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TaKaRa taq polymerase

Comparative amplification of HPV-16 DNA with <t>Taq</t> DNA polymerase and with LoTemp™ HiFi ® DNA polymerase . Lanes 1–8: HPV-16 plasmid DNA template at 85 × 10 1 , 85 × 10 2 , 85 × 10 3 , 85 × 10 4 , 85 × 10 5 , 85 × 10 6 , 85 × 10 7 and 85 × 10 8 copies per milliliter. NC: negative control. M: molecular marker. Arrows indicate the lower limit of detection by nested PCR (A) and by the 1 st primary PCR (B) with <t>Takara</t> Taq DNA polymerase and LoTemp™ HiFi ® DNA polymerase, respectively.

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1) Product Images from "Routine human papillomavirus genotyping by DNA sequencing in community hospital laboratories"

Article Title: Routine human papillomavirus genotyping by DNA sequencing in community hospital laboratories

Journal: Infectious Agents and Cancer

doi: 10.1186/1750-9378-2-11

Comparative amplification of HPV-16 DNA with Taq DNA polymerase and with LoTemp™ HiFi ® DNA polymerase . Lanes 1–8: HPV-16 plasmid DNA template at 85 × 10 1 , 85 × 10 2 , 85 × 10 3 , 85 × 10 4 , 85 × 10 5 , 85 × 10 6 , 85 × 10 7 and 85 × 10 8 copies per milliliter. NC: negative control. M: molecular marker. Arrows indicate the lower limit of detection by nested PCR (A) and by the 1 st primary PCR (B) with Takara Taq DNA polymerase and LoTemp™ HiFi ® DNA polymerase, respectively.

Figure Legend Snippet: Comparative amplification of HPV-16 DNA with Taq DNA polymerase and with LoTemp™ HiFi ® DNA polymerase . Lanes 1–8: HPV-16 plasmid DNA template at 85 × 10 1 , 85 × 10 2 , 85 × 10 3 , 85 × 10 4 , 85 × 10 5 , 85 × 10 6 , 85 × 10 7 and 85 × 10 8 copies per milliliter. NC: negative control. M: molecular marker. Arrows indicate the lower limit of detection by nested PCR (A) and by the 1 st primary PCR (B) with Takara Taq DNA polymerase and LoTemp™ HiFi ® DNA polymerase, respectively.

Techniques Used: Amplification, Plasmid Preparation, Negative Control, Marker, Nested PCR, Polymerase Chain Reaction

2) Product Images from "Molecular Identification of Ancylostoma caninum Isolated from Cats in Southern China Based on Complete ITS Sequence"

Article Title: Molecular Identification of Ancylostoma caninum Isolated from Cats in Southern China Based on Complete ITS Sequence

Journal: BioMed Research International

doi: 10.1155/2013/868050

PCR amplicons digested by restriction endonucleases Taq I. Lanes 1–53: random samples from 56 samples; P: PCR amplicon; M: DL-2000 DNA marker.

Figure Legend Snippet: PCR amplicons digested by restriction endonucleases Taq I. Lanes 1–53: random samples from 56 samples; P: PCR amplicon; M: DL-2000 DNA marker.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

3) Product Images from "Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium"

Article Title: Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium

Journal:

doi: 10.1128/AEM.68.1.365-371.2002

Schematic representation of a method for monitoring sthA expression in vivo. (A) Construction of pSTH16, which was used to insert gfp and the Kmr cassette downstream of sthA by double-crossover integration. A 2.5-kb fragment containing sthA (open box) and its upstream region (shaded box) was amplified from genomic DNA by using PyroBEST polymerase and primers UP1 (5′-ATGAGCGATACCCACTTCGGCTTC-3′) and UP2 (5′-TGTGGCGTGCACGACATGTTCGAT-3′). Adenine was added to the 3′ termini of the PCR products by using Taq polymerase, and the products were cloned into pT7Blue by the TA cloning procedure. A plasmid in which the orientation of the insert was opposite that of lacZ was then selected and designated pSTH11. A 0.8-kb Xba I- Hin dIII fragment, including promoterless gfp , was prepared from pUTminiTn 5 gfp and ligated into pSTH11 which had been digested with Xba I and Hin dIII to generate pSTH12. A 1.5-kb fragment that included the region downstream of the sthA gene (ORF and shaded boxes) was amplified from genomic DNA by using PyroBEST polymerase with primers DP3 (5′-TGAAGGCCTGCGGGACGACAGAAGC-3′) and DP4 (5′-TGCTCGTCGATGAAGCGGCTGGCCT-3′), and the PCR product was cloned into pT7Blue as described above. A plasmid in which the orientation of the insert was opposite that of lacZ was selected and designated pSTH13. A 1.3-kb Hin dIII- Sma I fragment carrying Kmr prepared from pSUP5011 was blunted and ligated into pSTH13 which had been digested with Bam HI and blunted in order to generate pSTH14. pSTH12 was digested with Eco RI and Hin dIII, and the 3.3-kb Eco RI- Hin dIII fragment containing sthA-gfp of pSTH12 was blunted and ligated into the Sma I site of pSTH14. A plasmid in which the insert was oriented properly was selected and designated pSTH15. Finally, a 1.8-kb Bam HI fragment carrying the mob site prepared from pSUP5011 was inserted into the Bam HI site of pSTH15, yielding pSTH16, in which gfp and the Kmr cassette were situated between sthA and its downstream ORF, without disrupting either gene. pol., polymerase. (B) Strategy for insertion of gfp and the Kmr cassette between sthA and its downstream ORF.

Figure Legend Snippet: Schematic representation of a method for monitoring sthA expression in vivo. (A) Construction of pSTH16, which was used to insert gfp and the Kmr cassette downstream of sthA by double-crossover integration. A 2.5-kb fragment containing sthA (open box) and its upstream region (shaded box) was amplified from genomic DNA by using PyroBEST polymerase and primers UP1 (5′-ATGAGCGATACCCACTTCGGCTTC-3′) and UP2 (5′-TGTGGCGTGCACGACATGTTCGAT-3′). Adenine was added to the 3′ termini of the PCR products by using Taq polymerase, and the products were cloned into pT7Blue by the TA cloning procedure. A plasmid in which the orientation of the insert was opposite that of lacZ was then selected and designated pSTH11. A 0.8-kb Xba I- Hin dIII fragment, including promoterless gfp , was prepared from pUTminiTn 5 gfp and ligated into pSTH11 which had been digested with Xba I and Hin dIII to generate pSTH12. A 1.5-kb fragment that included the region downstream of the sthA gene (ORF and shaded boxes) was amplified from genomic DNA by using PyroBEST polymerase with primers DP3 (5′-TGAAGGCCTGCGGGACGACAGAAGC-3′) and DP4 (5′-TGCTCGTCGATGAAGCGGCTGGCCT-3′), and the PCR product was cloned into pT7Blue as described above. A plasmid in which the orientation of the insert was opposite that of lacZ was selected and designated pSTH13. A 1.3-kb Hin dIII- Sma I fragment carrying Kmr prepared from pSUP5011 was blunted and ligated into pSTH13 which had been digested with Bam HI and blunted in order to generate pSTH14. pSTH12 was digested with Eco RI and Hin dIII, and the 3.3-kb Eco RI- Hin dIII fragment containing sthA-gfp of pSTH12 was blunted and ligated into the Sma I site of pSTH14. A plasmid in which the insert was oriented properly was selected and designated pSTH15. Finally, a 1.8-kb Bam HI fragment carrying the mob site prepared from pSUP5011 was inserted into the Bam HI site of pSTH15, yielding pSTH16, in which gfp and the Kmr cassette were situated between sthA and its downstream ORF, without disrupting either gene. pol., polymerase. (B) Strategy for insertion of gfp and the Kmr cassette between sthA and its downstream ORF.

Techniques Used: Expressing, In Vivo, Amplification, Polymerase Chain Reaction, Clone Assay, TA Cloning, Plasmid Preparation

4) Product Images from "Structural analysis of adenylate cyclases from Trypanosoma brucei in their monomeric state"

Article Title: Structural analysis of adenylate cyclases from Trypanosoma brucei in their monomeric state

Journal:

doi: 10.1093/emboj/20.3.433

Fig. 1. ( A ) Overall structure of GRESAG4.1-(A884–T1131). The Δ-subdomain between α3 and β4 that is absent in other ACs is highlighted in orange. ( B ) Topologies of tACs, DNA polymerase and NDP kinase. The first half of the GRESAG4.1 AC domain (β1–β4, α1, α2) superimposes at 73 Cα positions with Taq DNA polymerase (r.m.s.d. 1.9 Å) and at 42 positions with NDP kinase from D.melanogaster (r.m.s.d. 2.3 Å). The active center is only in ACs and DNA polymerases placed along β1–β4, while NDP kinases harbor the active site in a loop region. ( C ) σA-weighted 2 F obs – F calc electron density map of GRESAG4.1 at 1.46 Å resolution (contouring level 1.7 σ). ( D ) Dendrogram showing the phylogenetic relationship of ACs and GCs. For its calculation, 40 protein sequences of nucleotidyl cyclases were aligned using the structural alignment between the mammalian C1A and C2A domains and GRESAG4.1 and the multiple and pairwise sequence alignments as computed by PSI-Blast and Clustal_X . A non-redundant subset of 22 nucleotidyl cyclase sequences was selected from the resulting multiple sequence alignment to calculate the phylogenetic relationships using PAUPSCRIPT. The sequences of the AC domains of GRESAG4.1 and GRESAG4.3 correspond to the SWISSPROT entries cy41_trybb and cy43_trybb . Figure A and Figures – were made with MOLSCRIPT and RASTER3D .

Figure Legend Snippet: Fig. 1. ( A ) Overall structure of GRESAG4.1-(A884–T1131). The Δ-subdomain between α3 and β4 that is absent in other ACs is highlighted in orange. ( B ) Topologies of tACs, DNA polymerase and NDP kinase. The first half of the GRESAG4.1 AC domain (β1–β4, α1, α2) superimposes at 73 Cα positions with Taq DNA polymerase (r.m.s.d. 1.9 Å) and at 42 positions with NDP kinase from D.melanogaster (r.m.s.d. 2.3 Å). The active center is only in ACs and DNA polymerases placed along β1–β4, while NDP kinases harbor the active site in a loop region. ( C ) σA-weighted 2 F obs – F calc electron density map of GRESAG4.1 at 1.46 Å resolution (contouring level 1.7 σ). ( D ) Dendrogram showing the phylogenetic relationship of ACs and GCs. For its calculation, 40 protein sequences of nucleotidyl cyclases were aligned using the structural alignment between the mammalian C1A and C2A domains and GRESAG4.1 and the multiple and pairwise sequence alignments as computed by PSI-Blast and Clustal_X . A non-redundant subset of 22 nucleotidyl cyclase sequences was selected from the resulting multiple sequence alignment to calculate the phylogenetic relationships using PAUPSCRIPT. The sequences of the AC domains of GRESAG4.1 and GRESAG4.3 correspond to the SWISSPROT entries cy41_trybb and cy43_trybb . Figure A and Figures – were made with MOLSCRIPT and RASTER3D .

Techniques Used: Sequencing

DNA Extraction:

Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
Article Snippet: Paragraph title: DNA extraction, PCR amplification, and genotyping ... Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water.

Electrophoresis:

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: The PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62.2°C for 20 s and extension at 72°C for 25 s (the annealing temperature used for the amplification of Mu*8 was 62.9°C).

Amplification:

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: Paragraph title: Amplification of whole pHsh plasmid DNA by PPCP with simultaneous selection maker swapping ... The PPCP experiment shown in Fig. was performed in 20 μl reaction solution containing 50 ng pHsh-Amp (2.44 kb) as template, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 80 ng PPCP primer pair prepared by PCR as described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 and 0.5 mM NAD+ .

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: In the new strategy, we selected the ddNTP-blocked primer, which appears to have the best blocking effect , as the allele-specific primer to improve the specificity of PR-PCR. .. In addition, we largely decreased the amount of high-fidelity DNA polymerase to avoid the non-specific primer extension and added a routine amount of Taq DNA polymerase into the reaction to improve amplification efficiency. .. The major role of the tiny amount of high-fidelity DNA polymerase in the reaction is to activate primer extension by specifically removing the 3'-blocked nucleotide under the presence of a mismatch between the primer and the template ( ).

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: Then we selected 62.2°C as the annealing temperature of the modified PR-PCR to further optimize the amount of high-fidelity DNA polymerase. .. The results showed that the use of 0.1–0.15 U of high-fidelity DNA polymerase combined with 1 U of Taq DNA polymerase in per 20 μL reaction resulted in the best discrimination and amplification effects ( ). .. Thus, a mix of 0.1–0.15 U of high-fidelity DNA polymerase with 1 U of Taq DNA polymerase in per 20 μL reaction was selected for the subsequent experiments.

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: With each of these two plasmids serving as template, the same primer pair from pHsh-kan described above was used to construct random mutagenesis libraries by in situ error-prone PPCP. .. The amplification was performed over celA and celB using Taq DNA polymerase in the presence of 0.1 mM Mn2+ and Tma DNA ligase. .. The amplification products were transformed into competent E. coli cells, and the mutant libraries screened on the LB plates containing kanamycin and CMC.

Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
Article Snippet: After preliminary screening from universal plastid and nuclear primers, four cpDNA intergenic spacers, psb A-trn H (Chiang and Peng, ), psb M-trn D (Shaw et al., ), trn S-trn G (Shaw et al., ), and trn L-trn T (Taberlet et al., ) and three nuclear genes, GTP , guanosine triphosphate (GTP) gene (Salas-Leiva et al., ); PHYP , phytochrome P gene and PPRC , hypothetical protein gene (unpublished) were chose for complete analysis (Table ). .. Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water. .. PCR amplifications were performed in a thermocycler under the following conditions: an initial 5 min denaturation at 80°C, followed by 29 cycles of 1 min at 95°C, 1 min annealing at 50°C, and a 1.5 min extension at 65°C, and a final extension for 5 min at 65°C for cpDNA intergenic spacers.

Agarose Gel Electrophoresis:

In Situ:

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: Paragraph title: In situ error-prone PPCP mutagenesis ... Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ .

Polymerase Chain Reaction:

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: Both the wild-type and mutant templates (plasmids) were amplified using an allele-specific 3'-ddC-blocked forward primer and an unblocked reverse primer. .. The PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62.2°C for 20 s and extension at 72°C for 25 s (the annealing temperature used for the amplification of Mu*8 was 62.9°C).

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: PCR was performed in 50 μl reaction solution containing 1.25 U Pyrobest DNA polymerase (TaKaRa) for 30 cycles, each cycle consisting of incubation at 94°C for 40 s, 52°C for 40 s and 72°C for 1 min 10 s. The 894 bp dsDNA fragment generated was subsequently used as PPCP primer pair for amplifying whole pHsh plasmid DNA as described below. .. The PPCP experiment shown in Fig. was performed in 20 μl reaction solution containing 50 ng pHsh-Amp (2.44 kb) as template, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 80 ng PPCP primer pair prepared by PCR as described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 and 0.5 mM NAD+ . .. PCR was performed for 25 cycles with each cycle consisting of incubation at 94°C for 30 s (denaturation), 62°C for 30 s (annealing), 72°C for 1.5 min (extension) and 60°C for 2 min (ligation).

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: Two most common rifampin-resistant mutations, H526Y ( C AC > T AC) and S531L (T C G > T T G) [ ] in the rpoB gene of TB were detected using the modified PR-PCR. .. All reactions were performed in a total volume of 20 μL containing a mixture of 2 μL of (10×) Taq PCR buffer, 0.2 mM dNTPs, 1 U of Taq DNA polymerase, 0.15 U of PrimeSTAR HS DNA polymerase, 5 ng of gDNA (equal to approximately 1×106 copies of the TB genome) and 0.3 μM each of 3'-blocked forward primer and common reverse primer. .. The PCR cycling conditions consisted of pre-denaturation at 95°C for 1 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 59°C for 15 s and extension at 72°C for 15 s. To confirm the results obtained from the modified PR-PCR, a 493 bp fragment of the rpoB gene encompassing the two mutation sites from six TB strains was sequenced using the 454 GS-FLX system (Roche Diagnostics Corporation).

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: For other details, please see . (B) Gel electrophoresis of the amplicons obtained using the AS-PCR with 5 ng of genomic DNA. .. All reactions were performed in a total volume of 20 μL containing a mixture of 0.5 U of Taq DNA polymerase, 0.3 μM each of forward and reverse primer, 0.2 mM dNTPs, (1×) PCR Buffer and 5 ng (equal to approximately 1×106 copies) of template. .. The PCR cycling condition was pre-denaturation at 98°C for 2 min, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 15 s and extension at 72°C for 15 s. The positive (TB-526PC and TB-531PC) and negative control plasmids were both amplified, regardless of whether WT-F or Mu-F was used.

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: The extracted gDNA was eluted into 70 μL of (1×) TE buffer (pH 8.0) and then quantified using a Nanodrop-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). .. For the clinical samples, the modified PR-PCR was performed in a total volume of 25 μL reaction mixture including 20 ng of gDNA, 3.75 μL of (5×) PrimeSTAR buffer, 1.25 μL of (5×) Taq PCR buffer, 0.2 mM dNTPs, 0.2 U of PrimeSTAR HS DNA polymerase, 0.7 U of Taq DNA polymerase, 1 μL of DMSO and 0.3 μM each of reverse primer, fusion-blocked forward primer and adaptor. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min; 40 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 20 s and extension at 72°C for 18 s. The PCR products were visualized via 1.5% agarose gel electrophoresis.

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: In contrast, an increasing amount of circular DNA product was obtained with increasing amount of Tma DNA ligase added to the reaction mixture (lanes 2–5, Fig. ), indicating the need to determine the sufficient amount of DNA ligase required for optimal ligation to compensate for enzyme inactivation at high temperature during the denaturation step of the cycle. .. When Taq DNA polymerase is employed under error-prone conditions, e.g. in the presence of Mn2+ (mutagenic PCR), PPCP can be used for generating random mutagenesis libraries. .. Since mutagenesis occurs in situ during the PCR reaction, eliminating the need of a separate process for generating or sub-cloning mutant DNA, we designated the procedure as in situ error-prone PPCP.

Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
Article Snippet: After preliminary screening from universal plastid and nuclear primers, four cpDNA intergenic spacers, psb A-trn H (Chiang and Peng, ), psb M-trn D (Shaw et al., ), trn S-trn G (Shaw et al., ), and trn L-trn T (Taberlet et al., ) and three nuclear genes, GTP , guanosine triphosphate (GTP) gene (Salas-Leiva et al., ); PHYP , phytochrome P gene and PPRC , hypothetical protein gene (unpublished) were chose for complete analysis (Table ). .. Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water. .. PCR amplifications were performed in a thermocycler under the following conditions: an initial 5 min denaturation at 80°C, followed by 29 cycles of 1 min at 95°C, 1 min annealing at 50°C, and a 1.5 min extension at 65°C, and a final extension for 5 min at 65°C for cpDNA intergenic spacers.

Mutagenesis:

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: Paragraph title: In situ error-prone PPCP mutagenesis ... Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ .

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: In contrast, an increasing amount of circular DNA product was obtained with increasing amount of Tma DNA ligase added to the reaction mixture (lanes 2–5, Fig. ), indicating the need to determine the sufficient amount of DNA ligase required for optimal ligation to compensate for enzyme inactivation at high temperature during the denaturation step of the cycle. .. When Taq DNA polymerase is employed under error-prone conditions, e.g. in the presence of Mn2+ (mutagenic PCR), PPCP can be used for generating random mutagenesis libraries. .. Since mutagenesis occurs in situ during the PCR reaction, eliminating the need of a separate process for generating or sub-cloning mutant DNA, we designated the procedure as in situ error-prone PPCP.

Blocking Assay:

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: The ddNTP-blocked primer can not be extended by DNA polymerase because of the lack of free 3'-OH. .. We first performed AS-PCR assays with Taq DNA polymerase to test and compare the blocking efficiencies of the three kinds of inert primers mentioned above. .. A wild-type template that completely matched the allele-specific blocked primer and a mutant template that contained a C-T mismatch with the 3'-end of the allele-specific primer were used in the reactions ( ).

Purification:

Article Title: Single systemic administration of Ag85B of mycobacteria DNA inhibits allergic airway inflammation in a mouse model of asthma
Article Snippet: Total RNA was purified from OVA-stimulated or fetal calf serum (control)-stimulated spleen cells using Isogen (Nippon Gene Co, Ltd, Tokyo, Japan) following the manufacturer’s instructions. .. Polymerase chain reaction was performed in a total volume of 50 μL of 1 × polymerase chain reaction buffer (Takara Shuzo, Kyoto, Japan) containing 0.5–1.0 μg of complementary DNA, 0.25 mM of each deoxyribonucleotide triphosphate, 2 μM of each primer, and 2.5 U of Taq DNA polymerase (Takara Shuzo).

Real-time Polymerase Chain Reaction:

Article Title: Single systemic administration of Ag85B of mycobacteria DNA inhibits allergic airway inflammation in a mouse model of asthma
Article Snippet: Paragraph title: Detection of cytokine mRNA from lymphocytes using real-time polymerase chain reaction ... Polymerase chain reaction was performed in a total volume of 50 μL of 1 × polymerase chain reaction buffer (Takara Shuzo, Kyoto, Japan) containing 0.5–1.0 μg of complementary DNA, 0.25 mM of each deoxyribonucleotide triphosphate, 2 μM of each primer, and 2.5 U of Taq DNA polymerase (Takara Shuzo).

Modification:

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: These indicate that the modified PR-PCR can be used in long-fragment amplification as well. .. In conclusion, we developed a novel modified PR-PCR method that uses a ddNTP-blocked primer and an enzyme combination of a routine amount of Taq DNA polymerase and a tiny amount of high-fidelity DNA polymerase. .. This method can be used for easy detection of various mutation types, including point mutations, insertions and deletions, with high selectivity and sensitivity.

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: The major role of the tiny amount of high-fidelity DNA polymerase in the reaction is to activate primer extension by specifically removing the 3'-blocked nucleotide under the presence of a mismatch between the primer and the template ( ). .. 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 ( ). .. The result showed that when the amount of high-fidelity DNA polymerase was decreased to 0.05 U, only the mutant sequence could be slightly amplified , indicating that the decrease in the amount of high-fidelity DNA polymerase indeed inhibited the non-specific amplification of the wild-type sequence and enhanced the discrimination efficiency.

Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
Article Snippet: We extracted genomic DNA from dried leaves using the modified CTAB method (Doyle, ). .. Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water.

Selection:

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: Paragraph title: Amplification of whole pHsh plasmid DNA by PPCP with simultaneous selection maker swapping ... The PPCP experiment shown in Fig. was performed in 20 μl reaction solution containing 50 ng pHsh-Amp (2.44 kb) as template, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 80 ng PPCP primer pair prepared by PCR as described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 and 0.5 mM NAD+ .

Sequencing:

Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
Article Snippet: Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water. .. All PCR products of different DNA fragments were sequenced directly in both directions by the dideoxy chain-termination method, using an ABI 3730XL automated sequencer (made in Applied Biosystems) at Shanghai Meiji Biological Medicine and Technology Co Ltd.

other:

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: Thus, a mix of 0.1–0.15 U of high-fidelity DNA polymerase with 1 U of Taq DNA polymerase in per 20 μL reaction was selected for the subsequent experiments.

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: We further performed the routine PR-PCR assay using high-fidelity DNA polymerase instead of Taq DNA polymerase.

Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
Article Snippet: S1 Fig (a) The conventional AS-PCR mediated by Taq DNA polymerase. (b) The typical PR-PCR method mediated by high fidelity DNA polymerase and ddC-blocked primer.

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: Error-prone PPCP using Taq DNA polymerase in the presence of Mn2+ , Tma DNA ligase and the PPCP primer pair from pHsh-kan was performed over the xynA 1 region of pHsh-xynA 1 template (Fig. ).

Plasmid Preparation:

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: PCR of 30 cycles was performed in 50 μl of reaction solution containing 1.25 U Pyrobest DNA polymerase (TaKaRa), each cycle consisting of incubation at 94°C for 30 s, 54°C for 30 s and 72°C for 2 min. .. Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ . .. Endoglucanase gene celA was amplified by PCR using the genomic DNA of T. maritima as template and the following primers: 5′-CTGTGGTACTGATGACAAAACCGGGAACATC-3′ and 5′-GGGAAGCTTTCATTCTCTCACCTCCAGATC-3′.

Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
Article Snippet: Paragraph title: Amplification of whole pHsh plasmid DNA by PPCP with simultaneous selection maker swapping ... The PPCP experiment shown in Fig. was performed in 20 μl reaction solution containing 50 ng pHsh-Amp (2.44 kb) as template, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 80 ng PPCP primer pair prepared by PCR as described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 and 0.5 mM NAD+ .