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  • 99
    New England Biolabs apai
    Pulse-field gel electrophoresis patterns of ( a ) <t>XbaI-digested</t> genomic DNA of B. breve isolates and ( b ) <t>ApaI-digested</t> genomic DNA of L. plantarum isolates from human milk and infant faeces. The unedited versions of these images can be found as Supplementary Figures S3 and S4 .
    Apai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 961 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    apai - by Bioz Stars, 2020-03
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    99
    Thermo Fisher apai
    Structural- and chemical probing of <t>DNA</t> and DNA-PNA complex formation in pMP178(75 repeats). A) Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP178(75 repeats), was incubated in the absence (No PNA) or the presence of 10 μM of PNA (GAA-PNA or CTT-PNA) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Non-treated plasmid was used in a set of four different controls (C) of the PE reactions C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of GAA-PNA. C3= plasmid incubated in the presence of CTT-PNA. C4= plasmid not incubated. All samples were linearized by <t>ApaI</t> then subjected as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP178(75 repeats) and the PE reaction control (C4) are shown as references. The DNA fragments detected in a PE reaction using the GAA (R)-strand as template, and the right gel panel shows the DNA fragments detected in a PE reaction using the CTT (Y)-strand as template. The A 1 to A 9 nucleotides flanking the repeats of the R-strand and T 1 to T 12 flanking in the Y-strand and the middle point of the repeat sequence are indicated (M). B) Models showing the most predominant DNA and DNA-PNA structures formed at pMP178(75 repeats) in the absence or the presence of CTT-PNA or GAA-PNA respectively. They correspond to: 1. 5′3′3′-pyrimidine motif H-DNA, 2. CTT-PNA stabilized 5′3′3′-pyrimidine motif H-DNA, 3. Triplex-invasion, 4. Intermolecular triplex and 5. Duplex-invasion structure. Thin line= purine strand, thick line= pyrimidine strand, grey line= PNA. Regions modified by CAA (π) or cleaved by BQQ-OP (o) are indicated.
    Apai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 687 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    apai - by Bioz Stars, 2020-03
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    96
    GE Healthcare apai
    Structural- and chemical probing of <t>DNA</t> and DNA-PNA complex formation in pMP178(75 repeats). A) Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP178(75 repeats), was incubated in the absence (No PNA) or the presence of 10 μM of PNA (GAA-PNA or CTT-PNA) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Non-treated plasmid was used in a set of four different controls (C) of the PE reactions C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of GAA-PNA. C3= plasmid incubated in the presence of CTT-PNA. C4= plasmid not incubated. All samples were linearized by <t>ApaI</t> then subjected as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP178(75 repeats) and the PE reaction control (C4) are shown as references. The DNA fragments detected in a PE reaction using the GAA (R)-strand as template, and the right gel panel shows the DNA fragments detected in a PE reaction using the CTT (Y)-strand as template. The A 1 to A 9 nucleotides flanking the repeats of the R-strand and T 1 to T 12 flanking in the Y-strand and the middle point of the repeat sequence are indicated (M). B) Models showing the most predominant DNA and DNA-PNA structures formed at pMP178(75 repeats) in the absence or the presence of CTT-PNA or GAA-PNA respectively. They correspond to: 1. 5′3′3′-pyrimidine motif H-DNA, 2. CTT-PNA stabilized 5′3′3′-pyrimidine motif H-DNA, 3. Triplex-invasion, 4. Intermolecular triplex and 5. Duplex-invasion structure. Thin line= purine strand, thick line= pyrimidine strand, grey line= PNA. Regions modified by CAA (π) or cleaved by BQQ-OP (o) are indicated.
    Apai, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    apai  (Roche)
    91
    Roche apai
    <t>PFGE</t> dendrogram of L. monocytogenes harboring LmoJ2 and LmoJ3. PFGE with <t>ApaI</t> and AscI and profile clustering were performed as described in Materials and Methods. Isolates harboring LmoJ2 and LmoJ3 are indicated with filled and open circles, respectively.
    Apai, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega apai
    Structural- and chemical probing of <t>DNA</t> and DNA-PNA complex formation in pMP178(75 repeats). A) Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP178(75 repeats), was incubated in the absence (No PNA) or the presence of 10 μM of PNA (GAA-PNA or CTT-PNA) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Non-treated plasmid was used in a set of four different controls (C) of the PE reactions C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of GAA-PNA. C3= plasmid incubated in the presence of CTT-PNA. C4= plasmid not incubated. All samples were linearized by <t>ApaI</t> then subjected as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP178(75 repeats) and the PE reaction control (C4) are shown as references. The DNA fragments detected in a PE reaction using the GAA (R)-strand as template, and the right gel panel shows the DNA fragments detected in a PE reaction using the CTT (Y)-strand as template. The A 1 to A 9 nucleotides flanking the repeats of the R-strand and T 1 to T 12 flanking in the Y-strand and the middle point of the repeat sequence are indicated (M). B) Models showing the most predominant DNA and DNA-PNA structures formed at pMP178(75 repeats) in the absence or the presence of CTT-PNA or GAA-PNA respectively. They correspond to: 1. 5′3′3′-pyrimidine motif H-DNA, 2. CTT-PNA stabilized 5′3′3′-pyrimidine motif H-DNA, 3. Triplex-invasion, 4. Intermolecular triplex and 5. Duplex-invasion structure. Thin line= purine strand, thick line= pyrimidine strand, grey line= PNA. Regions modified by CAA (π) or cleaved by BQQ-OP (o) are indicated.
    Apai, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    apai  (TaKaRa)
    99
    TaKaRa apai
    <t>ApaI</t> PFGE profiles of representative A. <t>baumannii</t> Isolates. (A–J): isolates from Hospital-1 to -10, respectively. The 85% was set as a cutoff to define PFGE types. The dendrogram was generated by the BioNumerics software.
    Apai, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Promega apai enzyme
    <t>ApaI</t> PFGE profiles of representative A. <t>baumannii</t> Isolates. (A–J): isolates from Hospital-1 to -10, respectively. The 85% was set as a cutoff to define PFGE types. The dendrogram was generated by the BioNumerics software.
    Apai Enzyme, supplied by Promega, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher apai fd
    <t>ApaI</t> PFGE profiles of representative A. <t>baumannii</t> Isolates. (A–J): isolates from Hospital-1 to -10, respectively. The 85% was set as a cutoff to define PFGE types. The dendrogram was generated by the BioNumerics software.
    Apai Fd, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher apai enzyme
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore apai
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    GE Healthcare apai restriction enzyme
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Restriction Enzyme, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Roche apai restriction enzyme
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Restriction Enzyme, supplied by Roche, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    TaKaRa apai restriction enzyme
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Restriction Enzyme, supplied by TaKaRa, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Boehringer Mannheim apai
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher apai restriction enzyme
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Restriction Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Promega apai saci cut pgem7zf
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Saci Cut Pgem7zf, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa ecori apai cut pegfpn1
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Ecori Apai Cut Pegfpn1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 79/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa apai site
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
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    Ridom GmbH pfge apai
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Pfge Apai, supplied by Ridom GmbH, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega apai endonuclease
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Endonuclease, supplied by Promega, used in various techniques. Bioz Stars score: 79/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa ecori apai
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
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    TaKaRa apai digestion
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Digestion, supplied by TaKaRa, used in various techniques. Bioz Stars score: 82/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene sacii apai digested pbluescript sk
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
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    Thermo Fisher hindiii apai double digestion
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
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    Becton Dickinson apai a allele
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
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    MRC Ltd noti apai sites
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
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    Promega apai digested genomic dna
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Digested Genomic Dna, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Genewiz apai restriction sites
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Apai Restriction Sites, supplied by Genewiz, used in various techniques. Bioz Stars score: 81/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    TaKaRa psti apai digested pegfp c1
    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial <t>ApaI</t> restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using <t>SalI</t> and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.
    Psti Apai Digested Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pulse-field gel electrophoresis patterns of ( a ) XbaI-digested genomic DNA of B. breve isolates and ( b ) ApaI-digested genomic DNA of L. plantarum isolates from human milk and infant faeces. The unedited versions of these images can be found as Supplementary Figures S3 and S4 .

