Structured Review

TaKaRa template dna
<t>PCR</t> amplification of the complete genomes of 3 isolates. M: <t>DNA</t> marker 3; 1–3: GD150509, GD160403, and GD160607; A, B, C, D: 4 DNA fragments of the complete genome.
Template Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/template dna/product/TaKaRa
Average 97 stars, based on 100 article reviews
Price from $9.99 to $1999.99
template dna - by Bioz Stars, 2020-01
97/100 stars

Images

1) Product Images from "Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China"

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China

Journal: Viruses

doi: 10.3390/v10040194

PCR amplification of the complete genomes of 3 isolates. M: DNA marker 3; 1–3: GD150509, GD160403, and GD160607; A, B, C, D: 4 DNA fragments of the complete genome.
Figure Legend Snippet: PCR amplification of the complete genomes of 3 isolates. M: DNA marker 3; 1–3: GD150509, GD160403, and GD160607; A, B, C, D: 4 DNA fragments of the complete genome.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

PCR amplification of the complete genomes of 3 isolates. M: DNA marker DL5000; 1–3: GDFX0601, GDFX0602, and GDFX0603; A, B, C, D: 4 DNA fragments of the complete genome.
Figure Legend Snippet: PCR amplification of the complete genomes of 3 isolates. M: DNA marker DL5000; 1–3: GDFX0601, GDFX0602, and GDFX0603; A, B, C, D: 4 DNA fragments of the complete genome.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

2) Product Images from "Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams"

Article Title: Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams

Journal: PLoS ONE

doi: 10.1371/journal.pone.0171274

Electrophorograms of the RT-PCR products of NER genes in Ca . Vesicomyosocius okutanii (Vok) and the symbiont of C . pacifica (Cpac_S). A, Cpac_S; B, Vok. Expression of NER-related genes was analyzed by RT-PCR. Lane 1, uvrA ; lane 2, uvrB or corresponding DNA region; lane 3, uvrC or corresponding DNA region; lane 4, uvrD ; lane 5, uvrDp ; lane 6, recA ; lane 7, mfd ; lane 8, 16S rRNA gene; lane 9, 16S rRNA gene negative control (with RNase treatment before RT PCR). M, molecular markers. Primers and the predicted lengths of amplicons are shown in S3 Table . Red arrowheads indicate the position of a band where no signal was detected.
Figure Legend Snippet: Electrophorograms of the RT-PCR products of NER genes in Ca . Vesicomyosocius okutanii (Vok) and the symbiont of C . pacifica (Cpac_S). A, Cpac_S; B, Vok. Expression of NER-related genes was analyzed by RT-PCR. Lane 1, uvrA ; lane 2, uvrB or corresponding DNA region; lane 3, uvrC or corresponding DNA region; lane 4, uvrD ; lane 5, uvrDp ; lane 6, recA ; lane 7, mfd ; lane 8, 16S rRNA gene; lane 9, 16S rRNA gene negative control (with RNase treatment before RT PCR). M, molecular markers. Primers and the predicted lengths of amplicons are shown in S3 Table . Red arrowheads indicate the position of a band where no signal was detected.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

3) Product Images from "Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus"

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus

Journal:

doi: 10.1128/JCM.00027-08

Sensitivities of C-PCR (A), first-round N-PCR with external primers Tox-100 and Tox-303 (B), and second-round N-PCR with internal primers Tox-130 and Tox-200 (C) to detect V. vulnificus in plasmid DNA. Lanes: 1, 100-bp ladder marker (Bioneer); 2, negative
Figure Legend Snippet: Sensitivities of C-PCR (A), first-round N-PCR with external primers Tox-100 and Tox-303 (B), and second-round N-PCR with internal primers Tox-130 and Tox-200 (C) to detect V. vulnificus in plasmid DNA. Lanes: 1, 100-bp ladder marker (Bioneer); 2, negative

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Marker

Standard curves (5 × 10 8 to 5 × 10 0 copies/μl) from the Q-PCR assay. Plasmid DNA was used as the template. Circled numerals: 1, negative control (sterile distilled water); 2 to 10, plasmid DNA serially diluted from 5 ×
Figure Legend Snippet: Standard curves (5 × 10 8 to 5 × 10 0 copies/μl) from the Q-PCR assay. Plasmid DNA was used as the template. Circled numerals: 1, negative control (sterile distilled water); 2 to 10, plasmid DNA serially diluted from 5 ×

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Negative Control

4) Product Images from "The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection"

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection

Journal: Archives of Virology

doi: 10.1007/s00705-017-3563-2

( A ) PCR amplification of the env gene from DF-1/K cells. (M) DNA marker; (DF-1/K): Genomic DNA extracted from DF-1/K cells; (DF-1): Genomic DNA extracted from DF-1 cells. ( B ) Verification of the stability of the ALV-K env gene in DF-1/K cells during passage. (M) DNAMarker; Lanes 1-5 and 7-10: ALV-K env gene cell-culture passage levels 5, 15, 25, 30, 35, 40, 45, 50, 60, respectively; Lane 6: DF-1 cells (negative control)
Figure Legend Snippet: ( A ) PCR amplification of the env gene from DF-1/K cells. (M) DNA marker; (DF-1/K): Genomic DNA extracted from DF-1/K cells; (DF-1): Genomic DNA extracted from DF-1 cells. ( B ) Verification of the stability of the ALV-K env gene in DF-1/K cells during passage. (M) DNAMarker; Lanes 1-5 and 7-10: ALV-K env gene cell-culture passage levels 5, 15, 25, 30, 35, 40, 45, 50, 60, respectively; Lane 6: DF-1 cells (negative control)

Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Cell Culture, Negative Control

A) . PCR amplification of the ALV-K env gene in DF-1/K cells. (M) DNA marker; (1): RNA extracted from DF-1/K cells; (2): Genomic DNA extracted from DF-1/K cells; (3) RNA extracted from DF-1 cells. B . Levels of ALV-K env gene transcription in DF-1/K cells were determined by real-time RT-PCR with gene specific primers. DF-1 cells served as a negative control. Data are representative of two independent experiments, both performed in triplicate
Figure Legend Snippet: A) . PCR amplification of the ALV-K env gene in DF-1/K cells. (M) DNA marker; (1): RNA extracted from DF-1/K cells; (2): Genomic DNA extracted from DF-1/K cells; (3) RNA extracted from DF-1 cells. B . Levels of ALV-K env gene transcription in DF-1/K cells were determined by real-time RT-PCR with gene specific primers. DF-1 cells served as a negative control. Data are representative of two independent experiments, both performed in triplicate

Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Quantitative RT-PCR, Negative Control

5) Product Images from "An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species"

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species

Journal: PLoS ONE

doi: 10.1371/journal.pone.0131204

Exon-intron structure of the Ccnb1ip1 gene in the JF1 and C57BL/6 genomes. RT-PCR products from exon 1 to exon 5 were detected in an E9.5 embryo of JF1. Exon 1 of Ccnb1ip1 was located in the vicinity of the Ccnb1ip1 insulator (red box) and enhancer (blue box) in the JF1 genome. The sequences corresponding to exon 2 and exon 4 of the Ccnb1ip1 gene were found to be present, but those corresponding to exon 1 and exon 3 to be absent (deleted) in the B6 genome. The horizon bars (1 to 7) indicate genomic regions for which DNA methylation statuses were assessed ( Fig 1 ).
Figure Legend Snippet: Exon-intron structure of the Ccnb1ip1 gene in the JF1 and C57BL/6 genomes. RT-PCR products from exon 1 to exon 5 were detected in an E9.5 embryo of JF1. Exon 1 of Ccnb1ip1 was located in the vicinity of the Ccnb1ip1 insulator (red box) and enhancer (blue box) in the JF1 genome. The sequences corresponding to exon 2 and exon 4 of the Ccnb1ip1 gene were found to be present, but those corresponding to exon 1 and exon 3 to be absent (deleted) in the B6 genome. The horizon bars (1 to 7) indicate genomic regions for which DNA methylation statuses were assessed ( Fig 1 ).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, DNA Methylation Assay

6) Product Images from "cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis"

Article Title: cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki652

The Oxct2b core promoter binds specifically to CREM factor through an Oxct2b-CL motif. ( A ) The testis nuclear extract binds to the promoter (−50/−10) of the Oxct2b gene. Lane 1, without nuclear extract; lane 2, liver nuclear extract; lanes 3–7, testis nuclear extract without competitor (lane 3), in the presence of a 100-fold molar excess of unlabeled oligonucleotides 1 (lane 7), 2 (lane 4), 3 (lane 5) and 4 (lane 6), which are shown to the left as competitors. The arrow shows testis-specific protein binding. ( B ) The Oxct2b-CL element in the Oxct2b promoter (−50/−10) binds a testis-specific nuclear protein. A 100-fold molar excess of the unlabeled wild-type (wt) or mutated (mut) promoter fragment was used as the competitor. ( C ) The CREM factor binds to the Oxct2b promoter (−40/−20) in vitro . EMSAs were performed with testis nuclear extracts in the presence of anti-CREM, anti-CREB or anti-AP-2α antibodies, using a labeled promoter fragment that spanned −40 to −20. ( D ) The CREM factor binds to the Oxct2b promoter in germ cell nuclei. Formaldehyde-cross-linked chromatin prepared from testicular germ cells was immunoprecipitated with anti-CREM (lane 2) or anti-CREB (lane 3) antibodies. Fragments shown were amplified using PCR performed with specific primers for the Oxct2b promoter. A sample of the total input DNA (lane 1) was included in the PCR analysis. ( E ) The 5′-half of the Oxct2b-CL motif is responsible for binding the testis-specific protein. The nucleotide sequences of the wild-type probe (−40/−20), mutant competitor (m1–m5) and testis-specific ACEτ oligonucleotides are shown in the upper panel. The Oxct2b-CL motif and the positions of the mutations are shown in boldface and lowercase, respectively. The binding reactions were performed in the absence of unlabeled nucleotide (lane 1), and in the presence of a 1-fold (lanes 2, 5, 8, 11, 14, 16 and 18), 10-fold (lanes 3, 6, 9, 12, 15, 17 and 19) or 100-fold (lanes 4, 7, 10 and 13) molar excess of competitor. The arrow indicates the testis-specific DNA–protein complex.
Figure Legend Snippet: The Oxct2b core promoter binds specifically to CREM factor through an Oxct2b-CL motif. ( A ) The testis nuclear extract binds to the promoter (−50/−10) of the Oxct2b gene. Lane 1, without nuclear extract; lane 2, liver nuclear extract; lanes 3–7, testis nuclear extract without competitor (lane 3), in the presence of a 100-fold molar excess of unlabeled oligonucleotides 1 (lane 7), 2 (lane 4), 3 (lane 5) and 4 (lane 6), which are shown to the left as competitors. The arrow shows testis-specific protein binding. ( B ) The Oxct2b-CL element in the Oxct2b promoter (−50/−10) binds a testis-specific nuclear protein. A 100-fold molar excess of the unlabeled wild-type (wt) or mutated (mut) promoter fragment was used as the competitor. ( C ) The CREM factor binds to the Oxct2b promoter (−40/−20) in vitro . EMSAs were performed with testis nuclear extracts in the presence of anti-CREM, anti-CREB or anti-AP-2α antibodies, using a labeled promoter fragment that spanned −40 to −20. ( D ) The CREM factor binds to the Oxct2b promoter in germ cell nuclei. Formaldehyde-cross-linked chromatin prepared from testicular germ cells was immunoprecipitated with anti-CREM (lane 2) or anti-CREB (lane 3) antibodies. Fragments shown were amplified using PCR performed with specific primers for the Oxct2b promoter. A sample of the total input DNA (lane 1) was included in the PCR analysis. ( E ) The 5′-half of the Oxct2b-CL motif is responsible for binding the testis-specific protein. The nucleotide sequences of the wild-type probe (−40/−20), mutant competitor (m1–m5) and testis-specific ACEτ oligonucleotides are shown in the upper panel. The Oxct2b-CL motif and the positions of the mutations are shown in boldface and lowercase, respectively. The binding reactions were performed in the absence of unlabeled nucleotide (lane 1), and in the presence of a 1-fold (lanes 2, 5, 8, 11, 14, 16 and 18), 10-fold (lanes 3, 6, 9, 12, 15, 17 and 19) or 100-fold (lanes 4, 7, 10 and 13) molar excess of competitor. The arrow indicates the testis-specific DNA–protein complex.

