Structured Review

TaKaRa pcr mix
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.
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Images

1) Product Images from "Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain"

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

Journal: BMC Neuroscience

doi: 10.1186/1471-2202-15-114

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

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

2) Product Images from "Role and mechanism of miR-222 in arsenic-transformed cells for inducing tumor growth"

Article Title: Role and mechanism of miR-222 in arsenic-transformed cells for inducing tumor growth

Journal: Oncotarget

doi: 10.18632/oncotarget.7525

miR-222 expression is upregulated in As-T cells The expression levels of miR-222 were determined using RT-PCR ( A ) and RT-qPCR ( B ) in As-T cells and BEAS-2B (B2B) cells. The expression levels of miR-222 were normalized to U6 snRNA levels. Data are presented as mean ± SE. **indicates significant difference compared to that of control cells ( P
Figure Legend Snippet: miR-222 expression is upregulated in As-T cells The expression levels of miR-222 were determined using RT-PCR ( A ) and RT-qPCR ( B ) in As-T cells and BEAS-2B (B2B) cells. The expression levels of miR-222 were normalized to U6 snRNA levels. Data are presented as mean ± SE. **indicates significant difference compared to that of control cells ( P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Related Articles

Clone Assay:

Article Title: A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources
Article Snippet: For PCR reactions, 0.5 μM DNA template was added to 50 μL PCR mixture consisting of Pyrobest DNA polymerase (5 U/μL, 0.5 μL), 10× Pyrobest buffer II (Takara Bio Inc.), 1 μM of each primer ( http://mlst.ucc.ie/mlst/dbs/Senterica ), and dNTP mixture (2.5 mM each). .. After PCR product purification, the DNA sequences of clones were analyzed by the Custom Oligonucleotide Synthesis Manufacture Office (Seoul, South Korea).

Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain
Article Snippet: Paragraph title: Cloning and expression of GAD1 splicing isoforms in different mouse tissues ... 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).

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl. .. Purified PCR products were cloned into TA-vector, and amplified plasmids were analyzed for the DNA sequence.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min. .. Via In-Fusion HD cloning (Takara), the scCro-tag was fused to HRP together with the T7-tag (N-terminal), GSGGGS linker (between HRP and scCro-tag), HA-tag (after linker), FLAG-tag (C-terminal) and 6×His-tag (after FLAG-tag).

Amplification:

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl. .. Purified PCR products were cloned into TA-vector, and amplified plasmids were analyzed for the DNA sequence.

Article Title: Inapparent Streptococcus agalactiae infection in adult/commercial tilapia
Article Snippet: Five microliters of a DNA solution was used as the template in a final 25 μl PCR mixture that contained the following: 2 mM MgCl2 PCR buffer, 200 μM of dNTPs, 250 nM of primers (except for primers 1 and 16, which were used at a concentration of 400 nM), and 0.3 U of HotStart Taq DNA polymerase (TaKaRa, China). .. By using GBS reference strains that represented all recognized serotypes, UV transillumination of the amplified products on the agarose gels showed a two or three band pattern, each of which was specific to and characteristic of each serotype .

Article Title: ?? T cells assist ?? T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine
Article Snippet: The PCR mixture contained 5 μl reverse-transcribed RNA (100 ng total RNA), 5 μl of 10× buffer, 4 μl of dNTP (2·5 m m each), 2·5 μl of 20 m m up-primer and 2·5 μl of 20 m m down-primer, 0·4 μl of 5 U/ml Taq polymerase (TaKaRa, Ohtsu, Japan) and 30·6 μl of water. .. PCR amplification was performed employing a 35-cycle program (β-actin: 25-cycle) consisting of denaturation, annealing and extension: 1 min of denaturation at 94°C, 1 min of annealing at 60°C and 2 min of extension at 72°C for β-actin; 1 min of denaturation at 94°C, 1 min of annealing at 62°C and 2 min of extension at 72°C for IL-4 or IFN-γ; 1 min of denaturation at 94°C, 1 min of annealing at 55°C and 2 min of extension at 72°C for IL-10.

Article Title: PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets
Article Snippet: .. The templates were amplified separately in a 10 µl PCR mixture containing 0.25 U of ExTaq DNA polymerase (Takara) with 0.25 µM of each primer in the following temperature sequence: preheating at 94°C for 5 min; 35 cycles consisting of 94°C for 15 s, 50°C for 30 s and 72°C for 30 s; and an additional extension at 72°C for 7 min. .. The PCR products were extracted with phenol-chloroform, precipitated with ethanol, and dissolved in TE buffer.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany).

Article Title: Toll-Like Receptor 9-Dependent Activation of Bone Marrow-Derived Dendritic Cells by URA5 DNA from Cryptococcus neoformans
Article Snippet: The 5′, 3′, and intervening fragments of this 345-bp sequence were amplified using the following specific primers ( ): ACG GTG AGG GCG GTA CTA TG (forward) and GCT TCG CTT CAG GGG AGG C (reverse) for the 5′ 129-bp fragment (base numbers 1 to 129), GCC TGT CGA GCC TAT TAT TG (forward) and AAG ACC TCT GAA CAC CGT AC (reverse) for the 3′ 123-bp fragment (base numbers 223 to 345), and GCC TCC CCT GAA GCG AAG C (forward) and CAA TAA TAG GCT CGA CAG GC (reverse) for the intervening 133-bp fragment (base numbers 110 to 242). .. We added 1.0 μl of C. neoformans DNA solution to 49 μl of a PCR mixture (TaKaRa Ex Taq; TaKaRa, Shiga, Japan) which contained 1.0 μM (each) forward and reverse primers.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. The DNA templates T1 for CFPS of HRP/scCro fusion proteins were amplified from the plasmid (10 ng) mentioned above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2-F1-Fw1: 5′-ctgcc ccggg ttcct cattc tatct cgatc ccgcg aaatt aatac-3′ and P1-R1: 5′-ccact acgcc tccgc tttcc tctct atgtc cggat atagt tcctc ctttc ag-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. Coupling the DNA template and the scCro-binding hairpin with the microbeads The HRP/scCro fusions-encoding DNA templates T2 were amplified from the DNA templates T1 (10 ng) and modified with an amino group at the 5′ end of the anti-sense strand in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2: 5′-ctgcc ccggg ttcct cattc t-3′ and P1-NH2 : 5′ amino modified oligonucleotide, 5′-ccact acgcc tccgc tttcc tctct atg-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s and 70°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