    Journal: Scientific Reports

    Article Title: The Composition of Human Milk and Infant Faecal Microbiota Over the First Three Months of Life: A Pilot Study

    doi: 10.1038/srep40597

    Figure Lengend Snippet: Pulse-field gel electrophoresis patterns of ( a ) XbaI-digested genomic DNA of B. breve isolates and ( b ) ApaI-digested genomic DNA of L. plantarum isolates from human milk and infant faeces. The unedited versions of these images can be found as Supplementary Figures S3 and S4 .

    Article Snippet: The restriction enzyme XbaI was used to cleave bifidobacterial chromosomal DNA and ApaI (New England Biolabs, MA, United States) was used for Lactobacillus .

    Techniques: Nucleic Acid Electrophoresis

    a - d Restriction endonuclease digestion for FokI, ApaI, Bsm I and TaqI polymorphisms, M = Marker

    Journal: BMC Nephrology

    Article Title: Influence of vitamin D receptor polymorphisms on biochemical markers of mineral bone disorders in South African patients with chronic kidney disease

    doi: 10.1186/s12882-018-0831-7

    Figure Lengend Snippet: a - d Restriction endonuclease digestion for FokI, ApaI, Bsm I and TaqI polymorphisms, M = Marker

    Article Snippet: The PCR products were then digested with enzymes ApaI, BsmI, FokI, and TaqI (New England Biolabs, Beverly, MA, USA) according to the supplier’s protocol.

    Techniques: Marker

    Frequency of vitamin D deficiency according to BsmI (A), ApaI (B), and TaqI (C) SNPs in VDR gene grouped by presence of polymorphic allele. White bars express the frequency of vitamin D status ( deficiency or insufficiency/sufficiency) for the wild genotype of each VDR SNP. Gray bars express the frequency of vitamin D status for the polymorphic (in heterozygous plus homozygous) genotypes. Pearson chi-square test, * p

    Journal: BMC Pediatrics

    Article Title: Vitamin D deficiency in girls from South Brazil: a cross-sectional study on prevalence and association with vitamin D receptor gene variants

    doi: 10.1186/1471-2431-12-62

    Figure Lengend Snippet: Frequency of vitamin D deficiency according to BsmI (A), ApaI (B), and TaqI (C) SNPs in VDR gene grouped by presence of polymorphic allele. White bars express the frequency of vitamin D status ( deficiency or insufficiency/sufficiency) for the wild genotype of each VDR SNP. Gray bars express the frequency of vitamin D status for the polymorphic (in heterozygous plus homozygous) genotypes. Pearson chi-square test, * p

    Article Snippet: Protocol conditions consisted of denaturation at 95°C for 2 min followed by 35 cycles (95°C, 30sec; 59.2°C, 30sec; 72°C, 80sec) and final extension at 72°C for 5 min. PCR products were digested overnight by the restriction enzymes ApaI or TaqI (New England Biolabs, USA) at 37°C or 65°C, respectively.

    Techniques:

    Vitamin D levels according to BsmI (A), ApaI (B), and TaqI (C) SNPs in VDR gene grouped by presence of polymorphic allele. Values are expressed as means (central circle) and 95% CI (lower and upper limit). Two-tailed Student’s t -test, * p

    Journal: BMC Pediatrics

    Article Title: Vitamin D deficiency in girls from South Brazil: a cross-sectional study on prevalence and association with vitamin D receptor gene variants

    doi: 10.1186/1471-2431-12-62

    Figure Lengend Snippet: Vitamin D levels according to BsmI (A), ApaI (B), and TaqI (C) SNPs in VDR gene grouped by presence of polymorphic allele. Values are expressed as means (central circle) and 95% CI (lower and upper limit). Two-tailed Student’s t -test, * p

    Article Snippet: Protocol conditions consisted of denaturation at 95°C for 2 min followed by 35 cycles (95°C, 30sec; 59.2°C, 30sec; 72°C, 80sec) and final extension at 72°C for 5 min. PCR products were digested overnight by the restriction enzymes ApaI or TaqI (New England Biolabs, USA) at 37°C or 65°C, respectively.

    Techniques: Two Tailed Test

    Dendrogram produced by the unweighted-pair group method using average linkages showing clustering by Dice coefficient of the combined PFGE (AscI and ApaI) profiles of the 20 L. monocytogenes isolates obtained from animal cases analyzed in this study.

    Journal: Applied and Environmental Microbiology

    Article Title: Ruminant Rhombencephalitis-Associated Listeria monocytogenes Strains Constitute a Genetically Homogeneous Group Related to Human Outbreak Strains

    doi: 10.1128/AEM.00219-13

    Figure Lengend Snippet: Dendrogram produced by the unweighted-pair group method using average linkages showing clustering by Dice coefficient of the combined PFGE (AscI and ApaI) profiles of the 20 L. monocytogenes isolates obtained from animal cases analyzed in this study.

    Article Snippet: PFGE was performed by including genomic DNA in agarose plugs prior to digestion with the AscI and ApaI restriction enzymes (New England BioLabs), followed by PFGE with a Chef DR III system (Bio-Rad, Hercules, CA) , and pulsotypes were analyzed as described elsewhere ( ).

    Techniques: Produced

    Structural- and chemical probing of DNA and DNA-PNA complex formation in pMP178(75 repeats). A) Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP178(75 repeats), was incubated in the absence (No PNA) or the presence of 10 μM of PNA (GAA-PNA or CTT-PNA) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Non-treated plasmid was used in a set of four different controls (C) of the PE reactions C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of GAA-PNA. C3= plasmid incubated in the presence of CTT-PNA. C4= plasmid not incubated. All samples were linearized by ApaI then subjected as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP178(75 repeats) and the PE reaction control (C4) are shown as references. The DNA fragments detected in a PE reaction using the GAA (R)-strand as template, and the right gel panel shows the DNA fragments detected in a PE reaction using the CTT (Y)-strand as template. The A 1 to A 9 nucleotides flanking the repeats of the R-strand and T 1 to T 12 flanking in the Y-strand and the middle point of the repeat sequence are indicated (M). B) Models showing the most predominant DNA and DNA-PNA structures formed at pMP178(75 repeats) in the absence or the presence of CTT-PNA or GAA-PNA respectively. They correspond to: 1. 5′3′3′-pyrimidine motif H-DNA, 2. CTT-PNA stabilized 5′3′3′-pyrimidine motif H-DNA, 3. Triplex-invasion, 4. Intermolecular triplex and 5. Duplex-invasion structure. Thin line= purine strand, thick line= pyrimidine strand, grey line= PNA. Regions modified by CAA (π) or cleaved by BQQ-OP (o) are indicated.

    Journal: PLoS ONE

    Article Title: Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    doi: 10.1371/journal.pone.0165788

    Figure Lengend Snippet: Structural- and chemical probing of DNA and DNA-PNA complex formation in pMP178(75 repeats). A) Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP178(75 repeats), was incubated in the absence (No PNA) or the presence of 10 μM of PNA (GAA-PNA or CTT-PNA) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Non-treated plasmid was used in a set of four different controls (C) of the PE reactions C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of GAA-PNA. C3= plasmid incubated in the presence of CTT-PNA. C4= plasmid not incubated. All samples were linearized by ApaI then subjected as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP178(75 repeats) and the PE reaction control (C4) are shown as references. The DNA fragments detected in a PE reaction using the GAA (R)-strand as template, and the right gel panel shows the DNA fragments detected in a PE reaction using the CTT (Y)-strand as template. The A 1 to A 9 nucleotides flanking the repeats of the R-strand and T 1 to T 12 flanking in the Y-strand and the middle point of the repeat sequence are indicated (M). B) Models showing the most predominant DNA and DNA-PNA structures formed at pMP178(75 repeats) in the absence or the presence of CTT-PNA or GAA-PNA respectively. They correspond to: 1. 5′3′3′-pyrimidine motif H-DNA, 2. CTT-PNA stabilized 5′3′3′-pyrimidine motif H-DNA, 3. Triplex-invasion, 4. Intermolecular triplex and 5. Duplex-invasion structure. Thin line= purine strand, thick line= pyrimidine strand, grey line= PNA. Regions modified by CAA (π) or cleaved by BQQ-OP (o) are indicated.

    Article Snippet: The isolated DNA was digested using ApaI (Fermentas Fastdigest) and the enzyme was then inactivated, 5 min at 65°C.

    Techniques: Polyacrylamide Gel Electrophoresis, Cellular Antioxidant Activity Assay, Modification, Plasmid Preparation, Incubation, Sequencing

    BQQ-OP mediated DNA cleavage of H-DNA forming (GAA) 115 repeats in the presence of PNA. A) Schematic presentation of pMP179 and the H-DNA forming site. The two DNA fragments generated by triplex-specific cleavage followed by enzymatic digestion are indicated as X (3814 bp) and Y (3178 bp). B) Representative gel for pMP179(115 repeats) incubated with 10 μM PNA (CTT-PNA or GAA-PNA, lane 1 or 2, respectively), in the absence of ONs (lane 3) or with 10 μM ssDNA (CTT-DNA or GAA-DNA, lane 4 or 5, respectively) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). Triplex-specific cleavage was carried out in the presence of Cu 2+ and MPA, followed by digestion with ApaI. Reference supercoiled (SC) and linearized (Lin) pMP179(115 repeats) and a molecular weight DNA ladder (M) are also shown. C) Graph represents the percentage of BQQ-OP mediated cleavage obtained for pMP179(115 repeats) in the absence (indicate in the graph as pMP179) or in the presence of PNA- or DNA oligomers. Values indicate the ratio between the intensity of DNA double strand cleavage to the total band intensity from the respective lane and are expressed as mean±S.D. (n=3). No cleavage was obtained in the presence of GAA-PNA and therefore not included in the graph. ** P ≤0.01 in relation to plasmid in the absence of oligonucleotide (pMP179).

    Journal: PLoS ONE

    Article Title: Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    doi: 10.1371/journal.pone.0165788

    Figure Lengend Snippet: BQQ-OP mediated DNA cleavage of H-DNA forming (GAA) 115 repeats in the presence of PNA. A) Schematic presentation of pMP179 and the H-DNA forming site. The two DNA fragments generated by triplex-specific cleavage followed by enzymatic digestion are indicated as X (3814 bp) and Y (3178 bp). B) Representative gel for pMP179(115 repeats) incubated with 10 μM PNA (CTT-PNA or GAA-PNA, lane 1 or 2, respectively), in the absence of ONs (lane 3) or with 10 μM ssDNA (CTT-DNA or GAA-DNA, lane 4 or 5, respectively) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). Triplex-specific cleavage was carried out in the presence of Cu 2+ and MPA, followed by digestion with ApaI. Reference supercoiled (SC) and linearized (Lin) pMP179(115 repeats) and a molecular weight DNA ladder (M) are also shown. C) Graph represents the percentage of BQQ-OP mediated cleavage obtained for pMP179(115 repeats) in the absence (indicate in the graph as pMP179) or in the presence of PNA- or DNA oligomers. Values indicate the ratio between the intensity of DNA double strand cleavage to the total band intensity from the respective lane and are expressed as mean±S.D. (n=3). No cleavage was obtained in the presence of GAA-PNA and therefore not included in the graph. ** P ≤0.01 in relation to plasmid in the absence of oligonucleotide (pMP179).

    Article Snippet: The isolated DNA was digested using ApaI (Fermentas Fastdigest) and the enzyme was then inactivated, 5 min at 65°C.

    Techniques: Generated, Incubation, Molecular Weight, Plasmid Preparation

    Structural- and chemical probing of DNA and DNA-PNA complex formation at (GAA) 9 repeats. Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP141(9 repeats), was incubated in the absence (No PNA) or presence of 10 μM of GAA-PNA or CTT-PNA in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then either chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Untreated plasmid was used in a set of four different controls (C) of the PE reactions: C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of CTT-PNA. C3= plasmid incubated in the presence of GAA-PNA. C4= plasmid not incubated. All samples were linearized using ApaI and then used as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP141(9 repeats) and a PE reaction control (C4) are also shown. The left gel panel shows the DNA fragments obtained in a PE reaction using the GAA containing (R)-strand as template, and the right gel panel shows the DNA fragments obtained when using the CTT containing (Y)-strand as template. The A 1 to A 23 nucleotides flanking the repeats of the R-strand and T 1 to T 23 flanking in the Y-strand and the mid-point of the repeat sequence (M) are indicated.

    Journal: PLoS ONE

    Article Title: Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    doi: 10.1371/journal.pone.0165788

    Figure Lengend Snippet: Structural- and chemical probing of DNA and DNA-PNA complex formation at (GAA) 9 repeats. Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP141(9 repeats), was incubated in the absence (No PNA) or presence of 10 μM of GAA-PNA or CTT-PNA in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then either chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Untreated plasmid was used in a set of four different controls (C) of the PE reactions: C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of CTT-PNA. C3= plasmid incubated in the presence of GAA-PNA. C4= plasmid not incubated. All samples were linearized using ApaI and then used as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP141(9 repeats) and a PE reaction control (C4) are also shown. The left gel panel shows the DNA fragments obtained in a PE reaction using the GAA containing (R)-strand as template, and the right gel panel shows the DNA fragments obtained when using the CTT containing (Y)-strand as template. The A 1 to A 23 nucleotides flanking the repeats of the R-strand and T 1 to T 23 flanking in the Y-strand and the mid-point of the repeat sequence (M) are indicated.

    Article Snippet: The isolated DNA was digested using ApaI (Fermentas Fastdigest) and the enzyme was then inactivated, 5 min at 65°C.

    Techniques: Polyacrylamide Gel Electrophoresis, Cellular Antioxidant Activity Assay, Modification, Plasmid Preparation, Incubation, Sequencing

    BQQ-OP mediated DNA cleavage of H-DNA forming (GAA) 115 repeats in the presence of LNA-PS. A) Representative gel from pMP179(115 repeats) incubated with 10 μM LNA-PS (GAA-LNA-PS 1, 2 or 3 or ER-LNA-PS or CTT-LNA-PS, lane 1, 2, 3, 4 or 7, respectively), in the absence of oligonucleotide (lane 8) or with 10 μM ssDNA (CTT-DNA or GAA-DNA, lane 5 or 6, respectively) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). Triplex-specific cleavage was carried out in the presence of Cu 2+ and MPA, and the plasmid was subsequently digested using ApaI. The two obtained DNA fragments have approximately 3814 and 3178 bp. Reference supercoiled (SC) and linearized (Lin) pMP179(115 repeats) and a molecular weight DNA ladder (M) are also shown. B) Graph represents the percentage of BQQ-OP mediated cleavage for pMP179(115 repeats) in the absence (indicate in the graph as pMP179) or in the presence of LNA- or DNA oligomers. Values indicate the ratio between the intensity of DNA double strand cleavage to the total band intensity from the respective lane and are expressed as mean±S.D. (n=3). *** P ≤0.001, **** P

    Journal: PLoS ONE

    Article Title: Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    doi: 10.1371/journal.pone.0165788

    Figure Lengend Snippet: BQQ-OP mediated DNA cleavage of H-DNA forming (GAA) 115 repeats in the presence of LNA-PS. A) Representative gel from pMP179(115 repeats) incubated with 10 μM LNA-PS (GAA-LNA-PS 1, 2 or 3 or ER-LNA-PS or CTT-LNA-PS, lane 1, 2, 3, 4 or 7, respectively), in the absence of oligonucleotide (lane 8) or with 10 μM ssDNA (CTT-DNA or GAA-DNA, lane 5 or 6, respectively) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). Triplex-specific cleavage was carried out in the presence of Cu 2+ and MPA, and the plasmid was subsequently digested using ApaI. The two obtained DNA fragments have approximately 3814 and 3178 bp. Reference supercoiled (SC) and linearized (Lin) pMP179(115 repeats) and a molecular weight DNA ladder (M) are also shown. B) Graph represents the percentage of BQQ-OP mediated cleavage for pMP179(115 repeats) in the absence (indicate in the graph as pMP179) or in the presence of LNA- or DNA oligomers. Values indicate the ratio between the intensity of DNA double strand cleavage to the total band intensity from the respective lane and are expressed as mean±S.D. (n=3). *** P ≤0.001, **** P

    Article Snippet: The isolated DNA was digested using ApaI (Fermentas Fastdigest) and the enzyme was then inactivated, 5 min at 65°C.

    Techniques: Incubation, Plasmid Preparation, Molecular Weight

    PFGE dendrogram of L. monocytogenes harboring LmoJ2 and LmoJ3. PFGE with ApaI and AscI and profile clustering were performed as described in Materials and Methods. Isolates harboring LmoJ2 and LmoJ3 are indicated with filled and open circles, respectively.

    Journal: Applied and Environmental Microbiology

    Article Title: Two Novel Type II Restriction-Modification Systems Occupying Genomically Equivalent Locations on the Chromosomes of Listeria monocytogenes Strains

    doi: 10.1128/AEM.07203-11

    Figure Lengend Snippet: PFGE dendrogram of L. monocytogenes harboring LmoJ2 and LmoJ3. PFGE with ApaI and AscI and profile clustering were performed as described in Materials and Methods. Isolates harboring LmoJ2 and LmoJ3 are indicated with filled and open circles, respectively.

    Article Snippet: Pulsed-field gel electrophoresis (PFGE) was conducted with AscI (New England BioLabs, Ipswich, MA) and ApaI (Roche, Indianapolis, IN) as described previously , and BioNumerics (Applied Maths, Austin, TX) was employed for analysis of the PFGE profiles.

    Techniques:

    Structural- and chemical probing of DNA and DNA-PNA complex formation in pMP178(75 repeats). A) Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP178(75 repeats), was incubated in the absence (No PNA) or the presence of 10 μM of PNA (GAA-PNA or CTT-PNA) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Non-treated plasmid was used in a set of four different controls (C) of the PE reactions C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of GAA-PNA. C3= plasmid incubated in the presence of CTT-PNA. C4= plasmid not incubated. All samples were linearized by ApaI then subjected as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP178(75 repeats) and the PE reaction control (C4) are shown as references. The DNA fragments detected in a PE reaction using the GAA (R)-strand as template, and the right gel panel shows the DNA fragments detected in a PE reaction using the CTT (Y)-strand as template. The A 1 to A 9 nucleotides flanking the repeats of the R-strand and T 1 to T 12 flanking in the Y-strand and the middle point of the repeat sequence are indicated (M). B) Models showing the most predominant DNA and DNA-PNA structures formed at pMP178(75 repeats) in the absence or the presence of CTT-PNA or GAA-PNA respectively. They correspond to: 1. 5′3′3′-pyrimidine motif H-DNA, 2. CTT-PNA stabilized 5′3′3′-pyrimidine motif H-DNA, 3. Triplex-invasion, 4. Intermolecular triplex and 5. Duplex-invasion structure. Thin line= purine strand, thick line= pyrimidine strand, grey line= PNA. Regions modified by CAA (π) or cleaved by BQQ-OP (o) are indicated.

    Journal: PLoS ONE

    Article Title: Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    doi: 10.1371/journal.pone.0165788

    Figure Lengend Snippet: Structural- and chemical probing of DNA and DNA-PNA complex formation in pMP178(75 repeats). A) Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP178(75 repeats), was incubated in the absence (No PNA) or the presence of 10 μM of PNA (GAA-PNA or CTT-PNA) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Non-treated plasmid was used in a set of four different controls (C) of the PE reactions C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of GAA-PNA. C3= plasmid incubated in the presence of CTT-PNA. C4= plasmid not incubated. All samples were linearized by ApaI then subjected as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP178(75 repeats) and the PE reaction control (C4) are shown as references. The DNA fragments detected in a PE reaction using the GAA (R)-strand as template, and the right gel panel shows the DNA fragments detected in a PE reaction using the CTT (Y)-strand as template. The A 1 to A 9 nucleotides flanking the repeats of the R-strand and T 1 to T 12 flanking in the Y-strand and the middle point of the repeat sequence are indicated (M). B) Models showing the most predominant DNA and DNA-PNA structures formed at pMP178(75 repeats) in the absence or the presence of CTT-PNA or GAA-PNA respectively. They correspond to: 1. 5′3′3′-pyrimidine motif H-DNA, 2. CTT-PNA stabilized 5′3′3′-pyrimidine motif H-DNA, 3. Triplex-invasion, 4. Intermolecular triplex and 5. Duplex-invasion structure. Thin line= purine strand, thick line= pyrimidine strand, grey line= PNA. Regions modified by CAA (π) or cleaved by BQQ-OP (o) are indicated.

    Article Snippet: The isolated DNA was digested using ApaI (1 U, Promega) during 3 h at 37°C and then analyzed using 0.7% agarose gel electrophoresis (50 V, 1 h) and ethidium bromide or SYBR-safe (Life Technologies) staining.

    Techniques: Polyacrylamide Gel Electrophoresis, Cellular Antioxidant Activity Assay, Modification, Plasmid Preparation, Incubation, Sequencing

    BQQ-OP mediated DNA cleavage of H-DNA forming (GAA) 115 repeats in the presence of PNA. A) Schematic presentation of pMP179 and the H-DNA forming site. The two DNA fragments generated by triplex-specific cleavage followed by enzymatic digestion are indicated as X (3814 bp) and Y (3178 bp). B) Representative gel for pMP179(115 repeats) incubated with 10 μM PNA (CTT-PNA or GAA-PNA, lane 1 or 2, respectively), in the absence of ONs (lane 3) or with 10 μM ssDNA (CTT-DNA or GAA-DNA, lane 4 or 5, respectively) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). Triplex-specific cleavage was carried out in the presence of Cu 2+ and MPA, followed by digestion with ApaI. Reference supercoiled (SC) and linearized (Lin) pMP179(115 repeats) and a molecular weight DNA ladder (M) are also shown. C) Graph represents the percentage of BQQ-OP mediated cleavage obtained for pMP179(115 repeats) in the absence (indicate in the graph as pMP179) or in the presence of PNA- or DNA oligomers. Values indicate the ratio between the intensity of DNA double strand cleavage to the total band intensity from the respective lane and are expressed as mean±S.D. (n=3). No cleavage was obtained in the presence of GAA-PNA and therefore not included in the graph. ** P ≤0.01 in relation to plasmid in the absence of oligonucleotide (pMP179).

    Journal: PLoS ONE

    Article Title: Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    doi: 10.1371/journal.pone.0165788

    Figure Lengend Snippet: BQQ-OP mediated DNA cleavage of H-DNA forming (GAA) 115 repeats in the presence of PNA. A) Schematic presentation of pMP179 and the H-DNA forming site. The two DNA fragments generated by triplex-specific cleavage followed by enzymatic digestion are indicated as X (3814 bp) and Y (3178 bp). B) Representative gel for pMP179(115 repeats) incubated with 10 μM PNA (CTT-PNA or GAA-PNA, lane 1 or 2, respectively), in the absence of ONs (lane 3) or with 10 μM ssDNA (CTT-DNA or GAA-DNA, lane 4 or 5, respectively) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). Triplex-specific cleavage was carried out in the presence of Cu 2+ and MPA, followed by digestion with ApaI. Reference supercoiled (SC) and linearized (Lin) pMP179(115 repeats) and a molecular weight DNA ladder (M) are also shown. C) Graph represents the percentage of BQQ-OP mediated cleavage obtained for pMP179(115 repeats) in the absence (indicate in the graph as pMP179) or in the presence of PNA- or DNA oligomers. Values indicate the ratio between the intensity of DNA double strand cleavage to the total band intensity from the respective lane and are expressed as mean±S.D. (n=3). No cleavage was obtained in the presence of GAA-PNA and therefore not included in the graph. ** P ≤0.01 in relation to plasmid in the absence of oligonucleotide (pMP179).

    Article Snippet: The isolated DNA was digested using ApaI (1 U, Promega) during 3 h at 37°C and then analyzed using 0.7% agarose gel electrophoresis (50 V, 1 h) and ethidium bromide or SYBR-safe (Life Technologies) staining.

    Techniques: Generated, Incubation, Molecular Weight, Plasmid Preparation

    Structural- and chemical probing of DNA and DNA-PNA complex formation at (GAA) 9 repeats. Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP141(9 repeats), was incubated in the absence (No PNA) or presence of 10 μM of GAA-PNA or CTT-PNA in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then either chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Untreated plasmid was used in a set of four different controls (C) of the PE reactions: C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of CTT-PNA. C3= plasmid incubated in the presence of GAA-PNA. C4= plasmid not incubated. All samples were linearized using ApaI and then used as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP141(9 repeats) and a PE reaction control (C4) are also shown. The left gel panel shows the DNA fragments obtained in a PE reaction using the GAA containing (R)-strand as template, and the right gel panel shows the DNA fragments obtained when using the CTT containing (Y)-strand as template. The A 1 to A 23 nucleotides flanking the repeats of the R-strand and T 1 to T 23 flanking in the Y-strand and the mid-point of the repeat sequence (M) are indicated.

    Journal: PLoS ONE

    Article Title: Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    doi: 10.1371/journal.pone.0165788

    Figure Lengend Snippet: Structural- and chemical probing of DNA and DNA-PNA complex formation at (GAA) 9 repeats. Non-denaturing PAGE of DNA fragments mapped by BQQ-OP cleavage and chloroacetaldehyde (CAA) modification followed by primer extension (PE). Plasmid pMP141(9 repeats), was incubated in the absence (No PNA) or presence of 10 μM of GAA-PNA or CTT-PNA in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). The plasmid was then either chemically modified using 2% CAA or cleaved using 1 μM BQQ-OP. Untreated plasmid was used in a set of four different controls (C) of the PE reactions: C1= plasmid incubated in the absence of PNA. C2= plasmid incubated in the presence of CTT-PNA. C3= plasmid incubated in the presence of GAA-PNA. C4= plasmid not incubated. All samples were linearized using ApaI and then used as templates for the PE reaction. Sequence ladders using dideoxynucleotides (C=ddCTP, T=ddTTP, G=ddGTP and A=ddATP), linearized pMP141(9 repeats) and a PE reaction control (C4) are also shown. The left gel panel shows the DNA fragments obtained in a PE reaction using the GAA containing (R)-strand as template, and the right gel panel shows the DNA fragments obtained when using the CTT containing (Y)-strand as template. The A 1 to A 23 nucleotides flanking the repeats of the R-strand and T 1 to T 23 flanking in the Y-strand and the mid-point of the repeat sequence (M) are indicated.

    Article Snippet: The isolated DNA was digested using ApaI (1 U, Promega) during 3 h at 37°C and then analyzed using 0.7% agarose gel electrophoresis (50 V, 1 h) and ethidium bromide or SYBR-safe (Life Technologies) staining.

    Techniques: Polyacrylamide Gel Electrophoresis, Cellular Antioxidant Activity Assay, Modification, Plasmid Preparation, Incubation, Sequencing

    BQQ-OP mediated DNA cleavage of H-DNA forming (GAA) 115 repeats in the presence of LNA-PS. A) Representative gel from pMP179(115 repeats) incubated with 10 μM LNA-PS (GAA-LNA-PS 1, 2 or 3 or ER-LNA-PS or CTT-LNA-PS, lane 1, 2, 3, 4 or 7, respectively), in the absence of oligonucleotide (lane 8) or with 10 μM ssDNA (CTT-DNA or GAA-DNA, lane 5 or 6, respectively) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). Triplex-specific cleavage was carried out in the presence of Cu 2+ and MPA, and the plasmid was subsequently digested using ApaI. The two obtained DNA fragments have approximately 3814 and 3178 bp. Reference supercoiled (SC) and linearized (Lin) pMP179(115 repeats) and a molecular weight DNA ladder (M) are also shown. B) Graph represents the percentage of BQQ-OP mediated cleavage for pMP179(115 repeats) in the absence (indicate in the graph as pMP179) or in the presence of LNA- or DNA oligomers. Values indicate the ratio between the intensity of DNA double strand cleavage to the total band intensity from the respective lane and are expressed as mean±S.D. (n=3). *** P ≤0.001, **** P

    Journal: PLoS ONE

    Article Title: Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

    doi: 10.1371/journal.pone.0165788

    Figure Lengend Snippet: BQQ-OP mediated DNA cleavage of H-DNA forming (GAA) 115 repeats in the presence of LNA-PS. A) Representative gel from pMP179(115 repeats) incubated with 10 μM LNA-PS (GAA-LNA-PS 1, 2 or 3 or ER-LNA-PS or CTT-LNA-PS, lane 1, 2, 3, 4 or 7, respectively), in the absence of oligonucleotide (lane 8) or with 10 μM ssDNA (CTT-DNA or GAA-DNA, lane 5 or 6, respectively) in buffer (10 mM sodium cacodylate, 100 mM NaCl, 2 mM MgCl 2 , pH 7.5). Triplex-specific cleavage was carried out in the presence of Cu 2+ and MPA, and the plasmid was subsequently digested using ApaI. The two obtained DNA fragments have approximately 3814 and 3178 bp. Reference supercoiled (SC) and linearized (Lin) pMP179(115 repeats) and a molecular weight DNA ladder (M) are also shown. B) Graph represents the percentage of BQQ-OP mediated cleavage for pMP179(115 repeats) in the absence (indicate in the graph as pMP179) or in the presence of LNA- or DNA oligomers. Values indicate the ratio between the intensity of DNA double strand cleavage to the total band intensity from the respective lane and are expressed as mean±S.D. (n=3). *** P ≤0.001, **** P

    Article Snippet: The isolated DNA was digested using ApaI (1 U, Promega) during 3 h at 37°C and then analyzed using 0.7% agarose gel electrophoresis (50 V, 1 h) and ethidium bromide or SYBR-safe (Life Technologies) staining.

    Techniques: Incubation, Plasmid Preparation, Molecular Weight

    ApaI PFGE profiles of representative A. baumannii Isolates. (A–J): isolates from Hospital-1 to -10, respectively. The 85% was set as a cutoff to define PFGE types. The dendrogram was generated by the BioNumerics software.

    Journal: Frontiers in Microbiology

    Article Title: Molecular Epidemiology of Multi-Drug Resistant Acinetobacter baumannii Isolated in Shandong, China

    doi: 10.3389/fmicb.2016.01687

    Figure Lengend Snippet: ApaI PFGE profiles of representative A. baumannii Isolates. (A–J): isolates from Hospital-1 to -10, respectively. The 85% was set as a cutoff to define PFGE types. The dendrogram was generated by the BioNumerics software.

    Article Snippet: In brief, the chromosomal DNA of A. baumannii was digested with 60 U of ApaI (Takara, Dalian, China) in a 37°C water bath.

    Techniques: Generated, Software

    Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial ApaI restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using SalI and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.

    Journal: BMC Biotechnology

    Article Title: An asymmetric PCR-based, reliable and rapid single-tube native DNA engineering strategy

    doi: 10.1186/1472-6750-12-39

    Figure Lengend Snippet: Validation of ABI-REC through double-resistance reporter assay. ( A ) The design of double resistance reporter assay. The fusion of the 1.6 kb kan R cassette (from pIRES2-EGFP) with the 2.7 kb pUC19 plasmid (ampicillin resistance) renders the new recombinant plasmid resistant to both kanamycin and ampicillin. The plasmid grows on (Kan + Amp) LB plates. An artificial ApaI restriction site is introduced into the 5′ fusion site to precisely localize the insertion site. ( B ) An asymmetric bridge PCR efficiently fuses the Kan R cassette into the pUC19 plasmid and generates a hybrid fragment. The gradient concentration of the primer pR was assessed and identified as within the optimal range from 2 nM to 0.2 nM in the bridge PCR. A conventional two-primer PCR reaction was conducted in parallel to compare their amplification efficiency. This indicates that the quantity of pR primer is critical to the output of fused sequence in the bridge PCR reaction. ( C ) Double resistant colonies were found to grow on (Kan + Amp) LB plates. Bridge PCR products, purified fused sequences, two-primer PCR products (extension), and a mixture of purified Kan R cassettes with pUC19 (no extension) were transformed into DH5α competent cells. Double resistant colonies were present for bridge PCR products and purified fused sequence, whereas the latter two did not produce viable colonies. This implies that intramolecular recombination occurs within the fused sequence, producing double resistant plasmids that renders cells resistant to both kanamycin and ampicillin. pUC19 and pIRES2-EGFP plasmids were not able to grow on the double-drug plates, precluding the risk of random integration of Kan R cassette into the E.coli genome. ( D ) Plasmids of the single colonies in (Kan + Amp) LB plates were extracted and digested using SalI and ApaI. As shown in the electrophoresis gel, the 1.6 kb insert was released, indicating that the Kan R cassette had been fused into pUC19 at pre-determined site. ( E ) Sequencing of the single colonies revealed the insertion of the Kan R cassette and the presence of artificial ApaI restriction site, further proving that the insert of choice has been fused with target plasmid in a restriction- and ligase-free manner. The red rectangle represents the restriction sites.

    Article Snippet: Enzymes and reagents ApaI, SalI, DpnI, BglII and XbaI restriction nucleases, Taq DNA polymerase and dNTP were purchased from Fermentas (Lithuania).

    Techniques: Reporter Assay, Plasmid Preparation, Recombinant, Bridge PCR, Concentration Assay, Polymerase Chain Reaction, Amplification, Sequencing, Purification, Transformation Assay, Electrophoresis

    Effects of homology and insert length upon the efficiency of ABI-REC. ( A ) Homology was varied by increasing the length of P1R primer in the asymmetric bridge PCR reaction. The reactions with 20 bp and 25 bp P1R primers gave rise to maximum output of fused fragments. ( B ) Transformation of equal volumes of these PCR products into E.coli cells produced double resistant colonies with various numbers. Colony capacity varied, with colonies producing maximum target fragment output being more numerous. The number of colonies per plate was plotted against homology. Error bars indicate mean ± SD from three independent assays. ( C ) Inserts ranging from 1.6 kb to 4 kb were fused into pUC19 in asymmetric PCR reactions. Red arrows denote the fused fragments. ( D ) Transformation of equal volumes of these PCR products into E.coli cells produced various numbers of double resistant colonies. Quantitation of single colonies revealed that colony capacity varied, with colonies producing maximum target fragment output being more numerous. The number of colonies per plate was plotted against insert size. Error bars indicate mean ± SD from three independent assays. ( E ) ApaI and SalI restriction digestion of the plasmids extracted from the single colonies in ( D ). All plasmids released the inserts as designed. Please note that 2.5 kb insert is nearly identical to backbone pUC19, as one band was observed. In addition, the 4 kb insert has three ApaI sites, one of which is only 133 bp in size and therefore undetectable in the gel. A 367 bp band is denoted by a red arrow.

    Journal: BMC Biotechnology

    Article Title: An asymmetric PCR-based, reliable and rapid single-tube native DNA engineering strategy

    doi: 10.1186/1472-6750-12-39

    Figure Lengend Snippet: Effects of homology and insert length upon the efficiency of ABI-REC. ( A ) Homology was varied by increasing the length of P1R primer in the asymmetric bridge PCR reaction. The reactions with 20 bp and 25 bp P1R primers gave rise to maximum output of fused fragments. ( B ) Transformation of equal volumes of these PCR products into E.coli cells produced double resistant colonies with various numbers. Colony capacity varied, with colonies producing maximum target fragment output being more numerous. The number of colonies per plate was plotted against homology. Error bars indicate mean ± SD from three independent assays. ( C ) Inserts ranging from 1.6 kb to 4 kb were fused into pUC19 in asymmetric PCR reactions. Red arrows denote the fused fragments. ( D ) Transformation of equal volumes of these PCR products into E.coli cells produced various numbers of double resistant colonies. Quantitation of single colonies revealed that colony capacity varied, with colonies producing maximum target fragment output being more numerous. The number of colonies per plate was plotted against insert size. Error bars indicate mean ± SD from three independent assays. ( E ) ApaI and SalI restriction digestion of the plasmids extracted from the single colonies in ( D ). All plasmids released the inserts as designed. Please note that 2.5 kb insert is nearly identical to backbone pUC19, as one band was observed. In addition, the 4 kb insert has three ApaI sites, one of which is only 133 bp in size and therefore undetectable in the gel. A 367 bp band is denoted by a red arrow.

    Article Snippet: Enzymes and reagents ApaI, SalI, DpnI, BglII and XbaI restriction nucleases, Taq DNA polymerase and dNTP were purchased from Fermentas (Lithuania).

    Techniques: Bridge PCR, Transformation Assay, Polymerase Chain Reaction, Produced, Quantitation Assay