Techniques Used: Protein Binding, In Vitro, Labeling, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Binding Assay, Mutagenesis

7) Product Images from "BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles"

Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1038/mtna.2013.28

Overview of EV-BEAMing. EVs from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting DNA-coated beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.
Figure Legend Snippet: Overview of EV-BEAMing. EVs from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting DNA-coated beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Mutagenesis, FACS

Related Articles

Diagnostic Assay:

Article Title: Aberrant methylated EDNRB can act as a potential diagnostic biomarker in sporadic colorectal cancer while KISS1 is controversial
Article Snippet: The final volume of each reaction was 20 μl, including 2 μl of converted template DNA, 10 μl of master mix (SYBR Premix Ex Taq II, TAKARA, Japan), 1.2 ul of primer pair (5 pmol) and 6.8 μl of double distilled water. .. The condition of PCR amplification was followed in: holding step at 95 ˚C for 30 s and followed by 40 cycles of denaturation at 95 ˚C for 5 s, optimal annealing temperatures (TA ) of each primer pairs ( ) for 15 s and extension at 72 ˚C for 15 s. As soon as it was finished, HRM step with the continuous ramp rate of the acquisition of 0.3˚C per 15 sec was sequentially initiated in a rising temperature from 60˚C to 95˚C.

Clone Assay:

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection
Article Snippet: PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus
Article Snippet: Paragraph title: toxR cloning. ... PCR was conducted in 20-μl mixtures containing 1 μl of template DNA, 0.2 μl of 2.5 U of Takara Taq DNA polymerase (Takara Bio, Shiga, Japan), 1 μl each of 10 μM of forward primer and reverse primer, 2 μl of deoxynucleoside triphosphates (dNTPs), 2 μl of 10× PCR buffer, and 12.8 μl of water.

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China
Article Snippet: PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Article Title: cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis
Article Snippet: PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)]. .. PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)].

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: Paragraph title: Bacterial culture and cloning of the cspB Pi gene ... The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume.

Amplification:

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection
Article Snippet: The ALV-K env gene was amplified by PCR using the following gene-specific primers: P1: 5’-CGCGGATCC GCCACC ATGGAAGCCGTCATAAAG GCATTTCTGACTGGATAC-3’and P2:5’-AAGGAAAAAAGCGGCCGC TTACACTGCTCCATTTTCG-3’. .. PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus
Article Snippet: PCR was conducted in 20-μl mixtures containing 1 μl of template DNA, 0.2 μl of 2.5 U of Takara Taq DNA polymerase (Takara Bio, Shiga, Japan), 1 μl each of 10 μM of forward primer and reverse primer, 2 μl of deoxynucleoside triphosphates (dNTPs), 2 μl of 10× PCR buffer, and 12.8 μl of water. .. The PCR product was electrophoresed on a 1.2% agarose gel (SeaKem LE agarose) with ethidium bromide at 100 V (0.5× Tris-borate-EDTA buffer).

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China
Article Snippet: Paragraph title: 2.4. Genomic DNA Amplification and Sequencing ... PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Article Title: Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams
Article Snippet: Paragraph title: Amplification and sequencing of NER genes ... For the PCR, 25 μl of the reaction mixture contained 80 ng of template DNA, 2.5 μl of 10× LA Taq buffer (TaKaRa), 2.5 μl of dNTP mix (TaKaRa), 5 μl each of the 1 pmol/μl forward and reverse primer solutions, 0.13 μl of LA Taq polymerase solution (TaKaRa) and 8.87 μl of pure water was used.

Article Title: cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis
Article Snippet: For the transgenic analysis, the 600 bp genomic DNA fragment, that extends 566 bp upstream and 35 bp downstream of the transcriptional start site of the Oxct2b gene, was amplified by PCR from a genomic clone , using an upstream BglII site-containing primer and a downstream HindIII site-containing primer ( ). .. PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)].

Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles
Article Snippet: Amplification was done in a Veriti Thermal Cycler (Applied Biosystems) using the following conditions: 98 °C for 3 minutes; 10 cycles of 98 °C for 10 seconds, 68 °C for 15 seconds (−0.5 °C/cycle), 72 °C for 10 seconds; 30 cycles of 98 °C for 10 seconds, 62 °C for 15 seconds, 72 °C for 10 seconds; 1 cycle of 72 °C for 5 minutes. .. We set up 150 μl PCR reactions containing 10 μl 20 pmol/l template DNA, 2X TITANIUM Taq DNA Polymerase (Clontech, Mountain View, CA), 1X TITANIUM Taq buffer (Clontech), 1 mmol/l dNTP mix, 8.3 mmol/l MgCl2 (Invitrogen), 0.05 μmol/l universal forward primer (5′-tcccgcgaaattaatacgac-3′), 8 μmol/l nested reverse primer (5′-AATCAGTTGCTCTGTATTGATCC-3′), and 6 × 107 magnetic streptavidin beads (MyOne, Invitrogen) coated with a modified universal forward primer (5′-dual biotion-T-Spacer18-tcccgcgaaattaatacgac-3′).

Article Title: Aberrant methylated EDNRB can act as a potential diagnostic biomarker in sporadic colorectal cancer while KISS1 is controversial
Article Snippet: For this purpose, PCR amplification was performed on Step one plus Real-time PCR (ABI applied biosystems, USA) using sets of specific primers that are listed in . .. The final volume of each reaction was 20 μl, including 2 μl of converted template DNA, 10 μl of master mix (SYBR Premix Ex Taq II, TAKARA, Japan), 1.2 ul of primer pair (5 pmol) and 6.8 μl of double distilled water.

Article Title: Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations
Article Snippet: Paragraph title: PCR amplification ... PCR reactions were carried out in 25 μL containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2 , 250 μM each of dNTP, 0.2 μM of each primer, 100–200 ng of template DNA, and 0.25 U La Taq polymerase (TaKaRa).

Article Title: Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure
Article Snippet: Paragraph title: Amplification and sequencing of PvMSP-7E ... Secondary PCR contained PCR buffer, 200 μM dNTP, 0.2 μM of each primer, nuclease free water, 1 μL of template DNA from primary PCR and 1.25 units of ExTaq DNA polymerase (Takara, Seta, Japan) in a total volume of 30 μL.

Article Title: Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs
Article Snippet: At the end of the follow-up period, all broth was examined via polymerase chain reaction (PCR) for amplification of 16S rDNA gene. .. The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan).

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: The cspB Pi gene (GenBank WP_004570868) was obtained by PCR amplification. .. The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume.

Article Title: Genetic Diversity and Structure of Sinopodophyllum hexandrum (Royle) Ying in the Qinling Mountains, China
Article Snippet: An optimum reaction system was obtained by screening DNA, Mg2+ , dNTP, primer (UBC900 was used for preliminary test), and Taq DNA polymerase concentrations and annealing temperature, and reaction conditions. .. Each 20 µL amplification reaction consisted of 1×PCR buffer (10 mM Tris-HCl at pH 8.3, 50 mM L–1 KCl, 0.001% gelatin, and l.5 mmol L–1 MgCl2 ), 1.6 mmol L–1 dNTP mix, 0.6 µmol L–1 primer (UBC900 was used for preliminary test), 15 ng of template DNA, and 1.0 U Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China), using a cycling profile of initial 5 min at 94°C, followed by 45 cycles of 30 s at 94°C, 45 s annealing at 50°C, and 90 s extension at 72°C, ending with a final extension of 7 min at 72°C. .. The optimized PCR experiment conditions were applied for primer screening in a PTC100TM Programmable Thermal Controller (MJ Research, Waltham, MA, USA).

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: PCR amplification was performed in triplicate. .. Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M).

Article Title: Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure
Article Snippet: The complete coding region of PvMSP-7E (~1.1 kb) from each isolate was amplified by nested PCR using outer primers PvMSP-7F ( 5’-CATACCTTCGATACGTGTACTTC-3’ ) and PvMSP-7R ( 5’-CATTTCGCGTGTGCGTGTCTATG-3’ ) of the Salvador I strain (GenBank accession no. XM_001614084, chromosome 12: position 771164 to 772282) and the inner primers located 5’ and 3’ before and after the coding region of PvMSP-7E (PvMSP-7EF: 5’-AATCGCCACACATCGTCTGTG-3’ and PvMSP-7ER: 5’-ATTTCATCTTTACTGTTGGGCAC-3’ ) ( ). .. Primary PCR amplification was performed in a total volume of 15 μL containing PCR buffer, 200 μM dNTP, 0.2 μM of each primer, nuclease free water, 2 μL of template DNA and 1.25 units of TaKaRa LA Taq™ (Takara, Seta, Japan). .. The thermal cycling profiles for primary PCR contained a pre-amplification denaturation at 94°C, 60 s; followed by 35 cycles of denaturation at 96°C, 30 s; annealing at 50°C, 30 s; polymerization at 72°C, 7 minutes, and final elongation at 72°C, 10 minutes.

Mass Spectrometry:

Article Title: Aberrant methylated EDNRB can act as a potential diagnostic biomarker in sporadic colorectal cancer while KISS1 is controversial
Article Snippet: Paragraph title: Methylation-sensitive high resolution melting (MS-HRM) assay ... The final volume of each reaction was 20 μl, including 2 μl of converted template DNA, 10 μl of master mix (SYBR Premix Ex Taq II, TAKARA, Japan), 1.2 ul of primer pair (5 pmol) and 6.8 μl of double distilled water.

Positive Control:

Article Title: Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs
Article Snippet: The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan). .. The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan).

Synthesized:

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species
Article Snippet: Total RNAs from E9.5 embryos were isolated using AllPrep DNA/RNA Mini kit (QIAGEN). cDNA was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription kit (QIAGEN). .. Quantitative RT-PCR was carried out using 1/100 of the resultant cDNA as template DNA and the SYBR Premix Ex Taq (Takara) in a 25 μl reaction volume on the 7900HT Fast Real-Time PCR System (Applied Biosystems) under the following PCR cycle conditions: 95°C for 5 min, 40 cycles of [95°C for 30 s, 59°C for 30 s, 72°C for 30 s].

Article Title: Genetic Diversity and Structure of Sinopodophyllum hexandrum (Royle) Ying in the Qinling Mountains, China
Article Snippet: A total of 100 ISSR primers (synthesized by Shanghai Sheng Gong Biotechnology CO. LTD, China) were screened based on the primer set published by the Biotechnology Laboratory, University of British Columbia, Canada (UBC set No. 9) and the studies on Himalayan mayapple – . .. Each 20 µL amplification reaction consisted of 1×PCR buffer (10 mM Tris-HCl at pH 8.3, 50 mM L–1 KCl, 0.001% gelatin, and l.5 mmol L–1 MgCl2 ), 1.6 mmol L–1 dNTP mix, 0.6 µmol L–1 primer (UBC900 was used for preliminary test), 15 ng of template DNA, and 1.0 U Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China), using a cycling profile of initial 5 min at 94°C, followed by 45 cycles of 30 s at 94°C, 45 s annealing at 50°C, and 90 s extension at 72°C, ending with a final extension of 7 min at 72°C.

Quantitative RT-PCR:

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species
Article Snippet: Total RNAs from E9.5 embryos were isolated using AllPrep DNA/RNA Mini kit (QIAGEN). cDNA was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription kit (QIAGEN). .. Quantitative RT-PCR was carried out using 1/100 of the resultant cDNA as template DNA and the SYBR Premix Ex Taq (Takara) in a 25 μl reaction volume on the 7900HT Fast Real-Time PCR System (Applied Biosystems) under the following PCR cycle conditions: 95°C for 5 min, 40 cycles of [95°C for 30 s, 59°C for 30 s, 72°C for 30 s]. .. PCR primers used are listed in .

Article Title: Effect of CEACAM-1 knockdown in human colorectal cancer cells
Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... Genomic DNA was removed from the extracted total RNA via DNase I digestion, and then the total RNA was reverse transcribed to generate cDNA, according to the manufacturer's instructions (PrimeScript™ 1st strand cDNA Synthesis kit; Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed in triplicate in a 10 µl reaction mix consisting of 4 µl template DNA (0.05 µg/µl), 5 µl SYBR-Green (Takara Biotechnology Co., Ltd.), 0.2 µl each forward and reverse oligonucleotide (10 µM each) and 0.6 µl deionized water.

SYBR Green Assay:

Article Title: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells
Article Snippet: Copy numbers were determined in triplicate and are presented as ratios of individual copy numbers relative to the control. .. Double-stranded DNA fragments were prepared from the oligonucleotides by polymerization: a 10 μL reaction mix consisting of 4 μL template DNA (0.05 μg/μL), 5 μL SYBR Green (TAKARA, Dalian, China), 0.2 μL of each forward and reverse oligonucleotide (10 μM each), and 0.6 μL deionized water was subjected to heating at 95 °C for 3 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and elongation for 30 s at 72 °C, then 60 °C for 30 min for data acquisition. .. After approximately 10 passages, the expression levels of recombinant mRNA were analyzed.

Article Title: Effect of CEACAM-1 knockdown in human colorectal cancer cells
Article Snippet: Briefly, total RNA was extracted from CRC cells, using an RNeasy Mini kit (Invitrogen; Thermo Fisher Scientific, Inc.). .. Genomic DNA was removed from the extracted total RNA via DNase I digestion, and then the total RNA was reverse transcribed to generate cDNA, according to the manufacturer's instructions (PrimeScript™ 1st strand cDNA Synthesis kit; Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed in triplicate in a 10 µl reaction mix consisting of 4 µl template DNA (0.05 µg/µl), 5 µl SYBR-Green (Takara Biotechnology Co., Ltd.), 0.2 µl each forward and reverse oligonucleotide (10 µM each) and 0.6 µl deionized water. .. The primers used for qPCR were: CEACAM-1 forward, 5′-CAGGGGCTTCTGCTCACAGC-3′ and reverse, 5′-AGTTGCTTCTTCACAAGAT-3′; β-actin forward, 5′-GGCTGTGGAGACAAAAATGACCTC-3′ and reverse, 5′-AGGCTTGGGCTTGAATGGAGTC-3′.

Infection:

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China
Article Snippet: Total genomic DNA from infected DF-1 cells was used as a template for PCR amplification. .. PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Expressing:

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection
Article Snippet: PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. The ALV-K env PCR product was purified using the QIAEX II gel extraction kit (Qiagen, Hilden, Germany), sequenced (Invitrogen, Shanghai, China) and subcloned into the pMD-18T vector (TaKaRa, Dalian, China).

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species
Article Snippet: Quantitative RT-PCR was carried out using 1/100 of the resultant cDNA as template DNA and the SYBR Premix Ex Taq (Takara) in a 25 μl reaction volume on the 7900HT Fast Real-Time PCR System (Applied Biosystems) under the following PCR cycle conditions: 95°C for 5 min, 40 cycles of [95°C for 30 s, 59°C for 30 s, 72°C for 30 s]. .. PCR primers used are listed in .

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume. .. The PCR was performed in a DNAEngine thermal cycler (Bio-Rad Laboratories Inc., USA) using a cycling condition that consisted of an initial denaturation at 95 °C for 5 min and then 30 cycles with denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min. A final extension was performed at 72 °C for 5 min.

Article Title: Effect of CEACAM-1 knockdown in human colorectal cancer cells
Article Snippet: CEACAM-1 expression was analyzed via RT-qPCR, as previously described ( ). .. Genomic DNA was removed from the extracted total RNA via DNase I digestion, and then the total RNA was reverse transcribed to generate cDNA, according to the manufacturer's instructions (PrimeScript™ 1st strand cDNA Synthesis kit; Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed in triplicate in a 10 µl reaction mix consisting of 4 µl template DNA (0.05 µg/µl), 5 µl SYBR-Green (Takara Biotechnology Co., Ltd.), 0.2 µl each forward and reverse oligonucleotide (10 µM each) and 0.6 µl deionized water.

Modification:

Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles
Article Snippet: We performed the BEAMing PCR using a protocol slightly modified from that previously described. .. We set up 150 μl PCR reactions containing 10 μl 20 pmol/l template DNA, 2X TITANIUM Taq DNA Polymerase (Clontech, Mountain View, CA), 1X TITANIUM Taq buffer (Clontech), 1 mmol/l dNTP mix, 8.3 mmol/l MgCl2 (Invitrogen), 0.05 μmol/l universal forward primer (5′-tcccgcgaaattaatacgac-3′), 8 μmol/l nested reverse primer (5′-AATCAGTTGCTCTGTATTGATCC-3′), and 6 × 107 magnetic streptavidin beads (MyOne, Invitrogen) coated with a modified universal forward primer (5′-dual biotion-T-Spacer18-tcccgcgaaattaatacgac-3′). .. The aqueous reaction mixture was then combined with 600 μl of an oil/emulsifier mixture of 7% w/v ABIL WE09 (Degussa, Essen, GE), 20% v/v M3516 mineral oil (Sigma-Aldrich, St Louis, MO) and 73% v/v Tegosoft DEC (Degussa), and one 5 mm steel bead (Qiagen) in one well of a 96-deep-well plate (Abgene, Lafayette, CO).

Transformation Assay:

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China
Article Snippet: PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR products were analyzed on 1% agarose-Tris-acetate-EDTA (TAE) gels, stained with ethidium bromide and photographed using a gel documentation system (UVITEC, Cambridge, UK).

Transfection:

Article Title: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells
Article Snippet: Genomic DNA was isolated from 4 × 106 transfected CHO-S cells using a genomic DNA Mini Preparation kit with spin column (Beyotime). .. Double-stranded DNA fragments were prepared from the oligonucleotides by polymerization: a 10 μL reaction mix consisting of 4 μL template DNA (0.05 μg/μL), 5 μL SYBR Green (TAKARA, Dalian, China), 0.2 μL of each forward and reverse oligonucleotide (10 μM each), and 0.6 μL deionized water was subjected to heating at 95 °C for 3 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and elongation for 30 s at 72 °C, then 60 °C for 30 min for data acquisition.

Sequencing:

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection
Article Snippet: The primers contain protective bases, restriction enzyme cutting sites (italicized letters), a Kozak sequence (underlined letters) and a leader sequence (italicized letters) to improve translational efficiency. .. PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China
Article Snippet: Paragraph title: 2.4. Genomic DNA Amplification and Sequencing ... PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Article Title: Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams
Article Snippet: Paragraph title: Amplification and sequencing of NER genes ... For the PCR, 25 μl of the reaction mixture contained 80 ng of template DNA, 2.5 μl of 10× LA Taq buffer (TaKaRa), 2.5 μl of dNTP mix (TaKaRa), 5 μl each of the 1 pmol/μl forward and reverse primer solutions, 0.13 μl of LA Taq polymerase solution (TaKaRa) and 8.87 μl of pure water was used.

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species
Article Snippet: Quantitative RT-PCR was carried out using 1/100 of the resultant cDNA as template DNA and the SYBR Premix Ex Taq (Takara) in a 25 μl reaction volume on the 7900HT Fast Real-Time PCR System (Applied Biosystems) under the following PCR cycle conditions: 95°C for 5 min, 40 cycles of [95°C for 30 s, 59°C for 30 s, 72°C for 30 s]. .. The expression levels of Ccnb1ip1 and Parp2 were normalized by that of Gapdh .

Article Title: Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations
Article Snippet: A pair of oligonucleotide primers: TgRON10F (forward primer, 5′-ATgCCTgAGGTTAACTgC-3′) and TgRON10R (reverse primer, 5′-TTAAGAAGAGTCTTCTgTCGC-3′) were designed based on the TgRON10 gene sequence of T. gondii ME49 strain available in ToxoDB database (TgME49_261750). .. PCR reactions were carried out in 25 μL containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2 , 250 μM each of dNTP, 0.2 μM of each primer, 100–200 ng of template DNA, and 0.25 U La Taq polymerase (TaKaRa).

Article Title: Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure
Article Snippet: Paragraph title: Amplification and sequencing of PvMSP-7E ... Secondary PCR contained PCR buffer, 200 μM dNTP, 0.2 μM of each primer, nuclease free water, 1 μL of template DNA from primary PCR and 1.25 units of ExTaq DNA polymerase (Takara, Seta, Japan) in a total volume of 30 μL.

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: The forward primer sequence was CspBPi F1H, 5′-AGTAAGCTTATGGCAAAATCGCAGCAGACCT-3′, and the reverse primer was CspB Pi B150*B, 5′-CCGGATCCTTATATTTTGGTAACTTTAACTGCATTCATT-3′ (manufactured by Bioneer Co., Daejion, Korea). .. The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume.

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: Paragraph title: 2.3. 16S rRNA Gene Sequencing ... Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M).

Cell Culture:

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus
Article Snippet: Briefly, V. vulnificus ATCC 27562 was cultured in tryptic soy broth (Bacto) containing 2% NaCl, and genomic DNA for PCR was extracted using a QIAamp DNA mini kit (Qiagen, Hilden, Germany). .. PCR was conducted in 20-μl mixtures containing 1 μl of template DNA, 0.2 μl of 2.5 U of Takara Taq DNA polymerase (Takara Bio, Shiga, Japan), 1 μl each of 10 μM of forward primer and reverse primer, 2 μl of deoxynucleoside triphosphates (dNTPs), 2 μl of 10× PCR buffer, and 12.8 μl of water.

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: P. irgensii KOPRI 22228 was isolated from Arctic Sea sediments near Dasan Korean Arctic Station (Ny-Alesund, Norway), and cultured as previously reported. .. The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume.

Generated:

Article Title: cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis
Article Snippet: PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)]. .. PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)].

Article Title: Genetic Diversity and Structure of Sinopodophyllum hexandrum (Royle) Ying in the Qinling Mountains, China
Article Snippet: Each 20 µL amplification reaction consisted of 1×PCR buffer (10 mM Tris-HCl at pH 8.3, 50 mM L–1 KCl, 0.001% gelatin, and l.5 mmol L–1 MgCl2 ), 1.6 mmol L–1 dNTP mix, 0.6 µmol L–1 primer (UBC900 was used for preliminary test), 15 ng of template DNA, and 1.0 U Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China), using a cycling profile of initial 5 min at 94°C, followed by 45 cycles of 30 s at 94°C, 45 s annealing at 50°C, and 90 s extension at 72°C, ending with a final extension of 7 min at 72°C. .. Each 20 µL amplification reaction consisted of 1×PCR buffer (10 mM Tris-HCl at pH 8.3, 50 mM L–1 KCl, 0.001% gelatin, and l.5 mmol L–1 MgCl2 ), 1.6 mmol L–1 dNTP mix, 0.6 µmol L–1 primer (UBC900 was used for preliminary test), 15 ng of template DNA, and 1.0 U Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China), using a cycling profile of initial 5 min at 94°C, followed by 45 cycles of 30 s at 94°C, 45 s annealing at 50°C, and 90 s extension at 72°C, ending with a final extension of 7 min at 72°C.

Imaging:

Article Title: Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations
Article Snippet: PCR reactions were carried out in 25 μL containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2 , 250 μM each of dNTP, 0.2 μM of each primer, 100–200 ng of template DNA, and 0.25 U La Taq polymerase (TaKaRa). .. Each amplicon (6 μl) was examined on 1% (w/v) agarose gel to assess amplification efficiency.

Polymerase Chain Reaction:

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection
Article Snippet: The primers contain protective bases, restriction enzyme cutting sites (italicized letters), a Kozak sequence (underlined letters) and a leader sequence (italicized letters) to improve translational efficiency. .. PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR thermocycling profiles included an initial denaturation for 3 min at 94 °C, followed by 30 cycles of amplification (94 °C for 30 s, 55 °C for 1min and 72 °C for 2 min), as well as a final extension of 72 °C for 8 min.

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus
Article Snippet: The primers designed to target the toxR gene of V. vulnificus (GenBank accession no. ) are given in Table . .. PCR was conducted in 20-μl mixtures containing 1 μl of template DNA, 0.2 μl of 2.5 U of Takara Taq DNA polymerase (Takara Bio, Shiga, Japan), 1 μl each of 10 μM of forward primer and reverse primer, 2 μl of deoxynucleoside triphosphates (dNTPs), 2 μl of 10× PCR buffer, and 12.8 μl of water. .. PCR was performed with predenaturation at 94°C for 5 min followed by 39 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 72°C for 1 min by use of an Applied Biosystems Veriti 96-well thermal cycler (Applied Biosystems, Foster City, CA).

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China
Article Snippet: Total genomic DNA from infected DF-1 cells was used as a template for PCR amplification. .. PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR products were analyzed on 1% agarose-Tris-acetate-EDTA (TAE) gels, stained with ethidium bromide and photographed using a gel documentation system (UVITEC, Cambridge, UK).

Article Title: Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams
Article Snippet: Specificities of the designed primers were validated with PCR using DNA extracted from the gill tissues of P . okutanii and C . pacifica . .. For the PCR, 25 μl of the reaction mixture contained 80 ng of template DNA, 2.5 μl of 10× LA Taq buffer (TaKaRa), 2.5 μl of dNTP mix (TaKaRa), 5 μl each of the 1 pmol/μl forward and reverse primer solutions, 0.13 μl of LA Taq polymerase solution (TaKaRa) and 8.87 μl of pure water was used. .. The gene fragments were amplified with an ABI 9700 Thermal cycler using a protocol; 96°C for 2 min and 30 cycles of 96°C for 20 s, 56°C for 15 s and 72°C for 2 or 5 or 7 min, followed by extension at 72°C for 10 min. After amplification, 2 μl of the reaction product mixture was applied to 1% agarose electrophoresis gel and stained with an ethidium bromide solution to visualize the reaction products.

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species
Article Snippet: Total RNAs from E9.5 embryos were isolated using AllPrep DNA/RNA Mini kit (QIAGEN). cDNA was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription kit (QIAGEN). .. Quantitative RT-PCR was carried out using 1/100 of the resultant cDNA as template DNA and the SYBR Premix Ex Taq (Takara) in a 25 μl reaction volume on the 7900HT Fast Real-Time PCR System (Applied Biosystems) under the following PCR cycle conditions: 95°C for 5 min, 40 cycles of [95°C for 30 s, 59°C for 30 s, 72°C for 30 s]. .. PCR primers used are listed in .

Article Title: cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis
Article Snippet: For the transgenic analysis, the 600 bp genomic DNA fragment, that extends 566 bp upstream and 35 bp downstream of the transcriptional start site of the Oxct2b gene, was amplified by PCR from a genomic clone , using an upstream BglII site-containing primer and a downstream HindIII site-containing primer ( ). .. PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)]. .. The PCR conditions consisted of an initial denaturation step at 95°C for 3 min, followed by 30 cycles at 94°C for 1 min, at 60°C for 1 min and at 72°C for 1.5 min. After digestion with BglII and HindIII, the amplified fragment was subcloned into the BglII and HindIII sites of the pEGFP-1 vector (Clontech), to generate the −566/+35EGFP construct.

Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles
Article Snippet: We performed the BEAMing PCR using a protocol slightly modified from that previously described. .. We set up 150 μl PCR reactions containing 10 μl 20 pmol/l template DNA, 2X TITANIUM Taq DNA Polymerase (Clontech, Mountain View, CA), 1X TITANIUM Taq buffer (Clontech), 1 mmol/l dNTP mix, 8.3 mmol/l MgCl2 (Invitrogen), 0.05 μmol/l universal forward primer (5′-tcccgcgaaattaatacgac-3′), 8 μmol/l nested reverse primer (5′-AATCAGTTGCTCTGTATTGATCC-3′), and 6 × 107 magnetic streptavidin beads (MyOne, Invitrogen) coated with a modified universal forward primer (5′-dual biotion-T-Spacer18-tcccgcgaaattaatacgac-3′). .. The aqueous reaction mixture was then combined with 600 μl of an oil/emulsifier mixture of 7% w/v ABIL WE09 (Degussa, Essen, GE), 20% v/v M3516 mineral oil (Sigma-Aldrich, St Louis, MO) and 73% v/v Tegosoft DEC (Degussa), and one 5 mm steel bead (Qiagen) in one well of a 96-deep-well plate (Abgene, Lafayette, CO).

Article Title: Association of IL10 gene promoter polymorphisms with risks of gastric cancer and atrophic gastritis
Article Snippet: Genomic DNA was purified using a method described previously with some modifications., The primers were purchased from TaKaRa Biotechnology (Dalian, China). .. The total PCR reaction volume was 25 µl containing 2.5 µl of 10 × PCR buffer, 2.0 µl of 2.5 mM dNTP mixture, 1.0 µl of each upstream and downstream primer at 10 pmol/µl, 2.5 U或0.5 µL, 1.0 µl of the template DNA (TaKaRa Biotechnology), and an appropriate amount of ddH2 O. .. The PCR reaction was performed by preliminary denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 45 s; and 72°C for 40 s and then 72°C for 60 s using a Biometra TGradient 96 thermal cycler (Analytik Jena, Jena, Germany).

Article Title: Aberrant methylated EDNRB can act as a potential diagnostic biomarker in sporadic colorectal cancer while KISS1 is controversial
Article Snippet: For this purpose, PCR amplification was performed on Step one plus Real-time PCR (ABI applied biosystems, USA) using sets of specific primers that are listed in . .. The final volume of each reaction was 20 μl, including 2 μl of converted template DNA, 10 μl of master mix (SYBR Premix Ex Taq II, TAKARA, Japan), 1.2 ul of primer pair (5 pmol) and 6.8 μl of double distilled water.

Article Title: Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations
Article Snippet: A pair of oligonucleotide primers: TgRON10F (forward primer, 5′-ATgCCTgAGGTTAACTgC-3′) and TgRON10R (reverse primer, 5′-TTAAGAAGAGTCTTCTgTCGC-3′) were designed based on the TgRON10 gene sequence of T. gondii ME49 strain available in ToxoDB database (TgME49_261750). .. PCR reactions were carried out in 25 μL containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2 , 250 μM each of dNTP, 0.2 μM of each primer, 100–200 ng of template DNA, and 0.25 U La Taq polymerase (TaKaRa). .. The PCR reaction was carried out in a thermocycler (Bio-Rad) with an initial denaturation at 94 °C for 4 min, followed by 37 cycles of 94 °C for 30 sec (denaturation), 67.5 °C for 30 sec (annealing), 72 °C for 5 min (extension), and a final extension of 72 °C for 10 min. A negative control sample without gDNA was included in each PCR reaction.

Article Title: Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure
Article Snippet: The thermal cycling profiles for primary PCR contained a pre-amplification denaturation at 94°C, 60 s; followed by 35 cycles of denaturation at 96°C, 30 s; annealing at 50°C, 30 s; polymerization at 72°C, 7 minutes, and final elongation at 72°C, 10 minutes. .. Secondary PCR contained PCR buffer, 200 μM dNTP, 0.2 μM of each primer, nuclease free water, 1 μL of template DNA from primary PCR and 1.25 units of ExTaq DNA polymerase (Takara, Seta, Japan) in a total volume of 30 μL. .. The amplification profile for secondary PCR consisted of 94°C, 60 s; 30 cycles of 96°C, 30 s; 50°C, 30 s and 72°C, 2 minutes; and 72°C, 5 minutes.

Article Title: Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs
Article Snippet: A positive control (with DNA from standard strain of P. acnes provided by Guangzhou Type Culture Collection, ATCC #6919) and a negative control (sterile water as template) were included. .. The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan). .. The PCR conditions were as follows: 94°C preheating for 4 min, 94°C denaturation for 45 s, 35 cycles of 94°C denaturation for 45 s, 57°C annealing for 45 s, 72°C extension for 1 min, and a final extension for 7 min at 72°C.

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: The forward primer sequence was CspBPi F1H, 5′-AGTAAGCTTATGGCAAAATCGCAGCAGACCT-3′, and the reverse primer was CspB Pi B150*B, 5′-CCGGATCCTTATATTTTGGTAACTTTAACTGCATTCATT-3′ (manufactured by Bioneer Co., Daejion, Korea). .. The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume. .. The PCR was performed in a DNAEngine thermal cycler (Bio-Rad Laboratories Inc., USA) using a cycling condition that consisted of an initial denaturation at 95 °C for 5 min and then 30 cycles with denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min. A final extension was performed at 72 °C for 5 min.

Article Title: Effect of CEACAM-1 knockdown in human colorectal cancer cells
Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... Genomic DNA was removed from the extracted total RNA via DNase I digestion, and then the total RNA was reverse transcribed to generate cDNA, according to the manufacturer's instructions (PrimeScript™ 1st strand cDNA Synthesis kit; Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed in triplicate in a 10 µl reaction mix consisting of 4 µl template DNA (0.05 µg/µl), 5 µl SYBR-Green (Takara Biotechnology Co., Ltd.), 0.2 µl each forward and reverse oligonucleotide (10 µM each) and 0.6 µl deionized water.

Recombinant:

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China
Article Snippet: PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Cellular Antioxidant Activity Assay:

Article Title: Association of IL10 gene promoter polymorphisms with risks of gastric cancer and atrophic gastritis
Article Snippet: – The primers were as follows: IL10 gene –1082A/G (rs1800896) polymorphism: forward: 5ʹ-TCT TAC CTA TCC CTA CTT CC- 3ʹ, reverse: 5ʹ-CTC GCT GCA ACC CAA CTG GC-3ʹ; IL10 gene –819C/T (rs1800871) polymorphism: forward: 5ʹ-CAC TAC TAA GGC TTC CTT GGG A-3ʹ, reverse: 5ʹ-GTG AGC AAA CTG AGG CAC GAC AT-3ʹ; IL10 gene –592A/C (rs1800872) polymorphism: forward: 5ʹ-GGT GAG CAC TAC CTG ACT AGC-3ʹ, reverse: 5ʹ-CCT AGG TCA CAG TGA CGT GGG-3ʹ. .. The total PCR reaction volume was 25 µl containing 2.5 µl of 10 × PCR buffer, 2.0 µl of 2.5 mM dNTP mixture, 1.0 µl of each upstream and downstream primer at 10 pmol/µl, 2.5 U或0.5 µL, 1.0 µl of the template DNA (TaKaRa Biotechnology), and an appropriate amount of ddH2 O.

DNA Extraction:

Article Title: Association of IL10 gene promoter polymorphisms with risks of gastric cancer and atrophic gastritis
Article Snippet: The total PCR reaction volume was 25 µl containing 2.5 µl of 10 × PCR buffer, 2.0 µl of 2.5 mM dNTP mixture, 1.0 µl of each upstream and downstream primer at 10 pmol/µl, 2.5 U或0.5 µL, 1.0 µl of the template DNA (TaKaRa Biotechnology), and an appropriate amount of ddH2 O. .. The total PCR reaction volume was 25 µl containing 2.5 µl of 10 × PCR buffer, 2.0 µl of 2.5 mM dNTP mixture, 1.0 µl of each upstream and downstream primer at 10 pmol/µl, 2.5 U或0.5 µL, 1.0 µl of the template DNA (TaKaRa Biotechnology), and an appropriate amount of ddH2 O.

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: The template DNA was extracted from P. irgensii KOPRI 22228 cells using G-spin™ bacteria genomic DNA extraction kit (Intron Co., Korea), according to the protocol suggested by the manufacturer. .. The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume.

Fluorescence:

Article Title: Aberrant methylated EDNRB can act as a potential diagnostic biomarker in sporadic colorectal cancer while KISS1 is controversial
Article Snippet: The final volume of each reaction was 20 μl, including 2 μl of converted template DNA, 10 μl of master mix (SYBR Premix Ex Taq II, TAKARA, Japan), 1.2 ul of primer pair (5 pmol) and 6.8 μl of double distilled water. .. The melting curves of all PCR products were run on HRM software v.2.2 (ABI applied biosystems, USA).

Methylation:

Article Title: Aberrant methylated EDNRB can act as a potential diagnostic biomarker in sporadic colorectal cancer while KISS1 is controversial
Article Snippet: Paragraph title: Methylation-sensitive high resolution melting (MS-HRM) assay ... The final volume of each reaction was 20 μl, including 2 μl of converted template DNA, 10 μl of master mix (SYBR Premix Ex Taq II, TAKARA, Japan), 1.2 ul of primer pair (5 pmol) and 6.8 μl of double distilled water.

Isolation:

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species
Article Snippet: Total RNAs from E9.5 embryos were isolated using AllPrep DNA/RNA Mini kit (QIAGEN). cDNA was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription kit (QIAGEN). .. Quantitative RT-PCR was carried out using 1/100 of the resultant cDNA as template DNA and the SYBR Premix Ex Taq (Takara) in a 25 μl reaction volume on the 7900HT Fast Real-Time PCR System (Applied Biosystems) under the following PCR cycle conditions: 95°C for 5 min, 40 cycles of [95°C for 30 s, 59°C for 30 s, 72°C for 30 s].

Article Title: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells
Article Snippet: Genomic DNA was isolated from 4 × 106 transfected CHO-S cells using a genomic DNA Mini Preparation kit with spin column (Beyotime). .. Double-stranded DNA fragments were prepared from the oligonucleotides by polymerization: a 10 μL reaction mix consisting of 4 μL template DNA (0.05 μg/μL), 5 μL SYBR Green (TAKARA, Dalian, China), 0.2 μL of each forward and reverse oligonucleotide (10 μM each), and 0.6 μL deionized water was subjected to heating at 95 °C for 3 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and elongation for 30 s at 72 °C, then 60 °C for 30 min for data acquisition.

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: P. irgensii KOPRI 22228 was isolated from Arctic Sea sediments near Dasan Korean Arctic Station (Ny-Alesund, Norway), and cultured as previously reported. .. The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume.

Multiplexing:

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: Each sample was tagged by an index sequence at the 5′ end of the forward primer for multiplexing to allow simultaneous analyses of several samples in a single sequencing run. .. Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M).

Purification:

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection
Article Snippet: PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR thermocycling profiles included an initial denaturation for 3 min at 94 °C, followed by 30 cycles of amplification (94 °C for 30 s, 55 °C for 1min and 72 °C for 2 min), as well as a final extension of 72 °C for 8 min.

Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles
Article Snippet: The PCR products for each sample were then pooled and purified using a QIAquick PCR purification column (Qiagen) and the concentration determined using a 2100 Bioanalyzer with a DNA 1000 LabChip kit (Agilent). .. We set up 150 μl PCR reactions containing 10 μl 20 pmol/l template DNA, 2X TITANIUM Taq DNA Polymerase (Clontech, Mountain View, CA), 1X TITANIUM Taq buffer (Clontech), 1 mmol/l dNTP mix, 8.3 mmol/l MgCl2 (Invitrogen), 0.05 μmol/l universal forward primer (5′-tcccgcgaaattaatacgac-3′), 8 μmol/l nested reverse primer (5′-AATCAGTTGCTCTGTATTGATCC-3′), and 6 × 107 magnetic streptavidin beads (MyOne, Invitrogen) coated with a modified universal forward primer (5′-dual biotion-T-Spacer18-tcccgcgaaattaatacgac-3′).

Article Title: Association of IL10 gene promoter polymorphisms with risks of gastric cancer and atrophic gastritis
Article Snippet: Genomic DNA was purified using a method described previously with some modifications., The primers were purchased from TaKaRa Biotechnology (Dalian, China). .. The total PCR reaction volume was 25 µl containing 2.5 µl of 10 × PCR buffer, 2.0 µl of 2.5 mM dNTP mixture, 1.0 µl of each upstream and downstream primer at 10 pmol/µl, 2.5 U或0.5 µL, 1.0 µl of the template DNA (TaKaRa Biotechnology), and an appropriate amount of ddH2 O.

Article Title: Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure
Article Snippet: Secondary PCR contained PCR buffer, 200 μM dNTP, 0.2 μM of each primer, nuclease free water, 1 μL of template DNA from primary PCR and 1.25 units of ExTaq DNA polymerase (Takara, Seta, Japan) in a total volume of 30 μL. .. The PCR products were analyzed on 1% agarose gel electrophoresis, stained with ethidium bromide and visualized under UV transillumination.

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M). .. Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams
Article Snippet: The specific RT-PCR primer sets for NER genes were designed from aligned sequences of Vok and the C . pacifica symbiont (Cpac_S). .. For the PCR, 25 μl of the reaction mixture contained 80 ng of template DNA, 2.5 μl of 10× LA Taq buffer (TaKaRa), 2.5 μl of dNTP mix (TaKaRa), 5 μl each of the 1 pmol/μl forward and reverse primer solutions, 0.13 μl of LA Taq polymerase solution (TaKaRa) and 8.87 μl of pure water was used.

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species
Article Snippet: Paragraph title: Reverse transcription (RT)-PCR ... Quantitative RT-PCR was carried out using 1/100 of the resultant cDNA as template DNA and the SYBR Premix Ex Taq (Takara) in a 25 μl reaction volume on the 7900HT Fast Real-Time PCR System (Applied Biosystems) under the following PCR cycle conditions: 95°C for 5 min, 40 cycles of [95°C for 30 s, 59°C for 30 s, 72°C for 30 s].

Gas Chromatography:

Article Title: Association of IL10 gene promoter polymorphisms with risks of gastric cancer and atrophic gastritis
Article Snippet: – The primers were as follows: IL10 gene –1082A/G (rs1800896) polymorphism: forward: 5ʹ-TCT TAC CTA TCC CTA CTT CC- 3ʹ, reverse: 5ʹ-CTC GCT GCA ACC CAA CTG GC-3ʹ; IL10 gene –819C/T (rs1800871) polymorphism: forward: 5ʹ-CAC TAC TAA GGC TTC CTT GGG A-3ʹ, reverse: 5ʹ-GTG AGC AAA CTG AGG CAC GAC AT-3ʹ; IL10 gene –592A/C (rs1800872) polymorphism: forward: 5ʹ-GGT GAG CAC TAC CTG ACT AGC-3ʹ, reverse: 5ʹ-CCT AGG TCA CAG TGA CGT GGG-3ʹ. .. The total PCR reaction volume was 25 µl containing 2.5 µl of 10 × PCR buffer, 2.0 µl of 2.5 mM dNTP mixture, 1.0 µl of each upstream and downstream primer at 10 pmol/µl, 2.5 U或0.5 µL, 1.0 µl of the template DNA (TaKaRa Biotechnology), and an appropriate amount of ddH2 O.

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: PCR amplification was performed in triplicate. .. Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M). .. The PCR were performed as follows: initial denaturation at 98°C for 3 min; 27 cycles at 98°C for 30 s, 50°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. Three microliters of each PCR product was examined by agarose gel (2.0%) electrophoresis.

Construct:

Article Title: cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis
Article Snippet: Paragraph title: Reporter constructs ... PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)].

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M). .. Pooled triplicate reactions were purified using the AxyPrep DNA Gel Extraction Kit (Axygen, USA) and qualified through a QuantiFluor™-ST fluorometer (Promega) and an Agilent 2100 Bioanalyzer (Agilent, USA) according to the manufacturer's recommendations.

Gel Extraction:

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection
Article Snippet: PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR thermocycling profiles included an initial denaturation for 3 min at 94 °C, followed by 30 cycles of amplification (94 °C for 30 s, 55 °C for 1min and 72 °C for 2 min), as well as a final extension of 72 °C for 8 min.

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus
Article Snippet: PCR was conducted in 20-μl mixtures containing 1 μl of template DNA, 0.2 μl of 2.5 U of Takara Taq DNA polymerase (Takara Bio, Shiga, Japan), 1 μl each of 10 μM of forward primer and reverse primer, 2 μl of deoxynucleoside triphosphates (dNTPs), 2 μl of 10× PCR buffer, and 12.8 μl of water. .. The PCR product was electrophoresed on a 1.2% agarose gel (SeaKem LE agarose) with ethidium bromide at 100 V (0.5× Tris-borate-EDTA buffer).

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M). .. Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M).

Nested PCR:

Article Title: Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure
Article Snippet: The complete coding region of PvMSP-7E (~1.1 kb) from each isolate was amplified by nested PCR using outer primers PvMSP-7F ( 5’-CATACCTTCGATACGTGTACTTC-3’ ) and PvMSP-7R ( 5’-CATTTCGCGTGTGCGTGTCTATG-3’ ) of the Salvador I strain (GenBank accession no. XM_001614084, chromosome 12: position 771164 to 772282) and the inner primers located 5’ and 3’ before and after the coding region of PvMSP-7E (PvMSP-7EF: 5’-AATCGCCACACATCGTCTGTG-3’ and PvMSP-7ER: 5’-ATTTCATCTTTACTGTTGGGCAC-3’ ) ( ). .. Secondary PCR contained PCR buffer, 200 μM dNTP, 0.2 μM of each primer, nuclease free water, 1 μL of template DNA from primary PCR and 1.25 units of ExTaq DNA polymerase (Takara, Seta, Japan) in a total volume of 30 μL.

Activated Clotting Time Assay:

Article Title: Association of IL10 gene promoter polymorphisms with risks of gastric cancer and atrophic gastritis
Article Snippet: – The primers were as follows: IL10 gene –1082A/G (rs1800896) polymorphism: forward: 5ʹ-TCT TAC CTA TCC CTA CTT CC- 3ʹ, reverse: 5ʹ-CTC GCT GCA ACC CAA CTG GC-3ʹ; IL10 gene –819C/T (rs1800871) polymorphism: forward: 5ʹ-CAC TAC TAA GGC TTC CTT GGG A-3ʹ, reverse: 5ʹ-GTG AGC AAA CTG AGG CAC GAC AT-3ʹ; IL10 gene –592A/C (rs1800872) polymorphism: forward: 5ʹ-GGT GAG CAC TAC CTG ACT AGC-3ʹ, reverse: 5ʹ-CCT AGG TCA CAG TGA CGT GGG-3ʹ. .. The total PCR reaction volume was 25 µl containing 2.5 µl of 10 × PCR buffer, 2.0 µl of 2.5 mM dNTP mixture, 1.0 µl of each upstream and downstream primer at 10 pmol/µl, 2.5 U或0.5 µL, 1.0 µl of the template DNA (TaKaRa Biotechnology), and an appropriate amount of ddH2 O.

Plasmid Preparation:

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection
Article Snippet: Paragraph title: Plasmid construction ... PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China
Article Snippet: PCR was performed in a 50 µL reaction mixture that consisted of template DNA (5 µL), 10× PCR buffer (TaKaRa, Dalian, China), 1 µM of forward and reverse primers, 2 mM of MgCl2 , 100 mM of each dNTP, and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China). .. PCR products were analyzed on 1% agarose-Tris-acetate-EDTA (TAE) gels, stained with ethidium bromide and photographed using a gel documentation system (UVITEC, Cambridge, UK).

Article Title: cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis
Article Snippet: PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)]. .. The PCR conditions consisted of an initial denaturation step at 95°C for 3 min, followed by 30 cycles at 94°C for 1 min, at 60°C for 1 min and at 72°C for 1.5 min. After digestion with BglII and HindIII, the amplified fragment was subcloned into the BglII and HindIII sites of the pEGFP-1 vector (Clontech), to generate the −566/+35EGFP construct.

Article Title: CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance
Article Snippet: The PCR mixture consisted of 5 μl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris–HCl, pH 9.0), 2.5 mM MgCl2 , 0.2 mM of each dNTP, 200 nM of each primer, 1 μl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 μl volume. .. The PCR was performed in a DNAEngine thermal cycler (Bio-Rad Laboratories Inc., USA) using a cycling condition that consisted of an initial denaturation at 95 °C for 5 min and then 30 cycles with denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min. A final extension was performed at 72 °C for 5 min.

Software:

Article Title: Aberrant methylated EDNRB can act as a potential diagnostic biomarker in sporadic colorectal cancer while KISS1 is controversial
Article Snippet: The converted DNA was applied to amplify CpG rich regions which were predicted using MethPrimer online software, these regions were located in the upstream of EDNRB gene and downstream of KISS1 gene. .. The final volume of each reaction was 20 μl, including 2 μl of converted template DNA, 10 μl of master mix (SYBR Premix Ex Taq II, TAKARA, Japan), 1.2 ul of primer pair (5 pmol) and 6.8 μl of double distilled water.

Real-time Polymerase Chain Reaction:

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species
Article Snippet: Total RNAs from E9.5 embryos were isolated using AllPrep DNA/RNA Mini kit (QIAGEN). cDNA was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription kit (QIAGEN). .. Quantitative RT-PCR was carried out using 1/100 of the resultant cDNA as template DNA and the SYBR Premix Ex Taq (Takara) in a 25 μl reaction volume on the 7900HT Fast Real-Time PCR System (Applied Biosystems) under the following PCR cycle conditions: 95°C for 5 min, 40 cycles of [95°C for 30 s, 59°C for 30 s, 72°C for 30 s]. .. PCR primers used are listed in .

Article Title: Aberrant methylated EDNRB can act as a potential diagnostic biomarker in sporadic colorectal cancer while KISS1 is controversial
Article Snippet: For this purpose, PCR amplification was performed on Step one plus Real-time PCR (ABI applied biosystems, USA) using sets of specific primers that are listed in . .. The final volume of each reaction was 20 μl, including 2 μl of converted template DNA, 10 μl of master mix (SYBR Premix Ex Taq II, TAKARA, Japan), 1.2 ul of primer pair (5 pmol) and 6.8 μl of double distilled water.

Article Title: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells
Article Snippet: Paragraph title: qPCR analysis of gene copy number ... Double-stranded DNA fragments were prepared from the oligonucleotides by polymerization: a 10 μL reaction mix consisting of 4 μL template DNA (0.05 μg/μL), 5 μL SYBR Green (TAKARA, Dalian, China), 0.2 μL of each forward and reverse oligonucleotide (10 μM each), and 0.6 μL deionized water was subjected to heating at 95 °C for 3 min, 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and elongation for 30 s at 72 °C, then 60 °C for 30 min for data acquisition.

Article Title: Effect of CEACAM-1 knockdown in human colorectal cancer cells
Article Snippet: Briefly, total RNA was extracted from CRC cells, using an RNeasy Mini kit (Invitrogen; Thermo Fisher Scientific, Inc.). .. Genomic DNA was removed from the extracted total RNA via DNase I digestion, and then the total RNA was reverse transcribed to generate cDNA, according to the manufacturer's instructions (PrimeScript™ 1st strand cDNA Synthesis kit; Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed in triplicate in a 10 µl reaction mix consisting of 4 µl template DNA (0.05 µg/µl), 5 µl SYBR-Green (Takara Biotechnology Co., Ltd.), 0.2 µl each forward and reverse oligonucleotide (10 µM each) and 0.6 µl deionized water. .. The primers used for qPCR were: CEACAM-1 forward, 5′-CAGGGGCTTCTGCTCACAGC-3′ and reverse, 5′-AGTTGCTTCTTCACAAGAT-3′; β-actin forward, 5′-GGCTGTGGAGACAAAAATGACCTC-3′ and reverse, 5′-AGGCTTGGGCTTGAATGGAGTC-3′.

Negative Control:

Article Title: Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs
Article Snippet: The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan). .. The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan).

Agarose Gel Electrophoresis:

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus
Article Snippet: PCR was conducted in 20-μl mixtures containing 1 μl of template DNA, 0.2 μl of 2.5 U of Takara Taq DNA polymerase (Takara Bio, Shiga, Japan), 1 μl each of 10 μM of forward primer and reverse primer, 2 μl of deoxynucleoside triphosphates (dNTPs), 2 μl of 10× PCR buffer, and 12.8 μl of water. .. PCR was conducted in 20-μl mixtures containing 1 μl of template DNA, 0.2 μl of 2.5 U of Takara Taq DNA polymerase (Takara Bio, Shiga, Japan), 1 μl each of 10 μM of forward primer and reverse primer, 2 μl of deoxynucleoside triphosphates (dNTPs), 2 μl of 10× PCR buffer, and 12.8 μl of water.

Article Title: Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations
Article Snippet: PCR reactions were carried out in 25 μL containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2 , 250 μM each of dNTP, 0.2 μM of each primer, 100–200 ng of template DNA, and 0.25 U La Taq polymerase (TaKaRa). .. The PCR reaction was carried out in a thermocycler (Bio-Rad) with an initial denaturation at 94 °C for 4 min, followed by 37 cycles of 94 °C for 30 sec (denaturation), 67.5 °C for 30 sec (annealing), 72 °C for 5 min (extension), and a final extension of 72 °C for 10 min. A negative control sample without gDNA was included in each PCR reaction.

Article Title: Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure
Article Snippet: Secondary PCR contained PCR buffer, 200 μM dNTP, 0.2 μM of each primer, nuclease free water, 1 μL of template DNA from primary PCR and 1.25 units of ExTaq DNA polymerase (Takara, Seta, Japan) in a total volume of 30 μL. .. DNA amplification was performed by using a GeneAmp 9700 PCR thermal cycler (Applied Biosystems, Foster City, CA).

Article Title: Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs
Article Snippet: The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan). .. The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan).

Electrophoresis:

Article Title: Histological Identification of Propionibacterium acnes in Nonpyogenic Degenerated Intervertebral Discs
Article Snippet: The PCR was performed as a mixture of 20 μ L volume containing 0.8 μ L of each primer, 2 μ L of 2.5 mM MgCl2, 1.6 μ L of dNTP, 2 μ L of 10x Ex Taq Buffer, 0.2 μ L of 5 U/μ L Ex Taq, 2 μ L of template DNA, and 10.6 μ L of sterilized distilled water (Takara RR01AM, Japan). .. Then the amplified product (5 μ L) was collected and run on a 1% agarose gel containing 1 μ g/ml of ethidium bromide.

Transgenic Assay:

Article Title: cAMP-responsive element in TATA-less core promoter is essential for haploid-specific gene expression in mouse testis
Article Snippet: For the transgenic analysis, the 600 bp genomic DNA fragment, that extends 566 bp upstream and 35 bp downstream of the transcriptional start site of the Oxct2b gene, was amplified by PCR from a genomic clone , using an upstream BglII site-containing primer and a downstream HindIII site-containing primer ( ). .. PCR was performed with 200 ng of template DNA in 100 μl of reaction mixture [1 μM of each primer, 800 μM dNTPs, 1× reaction buffer and 1 U of Taq DNA polymerase (TaKaRa Biomedical)].

Ethanol Precipitation:

Article Title: Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams
Article Snippet: For the PCR, 25 μl of the reaction mixture contained 80 ng of template DNA, 2.5 μl of 10× LA Taq buffer (TaKaRa), 2.5 μl of dNTP mix (TaKaRa), 5 μl each of the 1 pmol/μl forward and reverse primer solutions, 0.13 μl of LA Taq polymerase solution (TaKaRa) and 8.87 μl of pure water was used. .. The gene fragments were amplified with an ABI 9700 Thermal cycler using a protocol; 96°C for 2 min and 30 cycles of 96°C for 20 s, 56°C for 15 s and 72°C for 2 or 5 or 7 min, followed by extension at 72°C for 10 min. After amplification, 2 μl of the reaction product mixture was applied to 1% agarose electrophoresis gel and stained with an ethidium bromide solution to visualize the reaction products.

Spectrophotometry:

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: The concentration and quality of the total DNA were determined by a NanoDrop ND 2000 spectrophotometer (Thermo Scientific, USA) and a Qubit 2.0 Fluorometer (Invitrogen, NY, USA). .. Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M).

Concentration Assay:

Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles
Article Snippet: The PCR products for each sample were then pooled and purified using a QIAquick PCR purification column (Qiagen) and the concentration determined using a 2100 Bioanalyzer with a DNA 1000 LabChip kit (Agilent). .. We set up 150 μl PCR reactions containing 10 μl 20 pmol/l template DNA, 2X TITANIUM Taq DNA Polymerase (Clontech, Mountain View, CA), 1X TITANIUM Taq buffer (Clontech), 1 mmol/l dNTP mix, 8.3 mmol/l MgCl2 (Invitrogen), 0.05 μmol/l universal forward primer (5′-tcccgcgaaattaatacgac-3′), 8 μmol/l nested reverse primer (5′-AATCAGTTGCTCTGTATTGATCC-3′), and 6 × 107 magnetic streptavidin beads (MyOne, Invitrogen) coated with a modified universal forward primer (5′-dual biotion-T-Spacer18-tcccgcgaaattaatacgac-3′).

Article Title: Effect of Freeze-Thaw on a Midtemperate Soil Bacterial Community and the Correlation Network of Its Members
Article Snippet: The concentration and quality of the total DNA were determined by a NanoDrop ND 2000 spectrophotometer (Thermo Scientific, USA) and a Qubit 2.0 Fluorometer (Invitrogen, NY, USA). .. Each reaction consisted of a total volume of 25 μ L containing 8.75 μ L of sterilized water, 5.0 μ L of 5× PCR buffer, 5.0 μ L of 5× PCR GC-high enhancer, 2.0 μ L of dNTP (2.5 mM), 2.0 μ L of template DNA (200 ng/μ L), 0.25 μ L of TaKaRa polymerase (5 U/μ L), and 1.0 μ L of each primer (10 μ M).

CTG Assay:

Article Title: Association of IL10 gene promoter polymorphisms with risks of gastric cancer and atrophic gastritis
Article Snippet: – The primers were as follows: IL10 gene –1082A/G (rs1800896) polymorphism: forward: 5ʹ-TCT TAC CTA TCC CTA CTT CC- 3ʹ, reverse: 5ʹ-CTC GCT GCA ACC CAA CTG GC-3ʹ; IL10 gene –819C/T (rs1800871) polymorphism: forward: 5ʹ-CAC TAC TAA GGC TTC CTT GGG A-3ʹ, reverse: 5ʹ-GTG AGC AAA CTG AGG CAC GAC AT-3ʹ; IL10 gene –592A/C (rs1800872) polymorphism: forward: 5ʹ-GGT GAG CAC TAC CTG ACT AGC-3ʹ, reverse: 5ʹ-CCT AGG TCA CAG TGA CGT GGG-3ʹ. .. The total PCR reaction volume was 25 µl containing 2.5 µl of 10 × PCR buffer, 2.0 µl of 2.5 mM dNTP mixture, 1.0 µl of each upstream and downstream primer at 10 pmol/µl, 2.5 U或0.5 µL, 1.0 µl of the template DNA (TaKaRa Biotechnology), and an appropriate amount of ddH2 O.

Article Title: Genetic Diversity and Structure of Sinopodophyllum hexandrum (Royle) Ying in the Qinling Mountains, China
Article Snippet: Each 20 µL amplification reaction consisted of 1×PCR buffer (10 mM Tris-HCl at pH 8.3, 50 mM L–1 KCl, 0.001% gelatin, and l.5 mmol L–1 MgCl2 ), 1.6 mmol L–1 dNTP mix, 0.6 µmol L–1 primer (UBC900 was used for preliminary test), 15 ng of template DNA, and 1.0 U Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China), using a cycling profile of initial 5 min at 94°C, followed by 45 cycles of 30 s at 94°C, 45 s annealing at 50°C, and 90 s extension at 72°C, ending with a final extension of 7 min at 72°C. .. The optimized PCR experiment conditions were applied for primer screening in a PTC100TM Programmable Thermal Controller (MJ Research, Waltham, MA, USA).

Marker:

Article Title: Sequence Variation in Rhoptry Neck Protein 10 Gene among Toxoplasma gondii Isolates from Different Hosts and Geographical Locations
Article Snippet: PCR reactions were carried out in 25 μL containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2 , 250 μM each of dNTP, 0.2 μM of each primer, 100–200 ng of template DNA, and 0.25 U La Taq polymerase (TaKaRa). .. Each amplicon (6 μl) was examined on 1% (w/v) agarose gel to assess amplification efficiency.

Staining:

Article Title: Polymorphism in merozoite surface protein-7E of Plasmodium vivax in Thailand: Natural selection related to protein secondary structure
Article Snippet: Secondary PCR contained PCR buffer, 200 μM dNTP, 0.2 μM of each primer, nuclease free water, 1 μL of template DNA from primary PCR and 1.25 units of ExTaq DNA polymerase (Takara, Seta, Japan) in a total volume of 30 μL. .. DNA amplification was performed by using a GeneAmp 9700 PCR thermal cycler (Applied Biosystems, Foster City, CA).

Article Title: Genetic Diversity and Structure of Sinopodophyllum hexandrum (Royle) Ying in the Qinling Mountains, China
Article Snippet: Each 20 µL amplification reaction consisted of 1×PCR buffer (10 mM Tris-HCl at pH 8.3, 50 mM L–1 KCl, 0.001% gelatin, and l.5 mmol L–1 MgCl2 ), 1.6 mmol L–1 dNTP mix, 0.6 µmol L–1 primer (UBC900 was used for preliminary test), 15 ng of template DNA, and 1.0 U Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China), using a cycling profile of initial 5 min at 94°C, followed by 45 cycles of 30 s at 94°C, 45 s annealing at 50°C, and 90 s extension at 72°C, ending with a final extension of 7 min at 72°C. .. Primers that generated scorable bands and high levels of polymorphisms were selected by genotyping all populations.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 80
    TaKaRa template plasmid dna
    Optimization of the <t>TTcDR</t> system. A ) Optimization of each component in the TTcDR reaction. TTcDR reactions were performed at the indicated concentrations of the various components with circular <t>DNA</t> (1 ng/μl) for 12 h at 30 °C. The amount of DNA product was measured as described in Fig. 2 . The original concentrations and the optimized concentrations are indicated by black and white arrowheads, respectively. NTPs include each nucleotide triphosphate (ATP:GTP:CTP:UTP = 3.75:2.5:1.25:1.25) and the same molarity of magnesium acetate. B ) Comparison of DNA produced by TTcDR before and after optimization. The TTcDR reaction was performed with 1 ng/μl DNA for 2 h at 30 °C. The compositions before and after optimization are described in the Methods section. The error bars indicate the standard error (n = 14). Original gel images are shown in Figure S2 .
    Template Plasmid Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/template plasmid dna/product/TaKaRa
    Average 80 stars, based on 154 article reviews
    Price from $9.99 to $1999.99
    template plasmid dna - by Bioz Stars, 2020-01
    80/100 stars
      Buy from Supplier

    99
    TaKaRa template cdna
    Exon/intron structure and alternative mRNA transcripts of mouse GAD1 gene. The new arrangement of mouse GAD1 exons and introns as determined after the analysis of genomic and <t>cDNA</t> sequence data using bioinformatics, 3′-RACE, <t>RT-PCR,</t> cloning and sequencing. Exons are shown as numbered boxes (red numbers represent alternatively spliced exons) and introns as lines. Large boxes indicate the coding DNA sequence and the small boxes the 5′- and 3′-untranslated regions. The red arrowheads are showing the locations of the alternative promoters. The schematic representation of GAD1 splicing isoforms in relation to the gene is shown below the gene structure. The length in base pairs and the position of start and stop codons are indicated above each isoform. Diagrams are not to scale.
    Template Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/template cdna/product/TaKaRa
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    template cdna - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    Image Search Results


    Optimization of the TTcDR system. A ) Optimization of each component in the TTcDR reaction. TTcDR reactions were performed at the indicated concentrations of the various components with circular DNA (1 ng/μl) for 12 h at 30 °C. The amount of DNA product was measured as described in Fig. 2 . The original concentrations and the optimized concentrations are indicated by black and white arrowheads, respectively. NTPs include each nucleotide triphosphate (ATP:GTP:CTP:UTP = 3.75:2.5:1.25:1.25) and the same molarity of magnesium acetate. B ) Comparison of DNA produced by TTcDR before and after optimization. The TTcDR reaction was performed with 1 ng/μl DNA for 2 h at 30 °C. The compositions before and after optimization are described in the Methods section. The error bars indicate the standard error (n = 14). Original gel images are shown in Figure S2 .

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Optimization of the TTcDR system. A ) Optimization of each component in the TTcDR reaction. TTcDR reactions were performed at the indicated concentrations of the various components with circular DNA (1 ng/μl) for 12 h at 30 °C. The amount of DNA product was measured as described in Fig. 2 . The original concentrations and the optimized concentrations are indicated by black and white arrowheads, respectively. NTPs include each nucleotide triphosphate (ATP:GTP:CTP:UTP = 3.75:2.5:1.25:1.25) and the same molarity of magnesium acetate. B ) Comparison of DNA produced by TTcDR before and after optimization. The TTcDR reaction was performed with 1 ng/μl DNA for 2 h at 30 °C. The compositions before and after optimization are described in the Methods section. The error bars indicate the standard error (n = 14). Original gel images are shown in Figure S2 .

    Article Snippet: The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Produced

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Article Snippet: The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Electrophoresis, Autoradiography, Marker, Purification

    Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Article Snippet: The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Synthesized, Incubation, SDS Page, Autoradiography, Produced

    Characterization of the optimized TTcDR reaction. A ) Cleavage of the TTcDR product by restriction enzymes. After the TTcDR reaction was conducted for 12 h at 30 °C, the indicated restriction enzymes were added and incubated for 1 h at 37 °C. An aliquot was used for 1% agarose gel electrophoresis and autoradiography. The sample treated with Pst I was purified using a DNA column (Life Technologies) before electrophoresis. B ) Time-course data for the translation of DNA polymerase during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 35 S]-methionine at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 10% SDS-PAGE and autoradiography. The error bars indicate the standard error (n = 4). C ) Time-course data for DNA replication during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 32 P]-dCTP at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 1% agarose gel electrophoresis and autoradiography. The error bars indicate the standard error (n = 4).

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Characterization of the optimized TTcDR reaction. A ) Cleavage of the TTcDR product by restriction enzymes. After the TTcDR reaction was conducted for 12 h at 30 °C, the indicated restriction enzymes were added and incubated for 1 h at 37 °C. An aliquot was used for 1% agarose gel electrophoresis and autoradiography. The sample treated with Pst I was purified using a DNA column (Life Technologies) before electrophoresis. B ) Time-course data for the translation of DNA polymerase during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 35 S]-methionine at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 10% SDS-PAGE and autoradiography. The error bars indicate the standard error (n = 4). C ) Time-course data for DNA replication during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 32 P]-dCTP at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 1% agarose gel electrophoresis and autoradiography. The error bars indicate the standard error (n = 4).

    Article Snippet: The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Incubation, Agarose Gel Electrophoresis, Electrophoresis, Autoradiography, Purification, SDS Page

    DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.

    Article Snippet: The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Purification, Plasmid Preparation

    ( A ) PCR amplification of the env gene from DF-1/K cells. (M) DNA marker; (DF-1/K): Genomic DNA extracted from DF-1/K cells; (DF-1): Genomic DNA extracted from DF-1 cells. ( B ) Verification of the stability of the ALV-K env gene in DF-1/K cells during passage. (M) DNAMarker; Lanes 1-5 and 7-10: ALV-K env gene cell-culture passage levels 5, 15, 25, 30, 35, 40, 45, 50, 60, respectively; Lane 6: DF-1 cells (negative control)

    Journal: Archives of Virology

    Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection

    doi: 10.1007/s00705-017-3563-2

    Figure Lengend Snippet: ( A ) PCR amplification of the env gene from DF-1/K cells. (M) DNA marker; (DF-1/K): Genomic DNA extracted from DF-1/K cells; (DF-1): Genomic DNA extracted from DF-1 cells. ( B ) Verification of the stability of the ALV-K env gene in DF-1/K cells during passage. (M) DNAMarker; Lanes 1-5 and 7-10: ALV-K env gene cell-culture passage levels 5, 15, 25, 30, 35, 40, 45, 50, 60, respectively; Lane 6: DF-1 cells (negative control)

    Article Snippet: PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

    Techniques: Polymerase Chain Reaction, Amplification, Marker, Cell Culture, Negative Control

    A) . PCR amplification of the ALV-K env gene in DF-1/K cells. (M) DNA marker; (1): RNA extracted from DF-1/K cells; (2): Genomic DNA extracted from DF-1/K cells; (3) RNA extracted from DF-1 cells. B . Levels of ALV-K env gene transcription in DF-1/K cells were determined by real-time RT-PCR with gene specific primers. DF-1 cells served as a negative control. Data are representative of two independent experiments, both performed in triplicate

    Journal: Archives of Virology

    Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection

    doi: 10.1007/s00705-017-3563-2

    Figure Lengend Snippet: A) . PCR amplification of the ALV-K env gene in DF-1/K cells. (M) DNA marker; (1): RNA extracted from DF-1/K cells; (2): Genomic DNA extracted from DF-1/K cells; (3) RNA extracted from DF-1 cells. B . Levels of ALV-K env gene transcription in DF-1/K cells were determined by real-time RT-PCR with gene specific primers. DF-1 cells served as a negative control. Data are representative of two independent experiments, both performed in triplicate

    Article Snippet: PCR was performed in a 50 µl reaction mixture that consisted of template DNA (5µL), 10×PCR buffer (TaKaRa, Dalian, China), 1µM each of forward and reverse primers, 2mM MgCl2 , 100mM of each deoxynucleoside triphosphate (dNTP), and 1 unit of LA TaqTM DNA Polymerase (TaKaRa, Dalian, China).

    Techniques: Polymerase Chain Reaction, Amplification, Marker, Quantitative RT-PCR, Negative Control

    Identification of Armored DNA by PCR Amplification . The PCR amplification products of the DNA extracted from lambda phages were analysed using ethidium-bromide stained 1% agarose gel. Lane 1: negative control with no template; Lane 2, 3, 4, 5, and 6: PCR products from positive chimeric phages.

    Journal: Virology Journal

    Article Title: Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

    doi: 10.1186/1743-422X-6-226

    Figure Lengend Snippet: Identification of Armored DNA by PCR Amplification . The PCR amplification products of the DNA extracted from lambda phages were analysed using ethidium-bromide stained 1% agarose gel. Lane 1: negative control with no template; Lane 2, 3, 4, 5, and 6: PCR products from positive chimeric phages.

    Article Snippet: PCR was performed in a 50-μl reaction volume, which contained 5 μl of the DNA template, 1× PCR Buffer (TaKaRa, Japan), 1.5 mM MgCl2 , 300 μM deoxynucleoside triphosphate, 1.5 μM of each primer (gt11-for and gt11-rev), and 1.25 U of PrimerStart Taq DNA polymerase (TaKaRa, Japan).

    Techniques: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis, Negative Control

    Durability of Armored DNA . Equal amounts of armored DNA and plasmid DNA (extracted from the armored DNA) were incubated with DNase I (0.1 units/μl) at 37°C for 60 min. After digestion, the samples were analysed by real-time PCR. The armored DNA was completely resistant to DNase I digestion, whereas plasmid DNA was degraded completely. Armored DNA and plasmid DNA indicate samples without DNase I digestion.

    Journal: Virology Journal

    Article Title: Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

    doi: 10.1186/1743-422X-6-226

    Figure Lengend Snippet: Durability of Armored DNA . Equal amounts of armored DNA and plasmid DNA (extracted from the armored DNA) were incubated with DNase I (0.1 units/μl) at 37°C for 60 min. After digestion, the samples were analysed by real-time PCR. The armored DNA was completely resistant to DNase I digestion, whereas plasmid DNA was degraded completely. Armored DNA and plasmid DNA indicate samples without DNase I digestion.

    Article Snippet: PCR was performed in a 50-μl reaction volume, which contained 5 μl of the DNA template, 1× PCR Buffer (TaKaRa, Japan), 1.5 mM MgCl2 , 300 μM deoxynucleoside triphosphate, 1.5 μM of each primer (gt11-for and gt11-rev), and 1.25 U of PrimerStart Taq DNA polymerase (TaKaRa, Japan).

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

    Exon/intron structure and alternative mRNA transcripts of mouse GAD1 gene. The new arrangement of mouse GAD1 exons and introns as determined after the analysis of genomic and cDNA sequence data using bioinformatics, 3′-RACE, RT-PCR, cloning and sequencing. Exons are shown as numbered boxes (red numbers represent alternatively spliced exons) and introns as lines. Large boxes indicate the coding DNA sequence and the small boxes the 5′- and 3′-untranslated regions. The red arrowheads are showing the locations of the alternative promoters. The schematic representation of GAD1 splicing isoforms in relation to the gene is shown below the gene structure. The length in base pairs and the position of start and stop codons are indicated above each isoform. Diagrams are not to scale.

    Journal: BMC Neuroscience

    Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

    doi: 10.1186/1471-2202-15-114

    Figure Lengend Snippet: Exon/intron structure and alternative mRNA transcripts of mouse GAD1 gene. The new arrangement of mouse GAD1 exons and introns as determined after the analysis of genomic and cDNA sequence data using bioinformatics, 3′-RACE, RT-PCR, cloning and sequencing. Exons are shown as numbered boxes (red numbers represent alternatively spliced exons) and introns as lines. Large boxes indicate the coding DNA sequence and the small boxes the 5′- and 3′-untranslated regions. The red arrowheads are showing the locations of the alternative promoters. The schematic representation of GAD1 splicing isoforms in relation to the gene is shown below the gene structure. The length in base pairs and the position of start and stop codons are indicated above each isoform. Diagrams are not to scale.

    Article Snippet: The PCR mix (25 μl) contained 2.5 μl template cDNA, 1 μM forward and reverse primers, 200 μM dNTPs (Takara), and 0.5 U Ex Taq HS (Takara).

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Clone Assay

    Expression analysis of mouse GAD1 splicing isoforms in adult brain. (A) RT-PCR analysis of the expression of GAD1 mRNA splicing isoforms in adult mouse brain by using a forward primer either in Exon1 or Exon2 and a specific reverse primer for each transcript. (B) Analysis of the expression of Isoforms 1 to 6 by using the PCR product of lanes Exon1 and Exon2 in (A) as template for nested PCR with specific primers for each isoform. (C) Gel electrophoresis of Isoforms 3/4 and 5/6 , amplified from mouse brain cDNA using specific forward primer and reverse primer, close to the position of the polyadenylation signal. Plasmid containing Isoforms 1/2 was used as a negative control with the primers for Isoforms 3/4 and 5/6 and as a positive control with primers amplifying the 3′-end of Isoforms 1/2 . (D) Southern blotting of the gel in (C) . The membrane was probed with Probe-GAD1 against 3′-region of Isoforms 1 and 2 . Ma, marker λ/HindIII-ϕX174/HaeIII.

    Journal: BMC Neuroscience

    Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

    doi: 10.1186/1471-2202-15-114

    Figure Lengend Snippet: Expression analysis of mouse GAD1 splicing isoforms in adult brain. (A) RT-PCR analysis of the expression of GAD1 mRNA splicing isoforms in adult mouse brain by using a forward primer either in Exon1 or Exon2 and a specific reverse primer for each transcript. (B) Analysis of the expression of Isoforms 1 to 6 by using the PCR product of lanes Exon1 and Exon2 in (A) as template for nested PCR with specific primers for each isoform. (C) Gel electrophoresis of Isoforms 3/4 and 5/6 , amplified from mouse brain cDNA using specific forward primer and reverse primer, close to the position of the polyadenylation signal. Plasmid containing Isoforms 1/2 was used as a negative control with the primers for Isoforms 3/4 and 5/6 and as a positive control with primers amplifying the 3′-end of Isoforms 1/2 . (D) Southern blotting of the gel in (C) . The membrane was probed with Probe-GAD1 against 3′-region of Isoforms 1 and 2 . Ma, marker λ/HindIII-ϕX174/HaeIII.

    Article Snippet: The PCR mix (25 μl) contained 2.5 μl template cDNA, 1 μM forward and reverse primers, 200 μM dNTPs (Takara), and 0.5 U Ex Taq HS (Takara).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Nested PCR, Nucleic Acid Electrophoresis, Amplification, Plasmid Preparation, Negative Control, Positive Control, Southern Blot, Marker

    Expression of GAD1 mRNA splicing isoforms and GAD2 in different mouse tissues and during development. The expression level of Isoforms 1/2 (A) , GAD2 (B) , Isoforms 3/4 (C) , Isoforms 5/6 (D) , Isoforms 7/8 (E) , Isoforms 9/10 (F) are compared by PCR amplification using mouse Multiple Tissue cDNA Panel I and cDNA from pancreas, small intestine, and large intestine as cDNA template. (G) Control PCR reaction to verify the specificity of the primers for Isoforms 1/2, 3/4 and 5/6 . In the control reaction each primer pair was tested with a plasmid containing each full length insert as a template. The template in lanes 1, 4 and 7 was plasmid containing Isoforms 1/2 as template; lanes 2, 5 and 8, plasmid containing Isoforms 5/6 ; and lanes 3, 6, 9 plasmid containing Isoforms 3/4 . Lanes (Ht) heart; (Br) brain; (Sp) spleen; (L) lung; (Li) liver; (Ms) muscle; (K) kidney; (Ts) testis; (E7) 7-day embryo; (E11) 11-day embryo; (E15) 15-day embryo; (E17) 17-day embryo; (P) pancreas; (SI) small intestine; (LI) large intestine; (N) no template control; (−) plasmid containing Isoforms 1/2 is used as template for the amplification; and Ma, marker ϕX174/HaeIII.

    Journal: BMC Neuroscience

    Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

    doi: 10.1186/1471-2202-15-114

    Figure Lengend Snippet: Expression of GAD1 mRNA splicing isoforms and GAD2 in different mouse tissues and during development. The expression level of Isoforms 1/2 (A) , GAD2 (B) , Isoforms 3/4 (C) , Isoforms 5/6 (D) , Isoforms 7/8 (E) , Isoforms 9/10 (F) are compared by PCR amplification using mouse Multiple Tissue cDNA Panel I and cDNA from pancreas, small intestine, and large intestine as cDNA template. (G) Control PCR reaction to verify the specificity of the primers for Isoforms 1/2, 3/4 and 5/6 . In the control reaction each primer pair was tested with a plasmid containing each full length insert as a template. The template in lanes 1, 4 and 7 was plasmid containing Isoforms 1/2 as template; lanes 2, 5 and 8, plasmid containing Isoforms 5/6 ; and lanes 3, 6, 9 plasmid containing Isoforms 3/4 . Lanes (Ht) heart; (Br) brain; (Sp) spleen; (L) lung; (Li) liver; (Ms) muscle; (K) kidney; (Ts) testis; (E7) 7-day embryo; (E11) 11-day embryo; (E15) 15-day embryo; (E17) 17-day embryo; (P) pancreas; (SI) small intestine; (LI) large intestine; (N) no template control; (−) plasmid containing Isoforms 1/2 is used as template for the amplification; and Ma, marker ϕX174/HaeIII.

    Article Snippet: The PCR mix (25 μl) contained 2.5 μl template cDNA, 1 μM forward and reverse primers, 200 μM dNTPs (Takara), and 0.5 U Ex Taq HS (Takara).

    Techniques: Expressing, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Mass Spectrometry, Marker