Article Title: Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide
Article Snippet: The PCR mixture contained 5 μl of the cDNA mixture, 2 μl of 10× PCR buffer, 0.2 mM deoxynucleoside triphosphates, 50 pmol of each primer, and 0.1 μl of Ex Taq DNA polymerase (Takara, Tokyo, Japan) in a total volume of 20 μl. .. Amplification was performed in a model MP TP3000 PCR thermal cycler (Takara) as follows: with CD14, 25 cycles of denaturation at 94°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 1 min and then a final extension at 72°C for 3 min; and with GAPDH, 25 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min and then a final extension at 72°C for 3 min. Amplified samples were visualized on 2.0% agarose gels stained with ethidium bromide and photographed under UV light.

Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma
Article Snippet: Methylation-specific PCR (MSP) was performed with 2 μL of bisulfite-modified DNA (100 ng/50 μl) and 48 μL of PCR mixture consisting of 10 × PCR Buffer (Mg2+ free), 25 mM MgCl2 , dNTP mixture (each 2.5 mM), sense primer (20 μM), antisense primer (20 μM), and TaKaRa EpiTaq HS (5 U/μL; TaKaRa). .. PCR amplification was conducted using 40 cycles (98 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s).

Positive Control:

Article Title: Inapparent Streptococcus agalactiae infection in adult/commercial tilapia
Article Snippet: Five microliters of a DNA solution was used as the template in a final 25 μl PCR mixture that contained the following: 2 mM MgCl2 PCR buffer, 200 μM of dNTPs, 250 nM of primers (except for primers 1 and 16, which were used at a concentration of 400 nM), and 0.3 U of HotStart Taq DNA polymerase (TaKaRa, China). .. A 688 bp band corresponded to a conserved fragment of the cpsL gene (capsular gene L), which was used as an internal positive control.

Synthesized:

Article Title: ?? T cells assist ?? T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine
Article Snippet: The PCR mixture contained 5 μl reverse-transcribed RNA (100 ng total RNA), 5 μl of 10× buffer, 4 μl of dNTP (2·5 m m each), 2·5 μl of 20 m m up-primer and 2·5 μl of 20 m m down-primer, 0·4 μl of 5 U/ml Taq polymerase (TaKaRa, Ohtsu, Japan) and 30·6 μl of water. .. The forward and reverse DNA primers were as follows: IL-4 primers: upstream, 5′-CGAAGAACACCACAGAGAGTGAGCT-3′; downstream, 5′-GACTCATTCATGGTGCAGCTTATCG-3′; IL-10 primers: upstream, 5′-ATGCAGGACTTTAAGGGTTACTTGGGTT-3′; downstream, 5′-ATTTCGGAGAGAGGTACAAACGAGGTTT-3′; IFN-γ primers: upstream, 5′-AGCGGCTGACTGAACTCAG ATTGAAG-3′; downstream, 5′-GTCACATGTTTCAGCTGTAT AGGG-3′ and β-actin primer; upstream, 5′-TGGAATCCTGTGG CATCCATGAAAC-3′; downstream, 5-TAAAACGTAGCTCCA GTAACAGTCCG-3′. β-actin, IFN-γ and IL-4 primers were synthesized by the phosphoramide method using a DNA synthesizer (model 392 PCR-MATA; Applied BIosystems, Inc., Foster City, CA, USA) and purified on a Sephadex G50 column (Pharmacia LKB Biotechnology, Gaithersburg, MD, USA) and by HPLC.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: Synthesis of templates for cell-free synthesis of HRP The plasmid pUCIDT-KAN: T7P-3HA-G8-HRP-HIS-T7T containing the designed hrp gene was synthesized by Integrated DNA Technologies (Coralville, IA, USA). .. The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min.

Construct:

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: The pRSET B vector (Life Technologies, Carlsbad, CA, USA) was used to construct the plasmids (pRSET-HRP-scCro and pRSET-scCro-HRP) for CFPS of the HRP/scCro fusions. .. The DNA templates T1 for CFPS of HRP/scCro fusion proteins were amplified from the plasmid (10 ng) mentioned above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2-F1-Fw1: 5′-ctgcc ccggg ttcct cattc tatct cgatc ccgcg aaatt aatac-3′ and P1-R1: 5′-ccact acgcc tccgc tttcc tctct atgtc cggat atagt tcctc ctttc ag-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 2 min, with an additional extension at 72°C for 7 min.

Article Title: Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide
Article Snippet: The primers for CD14 and GAPDH were constructed to generate fragments of 426 and 983 bp, respectively. .. The PCR mixture contained 5 μl of the cDNA mixture, 2 μl of 10× PCR buffer, 0.2 mM deoxynucleoside triphosphates, 50 pmol of each primer, and 0.1 μl of Ex Taq DNA polymerase (Takara, Tokyo, Japan) in a total volume of 20 μl.

Electrophoresis:

Article Title: Inapparent Streptococcus agalactiae infection in adult/commercial tilapia
Article Snippet: Five microliters of a DNA solution was used as the template in a final 25 μl PCR mixture that contained the following: 2 mM MgCl2 PCR buffer, 200 μM of dNTPs, 250 nM of primers (except for primers 1 and 16, which were used at a concentration of 400 nM), and 0.3 U of HotStart Taq DNA polymerase (TaKaRa, China). .. The PCR amplicons were analysed using electrophoresis in 1.5% agarose gels.

Nested PCR:

Article Title: Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load
Article Snippet: A nested PCR assay was used for highly sensitive and qualitative detection of HBV-DNA. .. Five microliters of DNA solution was mixed with 45 μl of PCR mixture containing 250 μM concentrations of each deoxynucleoside triphosphate (dNTP), 1× PCR buffer, 0.25 U of Taq DNA polymerase (Takara, Kyoto, Japan), and 0.25 μM concentrations of each primer pair.

Incubation:

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: A total of 10 μl of agarose beads containing 1 × TE and tissue fragments was formed in 250 μl of pre-chilled mineral oil, and then incubated at 50 °C overnight in 1000 μl of 200 μg/ml proteinase K, 10 mM Tris-HCl (pH 8.0) and 25 mM ethylene diamine tetraacetic acid. .. The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl.

Article Title: ?? T cells assist ?? T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine
Article Snippet: Total cellular RNA was extracted from 1 × 107 purified γδ or αβ T cells ( > 95% cell purity) that had been incubated at 37°C in complete medium for 24 h after separation by the guanidinium isothiocyanate method using RNA zolR (Biotex CS 101). .. The PCR mixture contained 5 μl reverse-transcribed RNA (100 ng total RNA), 5 μl of 10× buffer, 4 μl of dNTP (2·5 m m each), 2·5 μl of 20 m m up-primer and 2·5 μl of 20 m m down-primer, 0·4 μl of 5 U/ml Taq polymerase (TaKaRa, Ohtsu, Japan) and 30·6 μl of water.

Expressing:

Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain
Article Snippet: Paragraph title: Cloning and expression of GAD1 splicing isoforms in different mouse tissues ... 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).

Modification:

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. Coupling the DNA template and the scCro-binding hairpin with the microbeads The HRP/scCro fusions-encoding DNA templates T2 were amplified from the DNA templates T1 (10 ng) and modified with an amino group at the 5′ end of the anti-sense strand in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2: 5′-ctgcc ccggg ttcct cattc t-3′ and P1-NH2 : 5′ amino modified oligonucleotide, 5′-ccact acgcc tccgc tttcc tctct atg-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s and 70°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

High Performance Liquid Chromatography:

Article Title: ?? T cells assist ?? T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine
Article Snippet: The PCR mixture contained 5 μl reverse-transcribed RNA (100 ng total RNA), 5 μl of 10× buffer, 4 μl of dNTP (2·5 m m each), 2·5 μl of 20 m m up-primer and 2·5 μl of 20 m m down-primer, 0·4 μl of 5 U/ml Taq polymerase (TaKaRa, Ohtsu, Japan) and 30·6 μl of water. .. The forward and reverse DNA primers were as follows: IL-4 primers: upstream, 5′-CGAAGAACACCACAGAGAGTGAGCT-3′; downstream, 5′-GACTCATTCATGGTGCAGCTTATCG-3′; IL-10 primers: upstream, 5′-ATGCAGGACTTTAAGGGTTACTTGGGTT-3′; downstream, 5′-ATTTCGGAGAGAGGTACAAACGAGGTTT-3′; IFN-γ primers: upstream, 5′-AGCGGCTGACTGAACTCAG ATTGAAG-3′; downstream, 5′-GTCACATGTTTCAGCTGTAT AGGG-3′ and β-actin primer; upstream, 5′-TGGAATCCTGTGG CATCCATGAAAC-3′; downstream, 5-TAAAACGTAGCTCCA GTAACAGTCCG-3′. β-actin, IFN-γ and IL-4 primers were synthesized by the phosphoramide method using a DNA synthesizer (model 392 PCR-MATA; Applied BIosystems, Inc., Foster City, CA, USA) and purified on a Sephadex G50 column (Pharmacia LKB Biotechnology, Gaithersburg, MD, USA) and by HPLC.

Sequencing:

Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain
Article Snippet: All isoforms were cloned in pGEM-T Easy (Promega, Madison, WI) and sequenced with ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits (Applied Biosystems, Tokyo, Japan). .. 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).

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl. .. Purified PCR products were cloned into TA-vector, and amplified plasmids were analyzed for the DNA sequence.

Article Title: PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets
Article Snippet: .. The templates were amplified separately in a 10 µl PCR mixture containing 0.25 U of ExTaq DNA polymerase (Takara) with 0.25 µM of each primer in the following temperature sequence: preheating at 94°C for 5 min; 35 cycles consisting of 94°C for 15 s, 50°C for 30 s and 72°C for 30 s; and an additional extension at 72°C for 7 min. .. The PCR products were extracted with phenol-chloroform, precipitated with ethanol, and dissolved in TE buffer.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany).

Article Title: Toll-Like Receptor 9-Dependent Activation of Bone Marrow-Derived Dendritic Cells by URA5 DNA from Cryptococcus neoformans
Article Snippet: The 5′, 3′, and intervening fragments of this 345-bp sequence were amplified using the following specific primers ( ): ACG GTG AGG GCG GTA CTA TG (forward) and GCT TCG CTT CAG GGG AGG C (reverse) for the 5′ 129-bp fragment (base numbers 1 to 129), GCC TGT CGA GCC TAT TAT TG (forward) and AAG ACC TCT GAA CAC CGT AC (reverse) for the 3′ 123-bp fragment (base numbers 223 to 345), and GCC TCC CCT GAA GCG AAG C (forward) and CAA TAA TAG GCT CGA CAG GC (reverse) for the intervening 133-bp fragment (base numbers 110 to 242). .. We added 1.0 μl of C. neoformans DNA solution to 49 μl of a PCR mixture (TaKaRa Ex Taq; TaKaRa, Shiga, Japan) which contained 1.0 μM (each) forward and reverse primers.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. The DNA templates T1 for CFPS of HRP/scCro fusion proteins were amplified from the plasmid (10 ng) mentioned above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2-F1-Fw1: 5′-ctgcc ccggg ttcct cattc tatct cgatc ccgcg aaatt aatac-3′ and P1-R1: 5′-ccact acgcc tccgc tttcc tctct atgtc cggat atagt tcctc ctttc ag-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. Coupling the DNA template and the scCro-binding hairpin with the microbeads The HRP/scCro fusions-encoding DNA templates T2 were amplified from the DNA templates T1 (10 ng) and modified with an amino group at the 5′ end of the anti-sense strand in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2: 5′-ctgcc ccggg ttcct cattc t-3′ and P1-NH2 : 5′ amino modified oligonucleotide, 5′-ccact acgcc tccgc tttcc tctct atg-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s and 70°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma
Article Snippet: Methylation-specific PCR (MSP) was performed with 2 μL of bisulfite-modified DNA (100 ng/50 μl) and 48 μL of PCR mixture consisting of 10 × PCR Buffer (Mg2+ free), 25 mM MgCl2 , dNTP mixture (each 2.5 mM), sense primer (20 μM), antisense primer (20 μM), and TaKaRa EpiTaq HS (5 U/μL; TaKaRa). .. For parallel quality control, a plasmid containing a methylated LATS1 sequence and water without DNA template were used as positive and negative controls, respectively.

Concentration Assay:

Article Title: Inapparent Streptococcus agalactiae infection in adult/commercial tilapia
Article Snippet: .. Five microliters of a DNA solution was used as the template in a final 25 μl PCR mixture that contained the following: 2 mM MgCl2 PCR buffer, 200 μM of dNTPs, 250 nM of primers (except for primers 1 and 16, which were used at a concentration of 400 nM), and 0.3 U of HotStart Taq DNA polymerase (TaKaRa, China). .. The PCR amplicons were analysed using electrophoresis in 1.5% agarose gels.

Generated:

Article Title: A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources
Article Snippet: For PCR reactions, 0.5 μM DNA template was added to 50 μL PCR mixture consisting of Pyrobest DNA polymerase (5 U/μL, 0.5 μL), 10× Pyrobest buffer II (Takara Bio Inc.), 1 μM of each primer ( http://mlst.ucc.ie/mlst/dbs/Senterica ), and dNTP mixture (2.5 mM each). .. TIFF images of agarose gels were generated using the GelDoc XR+ gel documentation system (Bio-Rad) and Image Lab 3.0 Software (Bio-Rad).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain
Article Snippet: The primer pairs used in the RT-PCR analysis are listed in Figure . .. 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).

Article Title: Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide
Article Snippet: Paragraph title: Reverse transcriptase PCR (RT-PCR) assay. ... The PCR mixture contained 5 μl of the cDNA mixture, 2 μl of 10× PCR buffer, 0.2 mM deoxynucleoside triphosphates, 50 pmol of each primer, and 0.1 μl of Ex Taq DNA polymerase (Takara, Tokyo, Japan) in a total volume of 20 μl.

Recombinant:

Article Title: Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load
Article Snippet: The recombinant plasmid was purified and subsequently quantified by measuring the optical density at 260 nm. .. Five microliters of DNA solution was mixed with 45 μl of PCR mixture containing 250 μM concentrations of each deoxynucleoside triphosphate (dNTP), 1× PCR buffer, 0.25 U of Taq DNA polymerase (Takara, Kyoto, Japan), and 0.25 μM concentrations of each primer pair.

Cellular Antioxidant Activity Assay:

Article Title: Toll-Like Receptor 9-Dependent Activation of Bone Marrow-Derived Dendritic Cells by URA5 DNA from Cryptococcus neoformans
Article Snippet: The 5′, 3′, and intervening fragments of this 345-bp sequence were amplified using the following specific primers ( ): ACG GTG AGG GCG GTA CTA TG (forward) and GCT TCG CTT CAG GGG AGG C (reverse) for the 5′ 129-bp fragment (base numbers 1 to 129), GCC TGT CGA GCC TAT TAT TG (forward) and AAG ACC TCT GAA CAC CGT AC (reverse) for the 3′ 123-bp fragment (base numbers 223 to 345), and GCC TCC CCT GAA GCG AAG C (forward) and CAA TAA TAG GCT CGA CAG GC (reverse) for the intervening 133-bp fragment (base numbers 110 to 242). .. We added 1.0 μl of C. neoformans DNA solution to 49 μl of a PCR mixture (TaKaRa Ex Taq; TaKaRa, Shiga, Japan) which contained 1.0 μM (each) forward and reverse primers.

DNA Extraction:

Article Title: A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources
Article Snippet: DNA was extracted using an UltraClean Microbial DNA Isolation Kit. .. For PCR reactions, 0.5 μM DNA template was added to 50 μL PCR mixture consisting of Pyrobest DNA polymerase (5 U/μL, 0.5 μL), 10× Pyrobest buffer II (Takara Bio Inc.), 1 μM of each primer ( http://mlst.ucc.ie/mlst/dbs/Senterica ), and dNTP mixture (2.5 mM each).

Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma
Article Snippet: Paragraph title: DNA extraction and methylation-specific PCR ... Methylation-specific PCR (MSP) was performed with 2 μL of bisulfite-modified DNA (100 ng/50 μl) and 48 μL of PCR mixture consisting of 10 × PCR Buffer (Mg2+ free), 25 mM MgCl2 , dNTP mixture (each 2.5 mM), sense primer (20 μM), antisense primer (20 μM), and TaKaRa EpiTaq HS (5 U/μL; TaKaRa).

Methylation:

Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma
Article Snippet: .. Methylation-specific PCR (MSP) was performed with 2 μL of bisulfite-modified DNA (100 ng/50 μl) and 48 μL of PCR mixture consisting of 10 × PCR Buffer (Mg2+ free), 25 mM MgCl2 , dNTP mixture (each 2.5 mM), sense primer (20 μM), antisense primer (20 μM), and TaKaRa EpiTaq HS (5 U/μL; TaKaRa). .. PCR amplification was conducted using 40 cycles (98 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s).

Mutagenesis:

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: In view of these macroscopic features suggestive of GCPS or PHS, mutation of the GLI3 gene was analyzed. .. The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl.

Isolation:

Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma
Article Snippet: Genomic DNA was isolated from HCC tumor tissues, paired normal adjacent tissues, and HCC cells using the DNA Isolation kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. .. Methylation-specific PCR (MSP) was performed with 2 μL of bisulfite-modified DNA (100 ng/50 μl) and 48 μL of PCR mixture consisting of 10 × PCR Buffer (Mg2+ free), 25 mM MgCl2 , dNTP mixture (each 2.5 mM), sense primer (20 μM), antisense primer (20 μM), and TaKaRa EpiTaq HS (5 U/μL; TaKaRa).

Purification:

Article Title: A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources
Article Snippet: For PCR reactions, 0.5 μM DNA template was added to 50 μL PCR mixture consisting of Pyrobest DNA polymerase (5 U/μL, 0.5 μL), 10× Pyrobest buffer II (Takara Bio Inc.), 1 μM of each primer ( http://mlst.ucc.ie/mlst/dbs/Senterica ), and dNTP mixture (2.5 mM each). .. After PCR product purification, the DNA sequences of clones were analyzed by the Custom Oligonucleotide Synthesis Manufacture Office (Seoul, South Korea).

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl. .. Purified PCR products were cloned into TA-vector, and amplified plasmids were analyzed for the DNA sequence.

Article Title: Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA
Article Snippet: A 50-μl PCR mixture contained 100 ng template DNA, 25 μl 2× PrimeSTAR master mix (TaKaRa, Dalian, China), and 0.2 μmol−l of each primer. .. The resulting PCR product was purified using a DNA gel extraction kit (Axygen, Hangzhou, China) and ligated into the Ecl136II site of the pEB vector.

Article Title: Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load
Article Snippet: The recombinant plasmid was purified and subsequently quantified by measuring the optical density at 260 nm. .. Five microliters of DNA solution was mixed with 45 μl of PCR mixture containing 250 μM concentrations of each deoxynucleoside triphosphate (dNTP), 1× PCR buffer, 0.25 U of Taq DNA polymerase (Takara, Kyoto, Japan), and 0.25 μM concentrations of each primer pair.

Article Title: ?? T cells assist ?? T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine
Article Snippet: Total cellular RNA was extracted from 1 × 107 purified γδ or αβ T cells ( > 95% cell purity) that had been incubated at 37°C in complete medium for 24 h after separation by the guanidinium isothiocyanate method using RNA zolR (Biotex CS 101). .. The PCR mixture contained 5 μl reverse-transcribed RNA (100 ng total RNA), 5 μl of 10× buffer, 4 μl of dNTP (2·5 m m each), 2·5 μl of 20 m m up-primer and 2·5 μl of 20 m m down-primer, 0·4 μl of 5 U/ml Taq polymerase (TaKaRa, Ohtsu, Japan) and 30·6 μl of water.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany).

Article Title: Toll-Like Receptor 9-Dependent Activation of Bone Marrow-Derived Dendritic Cells by URA5 DNA from Cryptococcus neoformans
Article Snippet: We added 1.0 μl of C. neoformans DNA solution to 49 μl of a PCR mixture (TaKaRa Ex Taq; TaKaRa, Shiga, Japan) which contained 1.0 μM (each) forward and reverse primers. .. For the stimulation of BM-DCs, PCR products were purified using a QIAquick PCR purification kit (Qiagen, Germantown, MD).

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: These epitope tags can be used for the purification of the HRP fusion and the detection of the HRP fusion displayed on microbeads. .. The DNA templates T1 for CFPS of HRP/scCro fusion proteins were amplified from the plasmid (10 ng) mentioned above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2-F1-Fw1: 5′-ctgcc ccggg ttcct cattc tatct cgatc ccgcg aaatt aatac-3′ and P1-R1: 5′-ccact acgcc tccgc tttcc tctct atgtc cggat atagt tcctc ctttc ag-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 2 min, with an additional extension at 72°C for 7 min.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: Coupling the DNA template and the scCro-binding hairpin with the microbeads The HRP/scCro fusions-encoding DNA templates T2 were amplified from the DNA templates T1 (10 ng) and modified with an amino group at the 5′ end of the anti-sense strand in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2: 5′-ctgcc ccggg ttcct cattc t-3′ and P1-NH2 : 5′ amino modified oligonucleotide, 5′-ccact acgcc tccgc tttcc tctct atg-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s and 70°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

Polymerase Chain Reaction:

Article Title: A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources
Article Snippet: .. For PCR reactions, 0.5 μM DNA template was added to 50 μL PCR mixture consisting of Pyrobest DNA polymerase (5 U/μL, 0.5 μL), 10× Pyrobest buffer II (Takara Bio Inc.), 1 μM of each primer ( http://mlst.ucc.ie/mlst/dbs/Senterica ), and dNTP mixture (2.5 mM each). .. The PCR products were separated on 2% agarose gels.

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

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: .. The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl. .. The PCR products were electrophoresed in a 3% agarose gel and stained with ethidium bromide.

Article Title: Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA
Article Snippet: .. A 50-μl PCR mixture contained 100 ng template DNA, 25 μl 2× PrimeSTAR master mix (TaKaRa, Dalian, China), and 0.2 μmol−l of each primer. .. The reaction consisted of an initial denaturation step at 94°C for 5 min, 30 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 2 min 20 s, and final extension step at 72°C for 10 min.

Article Title: Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load
Article Snippet: .. Five microliters of DNA solution was mixed with 45 μl of PCR mixture containing 250 μM concentrations of each deoxynucleoside triphosphate (dNTP), 1× PCR buffer, 0.25 U of Taq DNA polymerase (Takara, Kyoto, Japan), and 0.25 μM concentrations of each primer pair. .. The primer sets for the HBV core region were 5"-TTTTTCACCTCTGCCTAATCATCT-3" (nt 1823 to 1846) and 5"-GTAGAAGAATAAAGCCCAGTAA-3" (nt 2505 to 2484) for the first round of PCR and 5"-TGTTCATGTCCTACTGTTCAAG-3" (nt 1849 to 1870) and 5"-AGTTTCCCACCTTATGAGTCCA-3" (nt 2483 to 2462) for the second round of PCR.

Article Title: Inapparent Streptococcus agalactiae infection in adult/commercial tilapia
Article Snippet: .. Five microliters of a DNA solution was used as the template in a final 25 μl PCR mixture that contained the following: 2 mM MgCl2 PCR buffer, 200 μM of dNTPs, 250 nM of primers (except for primers 1 and 16, which were used at a concentration of 400 nM), and 0.3 U of HotStart Taq DNA polymerase (TaKaRa, China). .. The PCR amplicons were analysed using electrophoresis in 1.5% agarose gels.

Article Title: ?? T cells assist ?? T cells in the adoptive transfer of contact hypersensitivity to para-phenylenediamine
Article Snippet: .. The PCR mixture contained 5 μl reverse-transcribed RNA (100 ng total RNA), 5 μl of 10× buffer, 4 μl of dNTP (2·5 m m each), 2·5 μl of 20 m m up-primer and 2·5 μl of 20 m m down-primer, 0·4 μl of 5 U/ml Taq polymerase (TaKaRa, Ohtsu, Japan) and 30·6 μl of water. .. Oligonucleotide primers specific for β-actin, IL-4, IL-10 or IFN‐γ were used.

Article Title: PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets
Article Snippet: .. The templates were amplified separately in a 10 µl PCR mixture containing 0.25 U of ExTaq DNA polymerase (Takara) with 0.25 µM of each primer in the following temperature sequence: preheating at 94°C for 5 min; 35 cycles consisting of 94°C for 15 s, 50°C for 30 s and 72°C for 30 s; and an additional extension at 72°C for 7 min. .. The PCR products were extracted with phenol-chloroform, precipitated with ethanol, and dissolved in TE buffer.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany).

Article Title: Toll-Like Receptor 9-Dependent Activation of Bone Marrow-Derived Dendritic Cells by URA5 DNA from Cryptococcus neoformans
Article Snippet: .. We added 1.0 μl of C. neoformans DNA solution to 49 μl of a PCR mixture (TaKaRa Ex Taq; TaKaRa, Shiga, Japan) which contained 1.0 μM (each) forward and reverse primers. .. The PCR products were electrophoresed in 2% agarose gels, stained with 0.5 μg/ml ethidium bromide, and observed with a UV transilluminator.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. The DNA templates T1 for CFPS of HRP/scCro fusion proteins were amplified from the plasmid (10 ng) mentioned above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2-F1-Fw1: 5′-ctgcc ccggg ttcct cattc tatct cgatc ccgcg aaatt aatac-3′ and P1-R1: 5′-ccact acgcc tccgc tttcc tctct atgtc cggat atagt tcctc ctttc ag-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. Coupling the DNA template and the scCro-binding hairpin with the microbeads The HRP/scCro fusions-encoding DNA templates T2 were amplified from the DNA templates T1 (10 ng) and modified with an amino group at the 5′ end of the anti-sense strand in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2: 5′-ctgcc ccggg ttcct cattc t-3′ and P1-NH2 : 5′ amino modified oligonucleotide, 5′-ccact acgcc tccgc tttcc tctct atg-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s and 70°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

Article Title: Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide
Article Snippet: .. The PCR mixture contained 5 μl of the cDNA mixture, 2 μl of 10× PCR buffer, 0.2 mM deoxynucleoside triphosphates, 50 pmol of each primer, and 0.1 μl of Ex Taq DNA polymerase (Takara, Tokyo, Japan) in a total volume of 20 μl. .. Amplification was performed in a model MP TP3000 PCR thermal cycler (Takara) as follows: with CD14, 25 cycles of denaturation at 94°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 1 min and then a final extension at 72°C for 3 min; and with GAPDH, 25 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min and then a final extension at 72°C for 3 min. Amplified samples were visualized on 2.0% agarose gels stained with ethidium bromide and photographed under UV light.

Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma
Article Snippet: .. Methylation-specific PCR (MSP) was performed with 2 μL of bisulfite-modified DNA (100 ng/50 μl) and 48 μL of PCR mixture consisting of 10 × PCR Buffer (Mg2+ free), 25 mM MgCl2 , dNTP mixture (each 2.5 mM), sense primer (20 μM), antisense primer (20 μM), and TaKaRa EpiTaq HS (5 U/μL; TaKaRa). .. PCR amplification was conducted using 40 cycles (98 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s).

Gel Extraction:

Article Title: Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA
Article Snippet: A 50-μl PCR mixture contained 100 ng template DNA, 25 μl 2× PrimeSTAR master mix (TaKaRa, Dalian, China), and 0.2 μmol−l of each primer. .. The resulting PCR product was purified using a DNA gel extraction kit (Axygen, Hangzhou, China) and ligated into the Ecl136II site of the pEB vector.

IA:

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: Synthesis of templates for cell-free synthesis of HRP The plasmid pUCIDT-KAN: T7P-3HA-G8-HRP-HIS-T7T containing the designed hrp gene was synthesized by Integrated DNA Technologies (Coralville, IA, USA). .. The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min.

Plasmid Preparation:

Article Title: Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA
Article Snippet: A 50-μl PCR mixture contained 100 ng template DNA, 25 μl 2× PrimeSTAR master mix (TaKaRa, Dalian, China), and 0.2 μmol−l of each primer. .. The resulting PCR product was purified using a DNA gel extraction kit (Axygen, Hangzhou, China) and ligated into the Ecl136II site of the pEB vector.

Article Title: Sensitive Enzyme Immunoassay for Hepatitis B Virus Core-Related Antigens and Their Correlation to Virus Load
Article Snippet: The recombinant plasmid was purified and subsequently quantified by measuring the optical density at 260 nm. .. Five microliters of DNA solution was mixed with 45 μl of PCR mixture containing 250 μM concentrations of each deoxynucleoside triphosphate (dNTP), 1× PCR buffer, 0.25 U of Taq DNA polymerase (Takara, Kyoto, Japan), and 0.25 μM concentrations of each primer pair.

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany).

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: .. The DNA templates T1 for CFPS of HRP/scCro fusion proteins were amplified from the plasmid (10 ng) mentioned above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara) and 0.5 μM of each primer (P2-F1-Fw1: 5′-ctgcc ccggg ttcct cattc tatct cgatc ccgcg aaatt aatac-3′ and P1-R1: 5′-ccact acgcc tccgc tttcc tctct atgtc cggat atagt tcctc ctttc ag-3′) using the following temperature sequence: preheating at 94°C for 1 min; followed by 20 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 2 min, with an additional extension at 72°C for 7 min. .. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen).

Article Title: miR-29c-3p regulates DNMT3B and LATS1 methylation to inhibit tumor progression in hepatocellular carcinoma
Article Snippet: Methylation-specific PCR (MSP) was performed with 2 μL of bisulfite-modified DNA (100 ng/50 μl) and 48 μL of PCR mixture consisting of 10 × PCR Buffer (Mg2+ free), 25 mM MgCl2 , dNTP mixture (each 2.5 mM), sense primer (20 μM), antisense primer (20 μM), and TaKaRa EpiTaq HS (5 U/μL; TaKaRa). .. For parallel quality control, a plasmid containing a methylated LATS1 sequence and water without DNA template were used as positive and negative controls, respectively.

Software:

Article Title: A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources
Article Snippet: For PCR reactions, 0.5 μM DNA template was added to 50 μL PCR mixture consisting of Pyrobest DNA polymerase (5 U/μL, 0.5 μL), 10× Pyrobest buffer II (Takara Bio Inc.), 1 μM of each primer ( http://mlst.ucc.ie/mlst/dbs/Senterica ), and dNTP mixture (2.5 mM each). .. TIFF images of agarose gels were generated using the GelDoc XR+ gel documentation system (Bio-Rad) and Image Lab 3.0 Software (Bio-Rad).

Multiplex Assay:

Article Title: Inapparent Streptococcus agalactiae infection in adult/commercial tilapia
Article Snippet: Paragraph title: Serotyping by multiplex PCR ... Five microliters of a DNA solution was used as the template in a final 25 μl PCR mixture that contained the following: 2 mM MgCl2 PCR buffer, 200 μM of dNTPs, 250 nM of primers (except for primers 1 and 16, which were used at a concentration of 400 nM), and 0.3 U of HotStart Taq DNA polymerase (TaKaRa, China).

Agarose Gel Electrophoresis:

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl. .. The PCR products were electrophoresed in a 3% agarose gel and stained with ethidium bromide.

Oligonucleotide Synthesis:

Article Title: A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources
Article Snippet: For PCR reactions, 0.5 μM DNA template was added to 50 μL PCR mixture consisting of Pyrobest DNA polymerase (5 U/μL, 0.5 μL), 10× Pyrobest buffer II (Takara Bio Inc.), 1 μM of each primer ( http://mlst.ucc.ie/mlst/dbs/Senterica ), and dNTP mixture (2.5 mM each). .. After PCR product purification, the DNA sequences of clones were analyzed by the Custom Oligonucleotide Synthesis Manufacture Office (Seoul, South Korea).

FLAG-tag:

Article Title: Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter
Article Snippet: The DNA template, which contained the gene of the mature HRP with tags, T7 promoter, and T7 terminator, was amplified from the plasmid (1 ng) described above in a 20-μL PCR mixture with 0.05 U/μL Pyrobest DNA polymerase (Takara, Shiga, Japan) and 0.5 μM of each primer (Fw1: 5′-cgatc ccgcg aaatt aatac-3′ and R1: 5′-tccgg atata gttcc tcctt tcag-3′) using the following temperature sequence: preheating at 94°C for 3 min; followed by 25 cycles of 94°C for 15 s, 50°C for 15 s, and 72°C for 90 s, with an additional extension at 72°C for 7 min. .. Via In-Fusion HD cloning (Takara), the scCro-tag was fused to HRP together with the T7-tag (N-terminal), GSGGGS linker (between HRP and scCro-tag), HA-tag (after linker), FLAG-tag (C-terminal) and 6×His-tag (after FLAG-tag).

Staining:

Article Title: Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas
Article Snippet: The PCR mixture contained Mighty AMP® DNA polymerase (Takara, Tokyo, Japan) and bead fragments in a final volume of 25 μl. .. The PCR products were electrophoresed in a 3% agarose gel and stained with ethidium bromide.

Article Title: Toll-Like Receptor 9-Dependent Activation of Bone Marrow-Derived Dendritic Cells by URA5 DNA from Cryptococcus neoformans
Article Snippet: We added 1.0 μl of C. neoformans DNA solution to 49 μl of a PCR mixture (TaKaRa Ex Taq; TaKaRa, Shiga, Japan) which contained 1.0 μM (each) forward and reverse primers. .. The PCR products were electrophoresed in 2% agarose gels, stained with 0.5 μg/ml ethidium bromide, and observed with a UV transilluminator.

Article Title: Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide
Article Snippet: The PCR mixture contained 5 μl of the cDNA mixture, 2 μl of 10× PCR buffer, 0.2 mM deoxynucleoside triphosphates, 50 pmol of each primer, and 0.1 μl of Ex Taq DNA polymerase (Takara, Tokyo, Japan) in a total volume of 20 μl. .. Amplification was performed in a model MP TP3000 PCR thermal cycler (Takara) as follows: with CD14, 25 cycles of denaturation at 94°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 1 min and then a final extension at 72°C for 3 min; and with GAPDH, 25 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min and then a final extension at 72°C for 3 min. Amplified samples were visualized on 2.0% agarose gels stained with ethidium bromide and photographed under UV light.

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    TaKaRa sybr green qrt pcr master mix
    PTEN is a direct target gene of miR-130b. ( A ) PTEN was detected by <t>qRT-</t> <t>PCR</t> in 29 BC tissues and was matched the adjacent noncancerous breast tissues. ( B ) The relationship between PTEN and miR-130b expression was explored by Spearman’s correlation in 29 paired BC tissues and adjacent noncancerous breast tissues. PTEN was inversely correlated with the miR-130b levels in BC tissues. ( C ) Relative PTEN expression in MCF-7 and MCF-7/ADR cells were detected by qRT-PCR (left panel) and western blot (right panel). ( D ) Effects of miR-130b mimics in MCF-7 cells and miR-130b inhibitor in MCF-7/ADR cells on the relative expression levels of PTEN were detected by qRT- PCR (left panel) and western blot (right panel). ( E ) PTEN expression was detected by immunofluorescence staining in these cells. Red fluorescence: PTEN, blue fluorescence: DAPI, staining for nuclear DNA. There were no significant differences between the control group and the blank group. ( F ) The relative luciferase activity of 293 T cells was detected after the pGL3-control vector, wt or mut PTEN 3′UTR genes were co-transfected with miR-NC or miR-130b mimics (*P
    Sybr Green Qrt Pcr Master Mix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa emerald amp gt pcr master mix
    Genotyping of Arabidopsis thaliana plants from GABI_462A04 line (a), SAIL_547_C10 line (b) and SALK_022236 line (c) with Emerald Amp GT <t>PCR</t> Master Mix. WT - <t>DNA</t> from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–12 and 1–4 - individual plants from each T-DNA line. Molecular weight DNA standards are shown on the left. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.
    Emerald Amp Gt Pcr Master Mix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa pcr master mix
    cDNA sequence of mRNA de novo transcribed from <t>TNFSF9</t> in HepG2 clone 9-1 (A) HepG2 clone 9-1 cells were incubated with 500 ng/ml LPS for the indicated times. cDNA was synthesized from total RNA, and TNFSF9 expression was quantified by real-time <t>PCR</t> as described in “Materials and Methods”. (B) HepG2 cells were incubated with 500 ng/ml LPS in the presence of 5-ethynyl uridine (EU) for 16 hrs. EU-labeled RNA was isolated as described in “Materials and Methods”. TNFSF9 cDNA was amplified by PCR and the PCR products were isolated and sequenced. This experiment is representative of four independent experiments.
    Pcr Master Mix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa qrt pcr mixture
    Overexpression of PpGLK1 in u/u tomato. (A) <t>qRT-PCR</t> and western blot analysis confirming PpGLK1 overexpression in u/u tomato. (B) Phenotype of PpGLK1 -overexpressing u/u tomato. (C) Chlorophyll contents in u/u tomato and PpGLK1 -overexpressing u/u tomato. (D) Chloroplast development in fruit observed by TEM in PpGLK1 -overexpressing u/u tomato fruit and u/u tomato fruit. Data is presented as the means ± SD, n = 3. Different letters above bars denote statistical significance according to one-way ANOVA and Duncan's test ( P
    Qrt Pcr Mixture, supplied by TaKaRa, used in various techniques. Bioz Stars score: 98/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PTEN is a direct target gene of miR-130b. ( A ) PTEN was detected by qRT- PCR in 29 BC tissues and was matched the adjacent noncancerous breast tissues. ( B ) The relationship between PTEN and miR-130b expression was explored by Spearman’s correlation in 29 paired BC tissues and adjacent noncancerous breast tissues. PTEN was inversely correlated with the miR-130b levels in BC tissues. ( C ) Relative PTEN expression in MCF-7 and MCF-7/ADR cells were detected by qRT-PCR (left panel) and western blot (right panel). ( D ) Effects of miR-130b mimics in MCF-7 cells and miR-130b inhibitor in MCF-7/ADR cells on the relative expression levels of PTEN were detected by qRT- PCR (left panel) and western blot (right panel). ( E ) PTEN expression was detected by immunofluorescence staining in these cells. Red fluorescence: PTEN, blue fluorescence: DAPI, staining for nuclear DNA. There were no significant differences between the control group and the blank group. ( F ) The relative luciferase activity of 293 T cells was detected after the pGL3-control vector, wt or mut PTEN 3′UTR genes were co-transfected with miR-NC or miR-130b mimics (*P

    Journal: Scientific Reports

    Article Title: MicroRNA-130b targets PTEN to mediate drug resistance and proliferation of breast cancer cells via the PI3K/Akt signaling pathway

    doi: 10.1038/srep41942

    Figure Lengend Snippet: PTEN is a direct target gene of miR-130b. ( A ) PTEN was detected by qRT- PCR in 29 BC tissues and was matched the adjacent noncancerous breast tissues. ( B ) The relationship between PTEN and miR-130b expression was explored by Spearman’s correlation in 29 paired BC tissues and adjacent noncancerous breast tissues. PTEN was inversely correlated with the miR-130b levels in BC tissues. ( C ) Relative PTEN expression in MCF-7 and MCF-7/ADR cells were detected by qRT-PCR (left panel) and western blot (right panel). ( D ) Effects of miR-130b mimics in MCF-7 cells and miR-130b inhibitor in MCF-7/ADR cells on the relative expression levels of PTEN were detected by qRT- PCR (left panel) and western blot (right panel). ( E ) PTEN expression was detected by immunofluorescence staining in these cells. Red fluorescence: PTEN, blue fluorescence: DAPI, staining for nuclear DNA. There were no significant differences between the control group and the blank group. ( F ) The relative luciferase activity of 293 T cells was detected after the pGL3-control vector, wt or mut PTEN 3′UTR genes were co-transfected with miR-NC or miR-130b mimics (*P

    Article Snippet: The qRT-PCR for the analysis of PTEN mRNA expression was performed using the SYBR Green qRT-PCR master mix (TaKaRa, Otsu, Shiga, Japan) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control.

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence, Luciferase, Activity Assay, Plasmid Preparation, Transfection

    Genotyping of Arabidopsis thaliana plants from GABI_462A04 line (a), SAIL_547_C10 line (b) and SALK_022236 line (c) with Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–12 and 1–4 - individual plants from each T-DNA line. Molecular weight DNA standards are shown on the left. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.

    Journal: BioNanoScience

    Article Title: Selection of efficient Taq DNA polymerase to optimize T-DNA genotyping method for rapid detection of mutant Arabidopsis thaliana plants

    doi: 10.1007/s12668-016-0253-6

    Figure Lengend Snippet: Genotyping of Arabidopsis thaliana plants from GABI_462A04 line (a), SAIL_547_C10 line (b) and SALK_022236 line (c) with Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–12 and 1–4 - individual plants from each T-DNA line. Molecular weight DNA standards are shown on the left. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.

    Article Snippet: The following DNA polymerases were tested: Phusion High-Fidelity DNA polymerase (ThermoFisher Scientific, USA, cat #F530S), Ex-Taq DNA polymerase (Clontech, USA, cat #RR001A), Taq DNA polymerase (SibEnzyme, Russia, cat #E331), