: The N-Terminal Part of the Enzyme Component (C2I) of the Binary Clostridium botulinum C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin
    Article Snippet: .. The resulting PCR product (1 μl) was cloned into pCR2.1 vector (Invitrogen, NV Leek, The Netherlands) according to the manufacturer’s instructions (Fig. A). .. For expression experiments, the C2I gene was excised with Bgl II/ Bsa BI and cloned into Bam HI/ Sma I-digested pGEX2T, resulting in plasmid pGEX2T-C2I. pGEX2T-C2IN was constructed by Bam HI digestion of pGEX2T-C2I.

    Sequencing:

    Article Title: Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity
    Article Snippet: .. The complete sequence of open reading frame 16 of HVS strain C488 , which codes for HVS-Bcl-2, was amplified by PCR, cloned with the pCR2.1 vector (Invitrogen, De Schelp, The Netherlands), and confirmed by sequencing by the Dye Dideoxy terminator method (ABI, Weiterstadt, Germany). .. HVS-Bcl-2 was excised with Asp 718 and Not I from pCR2.1 and then inserted into the eukaryotic expression vector pCEP4 (Invitrogen).

    Plasmid Preparation:

    Article Title: Characterization of a WaaF (RfaF) Homolog Expressed by Haemophilus ducreyi
    Article Snippet: .. This 1.5-kb fragment was ligated into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.) and used to transform E. coli DH5α. .. A plasmid (pBB12) from one of these transformants was used to electroporate the waaF mutant strain S. typhimurium 3789 ( ); the desired recombinant strain was selected with ampicillin.

    Article Title: Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA
    Article Snippet: .. Amplified fragments were cloned into the Eco RI site of the pCR2.1 vector (Invitrogen Corp.) to obtain plasmids pCR#8, pCR#11, and pCR#23 (Fig. ). .. The insert DNA in all these plasmids was sequenced by the dideoxy chain termination method with an Applied Biosystem automated DNA sequencer, and the sequence data were analyzed by using PC/GENE (Intelligenetics) software.

    Article Title: Coupled Integration of Human Immunodeficiency Virus Type 1 cDNA Ends by Purified Integrase In Vitro: Stimulation by the Viral Nucleocapsid Protein
    Article Snippet: .. The PCR product was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, Calif.). .. Plasmid pTA-LTRsupF, which contains a supF gene within the LTR sequence, was constructed as follows.

    Article Title: SipA Is Required for Pilus Formation in Streptococcus pyogenes Serotype M3 ▿
    Article Snippet: .. sipA2, tee3 , and srtC2 were amplified in different combinations (Fig. ) using primers located at the 5′ ends of sipA2 (sipA_F_BamHI), tee3 (Orf100_F_BamHI), and srtC2 (SrtC2_F_BamHI) and the 3′ ends of sipA2 (sipA_R_BamHI), tee3 (Orf100_R_BamHI), and srtC2 (SrtC2_R_BamHI) and were cloned into the pCR2.1 vector using the TOPO-TA cloning kit (Invitrogen). ..

    Article Title: Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity
    Article Snippet: .. The complete sequence of open reading frame 16 of HVS strain C488 , which codes for HVS-Bcl-2, was amplified by PCR, cloned with the pCR2.1 vector (Invitrogen, De Schelp, The Netherlands), and confirmed by sequencing by the Dye Dideoxy terminator method (ABI, Weiterstadt, Germany). .. HVS-Bcl-2 was excised with Asp 718 and Not I from pCR2.1 and then inserted into the eukaryotic expression vector pCEP4 (Invitrogen).

    Article Title: Involvement of the Haemophilus ducreyi gmhA Gene Product in Lipooligosaccharide Expression and Virulence
    Article Snippet: .. This 1-kb fragment was ligated into the pCR2.1 vector from a TA cloning kit (Invitrogen, Carlsbad, Calif.). .. The ligation reaction mixture was used to transform E. coli DH5α; transformants were selected on LB agar supplemented with ampicillin.

    Article Title: Penicillin-Binding Proteins in Leptospira interrogans
    Article Snippet: .. Amplicons were ligated with pCR2.1 vector (Invitrogen Corp., Carlsbad, Calif.) and used to transform E. coli INV F′. .. The resulting plasmid, p921-1, contained the pbpB gene downstream of the T7 promoter.

    Article Title: The N-Terminal Part of the Enzyme Component (C2I) of the Binary Clostridium botulinum C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin
    Article Snippet: .. The resulting PCR product (1 μl) was cloned into pCR2.1 vector (Invitrogen, NV Leek, The Netherlands) according to the manufacturer’s instructions (Fig. A). .. For expression experiments, the C2I gene was excised with Bgl II/ Bsa BI and cloned into Bam HI/ Sma I-digested pGEX2T, resulting in plasmid pGEX2T-C2I. pGEX2T-C2IN was constructed by Bam HI digestion of pGEX2T-C2I.

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  • 96
    Thermo Fisher topo ta cloning kit
    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged <t>PCR</t> products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a <t>TOPO-cloning</t> procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.
    Topo Ta Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/topo ta cloning kit/product/Thermo Fisher
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    topo ta cloning kit - by Bioz Stars, 2020-08
    96/100 stars
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    98
    Thermo Fisher plasmid dna
    Standard curves of <t>Saccharibacteria</t> qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid <t>DNA</t> carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.
    Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 7611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid dna/product/Thermo Fisher
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    93
    Thermo Fisher pcr2 1 vector
    Standard curves of <t>Saccharibacteria</t> qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid <t>DNA</t> carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.
    Pcr2 1 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr2 1 vector/product/Thermo Fisher
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    pcr2 1 vector - by Bioz Stars, 2020-08
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    Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged PCR products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a TOPO-cloning procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.

    Journal: Disease Models & Mechanisms

    Article Title: A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

    doi: 10.1242/dmm.026765

    Figure Lengend Snippet: Strategy for F1 mutation carrier screening and identification of their mutations. (A) Screening of F1 mutation carriers. Genotyping, mutation screening and sequencing in this strategy are illustrated by data from screening of pycr1a mutation carriers. F1 mutation carriers are screened by fin clipping, preparing DNA extracts and running PCRs followed by HMA and the sample results of screening six F1 fish are shown (2 and 6 are positive and marked with ‘+’). (B) Cloning restriction site-tagged PCR products from multiple mutation carriers. Positive mutation carriers are split into groups of four, separated in individual tanks and assigned identifiers (e.g. 1A) and the corresponding forward PCR primers with either Age I, Cla I or Sac II. PCRs with the assigned forward primers and common reverse primer were run on DNA extracts from F1 fish as per assignment, pooled and cloned using a TOPO-cloning procedure. (C) HMA analysis of pycr1a bacterial clones. HMA on colony PCR products from bacterial clones mixed with wild-type PCR products is performed and positive clones are identified. (D) Restriction analysis of M13 PCR products. PCR products from positive bacterial clones amplified with M13 primers were digested with enzymes, for which sites were inserted into forward PCR primers, and clones digestible with each of the enzymes are identified. (E) Sequencing analysis of selected pycr1a clones. The identified pycr1a plasmid clones corresponding to single F1 zebrafish were sequenced and analyzed both at the restriction site position and the mutation site showing complete agreement with previous assays.

    Article Snippet: The resulting PCRs from the same group of fish were checked on the gel, mixed, purified using QIAquick PCR purification kit (QIAGEN, 28104) and TOPO-cloned into pCR2.1-TOPO (Thermo Fisher Scientific, 450641).

    Techniques: Mutagenesis, Sequencing, Fluorescence In Situ Hybridization, Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

    Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Journal: Materials

    Article Title: Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge

    doi: 10.3390/ma11071129

    Figure Lengend Snippet: Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Article Snippet: Standard curves were constructed from 10-fold dilutions of plasmid DNA (pCR2.1-TOPO, Thermo Fisher Scientific) carrying partial Saccharibacteria 16S rRNA genes retrieved from a clone library described in section 3.4 with a known copy number.

    Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification

    Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Journal: Materials

    Article Title: Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge

    doi: 10.3390/ma11071129

    Figure Lengend Snippet: Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Article Snippet: Standard curves were constructed from 10-fold dilutions of plasmid DNA (pCR2.1-TOPO, Thermo Fisher Scientific) carrying partial Saccharibacteria 16S rRNA genes retrieved from a clone library described in section 3.4 with a known copy number.

    Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification