dna polymerase Toyobo Search Results


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  • 90
    Millipore kod xl dna polymerase
    Kod Xl Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Toyobo kod dna polymerase blunting high kit toyobo
    Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion <t>DNA</t> polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using <t>KOD</t> DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.
    Kod Dna Polymerase Blunting High Kit Toyobo, supplied by Toyobo, used in various techniques. Bioz Stars score: 90/100, based on 564 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo kod one toyobo dna polymerase
    Transactivation of the BLV LTR by various tax clones from BLV-infected cattle. (A and B) BLV tax genes were amplified by PCR with EX Taq (A) or <t>KOD-Plus</t> (B) <t>DNA</t> polymerase from the genomic DNAs of BLV-infected animals and from a molecular clone pBLV-IF and then subcloned into pME18Neo. 293T cells were transfected with the reporter plasmid pGV-BLTR, the reference plasmid pRL-SV40, and individual Tax-expressing plasmids. At 60 h after transfection, cells were recovered and the activities of firefly and Renilla luciferases were measured in lysates. For each sample, the firefly luciferase activity (pGV-BLTR) was normalized by reference to Renilla luciferase activity (pRL-SV40). The extent of transactivation (fold) was calculated by dividing the transactivation activity of the mutant Tax protein by that of wild-type Tax protein. (C) Summary of the results shown in panels A and B. The tax mutant clones were divided into three groups as follows, with wild-type activity being taken as 1: high, > 3-fold transactivation; medium, 3- to 0.5-fold transactivation; low,
    Kod One Toyobo Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 96/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 508 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Toyobo δtth dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    δtth Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 87/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo tth dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Tth Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo t4 dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    T4 Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 87/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo rtth dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Rtth Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Toyobo recombinanttaq dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Recombinanttaq Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 87/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo dna polymerase kodplus
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Dna Polymerase Kodplus, supplied by Toyobo, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo dna polymerase blend taq
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Dna Polymerase Blend Taq, supplied by Toyobo, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo rtaq dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Rtaq Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 189 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo revertra ace dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Revertra Ace Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo sybr green dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Sybr Green Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo thermus thermophilus dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Thermus Thermophilus Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 82/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo rtaq dna polymerase kit
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Rtaq Dna Polymerase Kit, supplied by Toyobo, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo kod plus2 dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Kod Plus2 Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo high fidelity thermophilic dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    High Fidelity Thermophilic Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore kod hotstart dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Kod Hotstart Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo kod hifi dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Kod Hifi Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 87/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo thermostable dna polymerase kod
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Thermostable Dna Polymerase Kod, supplied by Toyobo, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Toyobo phusion dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Phusion Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Toyobo thermococcus kodakaraensis fx dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Thermococcus Kodakaraensis Fx Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Toyobo kod1 dna polymerase
    Quantification of transcription levels by real-time <t>RT-PCR</t> ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known <t>DNA</t> fragment. The expression
    Kod1 Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Toyobo thermostable dna polymerase
    Transcription mechanism of <t>CgHIFα-like.</t> (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro <t>DNA</t> binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).
    Thermostable Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Toyobo andtaq dna polymerase
    Transcription mechanism of <t>CgHIFα-like.</t> (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro <t>DNA</t> binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).
    Andtaq Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Toyobo dna polymerase system
    Transcription mechanism of <t>CgHIFα-like.</t> (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro <t>DNA</t> binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).
    Dna Polymerase System, supplied by Toyobo, used in various techniques. Bioz Stars score: 95/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Toyobo dna polymerase kit
    Transcription mechanism of <t>CgHIFα-like.</t> (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro <t>DNA</t> binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).
    Dna Polymerase Kit, supplied by Toyobo, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Toyobo gotag dna polymerase
    Transcription mechanism of <t>CgHIFα-like.</t> (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro <t>DNA</t> binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).
    Gotag Dna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcr  (TaKaRa)
    99
    TaKaRa pcr
    Transcription mechanism of <t>CgHIFα-like.</t> (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro <t>DNA</t> binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).
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    85
    Toyobo tth dna polymerase autosequencer core kit
    Transcription mechanism of <t>CgHIFα-like.</t> (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro <t>DNA</t> binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).
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    Image Search Results


    Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Journal: Nucleic Acid Therapeutics

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

    doi: 10.1089/nat.2014.0513

    Figure Lengend Snippet: Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Amplification, Modification

    The G/C-rich sequence associated with a meiosis-specific DSB on chromosome IV of S. cerevisiae folds into intramolecular G-quadruplex or i-motif structures and blocks DNA synthesis. ( A ) Here is a schematic diagram showing the nucleotide sequence of G-rich (WT) and its corresponding mutant template. ( B ) The Taq polymerase stop assay was performed in the absence (lanes 1 and 6 ) or presence of increasing concentrations of KCl (10, 25, 50, and 100 mM; lanes 2 – 5 and lanes 7 – 10 , respectively). The solid triangle on top of the gel denotes increasing concentrations of KCl. ( C ) Here we show a diagram of C-rich (WT) and its corresponding mutant DNA template. ( D ) Given here is the KOD-Plus DNA polymerase (from Thermococcus kodakaraensis ) stop assay performed with WT i-motif and its corresponding mutant template at the indicated pH values. Lane 1 is the primer used in this study. ( E ) SWNTs stabilize the i-motif DNA structure at neutral pH, which, in turn, blocks primer extension by DNA polymerase.

    Journal: Biophysical Journal

    Article Title: Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks

    doi: 10.1016/j.bpj.2017.04.028

    Figure Lengend Snippet: The G/C-rich sequence associated with a meiosis-specific DSB on chromosome IV of S. cerevisiae folds into intramolecular G-quadruplex or i-motif structures and blocks DNA synthesis. ( A ) Here is a schematic diagram showing the nucleotide sequence of G-rich (WT) and its corresponding mutant template. ( B ) The Taq polymerase stop assay was performed in the absence (lanes 1 and 6 ) or presence of increasing concentrations of KCl (10, 25, 50, and 100 mM; lanes 2 – 5 and lanes 7 – 10 , respectively). The solid triangle on top of the gel denotes increasing concentrations of KCl. ( C ) Here we show a diagram of C-rich (WT) and its corresponding mutant DNA template. ( D ) Given here is the KOD-Plus DNA polymerase (from Thermococcus kodakaraensis ) stop assay performed with WT i-motif and its corresponding mutant template at the indicated pH values. Lane 1 is the primer used in this study. ( E ) SWNTs stabilize the i-motif DNA structure at neutral pH, which, in turn, blocks primer extension by DNA polymerase.

    Article Snippet: The annealing mixture was mixed with a reaction buffer (1 μ L) containing 1.5 mg/mL bovine serum albumin, 0.2 mM dNTPs, and 0.5 U of KOD-Plus DNA polymerase (Toyobo, Osaka, Japan).

    Techniques: Sequencing, DNA Synthesis, Mutagenesis

    Blocking efficiency of three kinds of blocked primers in PCR amplifications mediated by Taq DNA polymerase and high-fidelity DNA polymerase. (a) Primer and template sets. WT and Mu indicate wild-type and mutant types, respectively. The 3'-blocked forward primer completely matches the WT sequence but forms a mismatch with the Mu sequence (Mu*1) at the 3'-end. The reverse primer matches both the WT and Mu sequences. The 3'-terminal nucleotide of the blocked primer is blocked with-Pi or-Amino C6, or replaced with ddCTP. (b) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by Taq DNA polymerase. (c) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by high-fidelity DNA polymerase (KOD FX). (d) Discrimination efficiency of the typical proofreading-PCR medicated by various amounts (0.5, 1, 1.5 and 2 U) of KOD FX DNA polymerase using the ddC-blocked primer. All PCR amplifications were performed in a total volume of 20 μL containing 1 U of Taq or KOD FX DNA polymerase (Panel b and c) or various amounts of KOD FX DNA polymerase (Panel d). The cycling conditions consisted of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. NTC: no-template control.

    Journal: PLoS ONE

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    doi: 10.1371/journal.pone.0123468

    Figure Lengend Snippet: Blocking efficiency of three kinds of blocked primers in PCR amplifications mediated by Taq DNA polymerase and high-fidelity DNA polymerase. (a) Primer and template sets. WT and Mu indicate wild-type and mutant types, respectively. The 3'-blocked forward primer completely matches the WT sequence but forms a mismatch with the Mu sequence (Mu*1) at the 3'-end. The reverse primer matches both the WT and Mu sequences. The 3'-terminal nucleotide of the blocked primer is blocked with-Pi or-Amino C6, or replaced with ddCTP. (b) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by Taq DNA polymerase. (c) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by high-fidelity DNA polymerase (KOD FX). (d) Discrimination efficiency of the typical proofreading-PCR medicated by various amounts (0.5, 1, 1.5 and 2 U) of KOD FX DNA polymerase using the ddC-blocked primer. All PCR amplifications were performed in a total volume of 20 μL containing 1 U of Taq or KOD FX DNA polymerase (Panel b and c) or various amounts of KOD FX DNA polymerase (Panel d). The cycling conditions consisted of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. NTC: no-template control.

    Article Snippet: To determine the appropriate amount of high-fidelity DNA polymerase for the modified PR-PCR, we performed a preliminary experiment using a combination of 1 U of Taq DNA polymerase together with 0.05, 0.1 or 0.3 U of KOD FX DNA polymerase at an annealing temperature of 56°C in a 20 μL reaction ( ).

    Techniques: Blocking Assay, Polymerase Chain Reaction, Mutagenesis, Sequencing

    Optimization of annealing temperature and amount of high-fidelity DNA polymerase in the modified PR-PCR. (a) Discrimination efficiency of the modified PR-PCR for known mutations using a mixture of 1 U of Taq DNA polymerase and various amount (0.05, 0.1 and 0.3 U) of KOD FX DNA polymerase in the reaction. The PCR amplifications were performed in a total volume of 20 μL under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. (b) Optimization of annealing temperature for the modified PR-PCR using a mixture of 1 U of Taq DNA polymerase and 0.1 U of KOD FX DNA polymerase. As the optimal discrimination efficiency was obtained when the annealing temperature was within a scope of 61.8°C–62.6°C, the results from 50°C to 61.2°C were not shown. The temperature 62.2°C was selected for subsequent experiments. (c) Optimization of amount of high-fidelity DNA polymerase for the modified PR-PCR with an input of 1 U of Taq DNA polymerase at an annealing temperature of 62.2°C.

    Journal: PLoS ONE

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    doi: 10.1371/journal.pone.0123468

    Figure Lengend Snippet: Optimization of annealing temperature and amount of high-fidelity DNA polymerase in the modified PR-PCR. (a) Discrimination efficiency of the modified PR-PCR for known mutations using a mixture of 1 U of Taq DNA polymerase and various amount (0.05, 0.1 and 0.3 U) of KOD FX DNA polymerase in the reaction. The PCR amplifications were performed in a total volume of 20 μL under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. (b) Optimization of annealing temperature for the modified PR-PCR using a mixture of 1 U of Taq DNA polymerase and 0.1 U of KOD FX DNA polymerase. As the optimal discrimination efficiency was obtained when the annealing temperature was within a scope of 61.8°C–62.6°C, the results from 50°C to 61.2°C were not shown. The temperature 62.2°C was selected for subsequent experiments. (c) Optimization of amount of high-fidelity DNA polymerase for the modified PR-PCR with an input of 1 U of Taq DNA polymerase at an annealing temperature of 62.2°C.

    Article Snippet: To determine the appropriate amount of high-fidelity DNA polymerase for the modified PR-PCR, we performed a preliminary experiment using a combination of 1 U of Taq DNA polymerase together with 0.05, 0.1 or 0.3 U of KOD FX DNA polymerase at an annealing temperature of 56°C in a 20 μL reaction ( ).

    Techniques: Modification, Polymerase Chain Reaction

    Successive incorporation of 2′,4′-bridged nucleotides using analogue 3 with various DNA polymerases; KOD Dash (lane 2), wild type KOD (lane 3), KOD 1 (lane 4), KOD 2 (lane 5), KOD 3 (lane 6), KOD 4 (lane 7), KOD 5 (lane 8), KOD 6 (lane 9), KOD 7 (lane 10), KOD 8 (lane 11), and Vent(exo-) (lane 12). Primer extension reactions were performed for 1 h at 74°C under enzyme concentration of 0.4 U/μL. Primer P2 only migrated in lane 1.

    Journal: Molecules

    Article Title: Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates

    doi: 10.3390/molecules15118229

    Figure Lengend Snippet: Successive incorporation of 2′,4′-bridged nucleotides using analogue 3 with various DNA polymerases; KOD Dash (lane 2), wild type KOD (lane 3), KOD 1 (lane 4), KOD 2 (lane 5), KOD 3 (lane 6), KOD 4 (lane 7), KOD 5 (lane 8), KOD 6 (lane 9), KOD 7 (lane 10), KOD 8 (lane 11), and Vent(exo-) (lane 12). Primer extension reactions were performed for 1 h at 74°C under enzyme concentration of 0.4 U/μL. Primer P2 only migrated in lane 1.

    Article Snippet: Wild-type KOD DNA polymerase (Toyobo), KOD Dash DNA polymerase (Toyobo), and Vent(exo-) DNA polymerase (New England Biolabs) were also used as reference materials; Vent(exo-) is a genetically engineered form of the native DNA polymerase, which also belongs to family B, from Thermococcus litoralis [ , ].

    Techniques: Concentration Assay

    Proofreading DNA polymerases but not Taq DNA polymerase bind to Olig2.5. ( A ) Biotin-labeled oligonucleotide Bio-Olig2.5 was subjected to electrophoresis mobility shift assay. All DNAPs except Taq caused band retardation. The retarded bands caused by GXL, Q5 and KOD were disappeared by adding 1000 × excess amount of specific competitor N10 but not non-specific competitor MutL2-16. ( B ) Taq didn’t cause any band shift even used at 1000 nM. A shifted band appeared when KOD was used at 10 nM approximately half of the Bio-Olig2.5 was shifted when KOD was at 40 nM. Full-length membrane exoposure image is presented in Supplementary Figure 2 .

    Journal: Scientific Reports

    Article Title: TT(N)mGCCTC inhibits archaeal family B DNA polymerases

    doi: 10.1038/s41598-018-20127-4

    Figure Lengend Snippet: Proofreading DNA polymerases but not Taq DNA polymerase bind to Olig2.5. ( A ) Biotin-labeled oligonucleotide Bio-Olig2.5 was subjected to electrophoresis mobility shift assay. All DNAPs except Taq caused band retardation. The retarded bands caused by GXL, Q5 and KOD were disappeared by adding 1000 × excess amount of specific competitor N10 but not non-specific competitor MutL2-16. ( B ) Taq didn’t cause any band shift even used at 1000 nM. A shifted band appeared when KOD was used at 10 nM approximately half of the Bio-Olig2.5 was shifted when KOD was at 40 nM. Full-length membrane exoposure image is presented in Supplementary Figure 2 .

    Article Snippet: KOD DNA polymerase (KOD) was purchased from TOYOBO Biotech (Shanghai, China).

    Techniques: Labeling, Electrophoresis, Mobility Shift, Electrophoretic Mobility Shift Assay

    Olig2.5 inhibited archaeal family B DNA polymerases. An E. coli DH5α colony harbouring the 2.6 kb upstream sequence of Olig2 in pGL3-Basic vector was cultured and 1 μl of the culture was directly used as template for PCR. Olig2F and OligTSSR were used as primers to amplify the 2 kb fragment. Olig2.5 was added to see its inhibition to various DNAP. DNAP: DNA polymerase. LA: LA Taq DNAP. Q5: Q5 High-Fidelity DNAP. PS: PrimeSTAR HS DNAP. GXL: PrimeSTAR GXL DNAP. Phusion: Phusion High-Fidelity DNAP. Cobuddy: Cobuddy Super Fidelity DNAP. KOD: KOD DNAP.

    Journal: Scientific Reports

    Article Title: TT(N)mGCCTC inhibits archaeal family B DNA polymerases

    doi: 10.1038/s41598-018-20127-4

    Figure Lengend Snippet: Olig2.5 inhibited archaeal family B DNA polymerases. An E. coli DH5α colony harbouring the 2.6 kb upstream sequence of Olig2 in pGL3-Basic vector was cultured and 1 μl of the culture was directly used as template for PCR. Olig2F and OligTSSR were used as primers to amplify the 2 kb fragment. Olig2.5 was added to see its inhibition to various DNAP. DNAP: DNA polymerase. LA: LA Taq DNAP. Q5: Q5 High-Fidelity DNAP. PS: PrimeSTAR HS DNAP. GXL: PrimeSTAR GXL DNAP. Phusion: Phusion High-Fidelity DNAP. Cobuddy: Cobuddy Super Fidelity DNAP. KOD: KOD DNAP.

    Article Snippet: KOD DNA polymerase (KOD) was purchased from TOYOBO Biotech (Shanghai, China).

    Techniques: Sequencing, Plasmid Preparation, Cell Culture, Polymerase Chain Reaction, Inhibition

    Transactivation of the BLV LTR by various tax clones from BLV-infected cattle. (A and B) BLV tax genes were amplified by PCR with EX Taq (A) or KOD-Plus (B) DNA polymerase from the genomic DNAs of BLV-infected animals and from a molecular clone pBLV-IF and then subcloned into pME18Neo. 293T cells were transfected with the reporter plasmid pGV-BLTR, the reference plasmid pRL-SV40, and individual Tax-expressing plasmids. At 60 h after transfection, cells were recovered and the activities of firefly and Renilla luciferases were measured in lysates. For each sample, the firefly luciferase activity (pGV-BLTR) was normalized by reference to Renilla luciferase activity (pRL-SV40). The extent of transactivation (fold) was calculated by dividing the transactivation activity of the mutant Tax protein by that of wild-type Tax protein. (C) Summary of the results shown in panels A and B. The tax mutant clones were divided into three groups as follows, with wild-type activity being taken as 1: high, > 3-fold transactivation; medium, 3- to 0.5-fold transactivation; low,

    Journal: Journal of Virology

    Article Title: The Region between Amino Acids 245 and 265 of the Bovine Leukemia Virus (BLV) Tax Protein Restricts Transactivation Not Only via the BLV Enhancer but Also via Other Retrovirus Enhancers

    doi:

    Figure Lengend Snippet: Transactivation of the BLV LTR by various tax clones from BLV-infected cattle. (A and B) BLV tax genes were amplified by PCR with EX Taq (A) or KOD-Plus (B) DNA polymerase from the genomic DNAs of BLV-infected animals and from a molecular clone pBLV-IF and then subcloned into pME18Neo. 293T cells were transfected with the reporter plasmid pGV-BLTR, the reference plasmid pRL-SV40, and individual Tax-expressing plasmids. At 60 h after transfection, cells were recovered and the activities of firefly and Renilla luciferases were measured in lysates. For each sample, the firefly luciferase activity (pGV-BLTR) was normalized by reference to Renilla luciferase activity (pRL-SV40). The extent of transactivation (fold) was calculated by dividing the transactivation activity of the mutant Tax protein by that of wild-type Tax protein. (C) Summary of the results shown in panels A and B. The tax mutant clones were divided into three groups as follows, with wild-type activity being taken as 1: high, > 3-fold transactivation; medium, 3- to 0.5-fold transactivation; low,

    Article Snippet: Approximately 10 to 100 ng of genomic or plasmid DNA was amplified in a 30-μl reaction mixture that contained 1× EX Taq buffer (Takara Shuzo, Ohtsu, Japan) or 1× KOD-Plus buffer containing 1 mM MgSO4 , (Toyobo, Kyoto, Japan), 0.25 mM each deoxynucleoside triphosphate, 0.33 μM each oligonucleotide primer, and 1 U of EX Taq (Takara Shuzo) or KOD-Plus™ (Toyobo) DNA polymerase.

    Techniques: Clone Assay, Infection, Amplification, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Mutagenesis

    Quantification of transcription levels by real-time RT-PCR ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known DNA fragment. The expression

    Journal: The Journal of Biological Chemistry

    Article Title: Chondroitin Sulfate Synthase-2/Chondroitin Polymerizing Factor Has Two Variants with Distinct Function *

    doi: 10.1074/jbc.M110.109553

    Figure Lengend Snippet: Quantification of transcription levels by real-time RT-PCR ( A ) and identification of the CSS2 variant as protein ( B ). A , standard curves for the CSS2 variants and GAPDH were generated by serial dilution of each concentration-known DNA fragment. The expression

    Article Snippet: The PCR was carried out using KOD -Plus DNA polymerase (Toyobo Biochemicals) with a program of 35 cycles of 98 °C for 10 s, 75 °C for 30 s, and 68 °C for 5 min.

    Techniques: Quantitative RT-PCR, Variant Assay, Generated, Serial Dilution, Concentration Assay, Expressing

    Electrophoresis patterns obtained by EAO-genotyping PCR. A total of 33 strains representing 33 EAOgs were analysed using three sets of PCR primers designed in this study. Strain names are indicated in parentheses. Strains belonging to the remaining seven EAOgs were not available in our laboratory. Arrowheads 1 and 2 indicate the bands derived from the E. albertii -specific primer pairs E_al_1_OF/OR and E_al_1_NF/NR, respectively. Lane M, 100 bp DNA ladder.

    Journal: Microbial Genomics

    Article Title: O-antigen biosynthesis gene clusters of Escherichia albertii: their diversity and similarity to Escherichia coli gene clusters and the development of an O-genotyping method

    doi: 10.1099/mgen.0.000314

    Figure Lengend Snippet: Electrophoresis patterns obtained by EAO-genotyping PCR. A total of 33 strains representing 33 EAOgs were analysed using three sets of PCR primers designed in this study. Strain names are indicated in parentheses. Strains belonging to the remaining seven EAOgs were not available in our laboratory. Arrowheads 1 and 2 indicate the bands derived from the E. albertii -specific primer pairs E_al_1_OF/OR and E_al_1_NF/NR, respectively. Lane M, 100 bp DNA ladder.

    Article Snippet: KOD -Multi and Epi- DNA polymerase (Toyobo) was used for PCR.

    Techniques: Electrophoresis, Polymerase Chain Reaction, Derivative Assay

    Selection of biallelic mutants using two different colors of fluorescence. (a) Fluorescence of F 2 embryos derived from mating with each F 1 monoallelic mutants harboring either the GFP or the RFP gene in the gap43 locus. Each of the F 2 embryos were categorized into the following four groups; no fluorescence (G−/R–), green fluorescence (G+/R–), red fluorescence (G−/R+), and both green and red fluorescence (G+/R+). The images were captured at 4 days post fertilization (dpf). White arrowheads show fluorescence in the central nervous system (CNS), while white arrows show autofluorescence of bacteria on the surface of the egg membrane. (b, c) PCR genotyping of the F 2 embryos derived from mating between the F 1 monoallelic mutants. (b) The EF1-α fragment (519 bp) was used to confirm genomic DNA extraction, and was detected in all samples. The gap43 fragment (551 bp) was detected from the groups (G−/R–), (G+/R–), and (G−/R+). The GFP fragment (749 bp) was detected from (G+/R–) and (G+/R+) groups, while the RFP fragment (698 bp) was detected from (G−/R+) and (G+/R+) groups. In (G+/R+), no PCR product of gap43 was detected. (c) Design of PCR primers to detect the targeted gene integration into the gap43 locus. (Upper): Genomic structure of the intact gap43 locus. The size of the PCR product amplified using primer pair (GAP43-for-Seq-Fw and GAP43-Rv) is 551 bp. The latter primer is 20 bp in size and is shown as a red arrow. The reverse primer is designed on the boundary between upstream and downstream long homology arms, so that this primer pairs can amplify the intact locus but not the GFP- or RFP-integrated loci. (Middle): Genomic structure of gap43 locus at which the donor plasmid harboring the GFP gene is integrated. The size of the PCR product amplified using the primer pair (GAP43-for-Seq-Fw and mAG-Rv) is 749 bp. (Lower): Genomic structure of the gap43 gene at which the donor plasmid harboring the RFP gene is integrated. The size of the PCR product amplified using the primer pair (GAP43-for-Seq-Fw and tdTomato-Rv) is 698 bp

    Journal: Zoological Letters

    Article Title: An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)

    doi: 10.1186/s40851-017-0071-x

    Figure Lengend Snippet: Selection of biallelic mutants using two different colors of fluorescence. (a) Fluorescence of F 2 embryos derived from mating with each F 1 monoallelic mutants harboring either the GFP or the RFP gene in the gap43 locus. Each of the F 2 embryos were categorized into the following four groups; no fluorescence (G−/R–), green fluorescence (G+/R–), red fluorescence (G−/R+), and both green and red fluorescence (G+/R+). The images were captured at 4 days post fertilization (dpf). White arrowheads show fluorescence in the central nervous system (CNS), while white arrows show autofluorescence of bacteria on the surface of the egg membrane. (b, c) PCR genotyping of the F 2 embryos derived from mating between the F 1 monoallelic mutants. (b) The EF1-α fragment (519 bp) was used to confirm genomic DNA extraction, and was detected in all samples. The gap43 fragment (551 bp) was detected from the groups (G−/R–), (G+/R–), and (G−/R+). The GFP fragment (749 bp) was detected from (G+/R–) and (G+/R+) groups, while the RFP fragment (698 bp) was detected from (G−/R+) and (G+/R+) groups. In (G+/R+), no PCR product of gap43 was detected. (c) Design of PCR primers to detect the targeted gene integration into the gap43 locus. (Upper): Genomic structure of the intact gap43 locus. The size of the PCR product amplified using primer pair (GAP43-for-Seq-Fw and GAP43-Rv) is 551 bp. The latter primer is 20 bp in size and is shown as a red arrow. The reverse primer is designed on the boundary between upstream and downstream long homology arms, so that this primer pairs can amplify the intact locus but not the GFP- or RFP-integrated loci. (Middle): Genomic structure of gap43 locus at which the donor plasmid harboring the GFP gene is integrated. The size of the PCR product amplified using the primer pair (GAP43-for-Seq-Fw and mAG-Rv) is 749 bp. (Lower): Genomic structure of the gap43 gene at which the donor plasmid harboring the RFP gene is integrated. The size of the PCR product amplified using the primer pair (GAP43-for-Seq-Fw and tdTomato-Rv) is 698 bp

    Article Snippet: The junction regions of the target site on the host genome and introduced gene were amplified by PCR using the following primer pairs: acta1-for-Seq-Fw and mAG-Rv, mAG-Fw and acta1-for-Seq-Rv, or GAP43-for-Seq-FW and GAP43-for Seq-RV (Additional file : Table S4), and KOD -plus- Neo DNA polymerase (Toyobo).

    Techniques: Selection, Fluorescence, Derivative Assay, Polymerase Chain Reaction, DNA Extraction, Amplification, Plasmid Preparation

    Typical mass spectra of four biomarker proteins in E. coli . A) MALDI mass spectra of non-EHEC E. coli strain NBRC12713 and EHEC E. coli strain O157 GTC 14513. Three biomarker peaks, HdeB ( m/z 9066.2 [M+H] + ), ribosomal protein S15 ( m/z 10138.6/10166.6 [M+H] + ) and L25 ( m/z 10676.4/10694.4 [M+H] + ), measured using sinapic acid as the matrix, are shown. B) Biomarker peak of DNA binding protein H-NS ( m/z 15409.4/15425.4 [M+H] + ) for strains O26, O111 and other E. coli .

    Journal: PLoS ONE

    Article Title: Discrimination of Escherichia coli O157, O26 and O111 from Other Serovars by MALDI-TOF MS Based on the S10-GERMS Method

    doi: 10.1371/journal.pone.0113458

    Figure Lengend Snippet: Typical mass spectra of four biomarker proteins in E. coli . A) MALDI mass spectra of non-EHEC E. coli strain NBRC12713 and EHEC E. coli strain O157 GTC 14513. Three biomarker peaks, HdeB ( m/z 9066.2 [M+H] + ), ribosomal protein S15 ( m/z 10138.6/10166.6 [M+H] + ) and L25 ( m/z 10676.4/10694.4 [M+H] + ), measured using sinapic acid as the matrix, are shown. B) Biomarker peak of DNA binding protein H-NS ( m/z 15409.4/15425.4 [M+H] + ) for strains O26, O111 and other E. coli .

    Article Snippet: In brief, respective regions of ribosomal protein-encoding genes (≈5 kbp) or genes encoding biomarker proteins were amplified using high-fidelity DNA polymerase, KOD plus (Toyobo, Osaka, Japan), and primers designed against the consensus DNA sequences up- and down-stream of the target regions in the E. coli genome sequences in the NCBI database.

    Techniques: Biomarker Assay, Binding Assay

    Transcription mechanism of CgHIFα-like. (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro DNA binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).

    Journal: PLoS ONE

    Article Title: Characterization of CgHIFα-Like, a Novel bHLH-PAS Transcription Factor Family Member, and Its Role under Hypoxia Stress in the Pacific Oyster Crassostrea gigas

    doi: 10.1371/journal.pone.0166057

    Figure Lengend Snippet: Transcription mechanism of CgHIFα-like. (A) Transactivation activities of CgHIFα-like. HEK 293T cells were transfected with the indicated pcDNA-CgHIFαlike, pcDNA-CgHIFα, or empty vector (control) plasmids together with p2.1 (solid bars) or the p2.4 (open bars) reporter plasmids; pRL-SV40 ( Renilla ) was cotransfected as an internal control. Transactivation activity is expressed as fold increase over the empty vector group. Data are displayed as mean ± SD of independent triplicate experiments. (B) Electrophoretic mobility shift assay for in vitro DNA binding of CgHIFα-like and CgHIFα. EMSA was performed using the recombinant proteins from the prokaryotic expression system. Lane 1 (Ck) was a negative control, and contained only the biotin Ew probe; Lane 2 (S) contained Ew probe as well as CgHIFαlike-His protein sample. For competition experiments, a 200 M excess of unlabeled human HRE Ew ( lane 3 ) or mutant oligonucleotides Em ( lane 4 ) of erythropoietin gene was used. For supershift experiment, binding reaction mixtures were incubated with a monoclonal anti-His antibody Ab ( lane 6 ) or the preimmune serum IgG ( lane 5 ).

    Article Snippet: Plasmid Construction The full-length CgHIFα-like cDNA was amplified by PCR using the thermostable DNA polymerase from Thermococcus kodakaraensis (KOD plus DNA polymerase; Toyobo, Japan).

    Techniques: Transfection, Plasmid Preparation, Activity Assay, Electrophoretic Mobility Shift Assay, In Vitro, Binding Assay, Recombinant, Expressing, Negative Control, Mutagenesis, Incubation

    The workflow of the rapid detection method for Mycoplasma , Ureaplasma , other bacteria and fungi in amniotic fluid samples. Using this PCR-based method, pathogens can be detected within three hours of amniotic fluid sample collection. To prevent the occurrence of unreliable results in PCR-based assaying of amniotic samples for bacterial pathogens, eukaryote-made thermostable DNA polymerase (or Taq polymerase), which is free from bacterial DNA contamination, is used in combination with bacterial universal primers (along with two specific primers in the second, nested PCR). In contrast, for fungal pathogens, conventional bacterially-made thermostable DNA polymerase (or Taq polymerase), which is usually free from fungal DNA contamination, is used in combination with fungal universal primers.

    Journal: PLoS ONE

    Article Title: Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases

    doi: 10.1371/journal.pone.0129032

    Figure Lengend Snippet: The workflow of the rapid detection method for Mycoplasma , Ureaplasma , other bacteria and fungi in amniotic fluid samples. Using this PCR-based method, pathogens can be detected within three hours of amniotic fluid sample collection. To prevent the occurrence of unreliable results in PCR-based assaying of amniotic samples for bacterial pathogens, eukaryote-made thermostable DNA polymerase (or Taq polymerase), which is free from bacterial DNA contamination, is used in combination with bacterial universal primers (along with two specific primers in the second, nested PCR). In contrast, for fungal pathogens, conventional bacterially-made thermostable DNA polymerase (or Taq polymerase), which is usually free from fungal DNA contamination, is used in combination with fungal universal primers.

    Article Snippet: During the PCR, the PCR reaction mixture (20 μL) contained 2 μL of DNA template or 2 μL (8.0 ng/μL) of DNA extracted from Candida albicans as a positive control, or distilled water (NAKALAI TESQUE, INC.) as a negative control in 50 mM KCl, 2.5 mM MgCl2 , 10 mM Tris-HCl (pH 8.3), 200 μM of each deoxynucleoside triphosphate (dNTP), 0.25 μM each of Fungal universal primer, 1×EvaGreen (Biotium Inc.), and 2.0 units (0.4 μL) of conventional thermostable DNA polymerase (r-Taq: Toyobo, Osaka, Japan) supplemented with stock buffer solution.

    Techniques: Polymerase Chain Reaction, Nested PCR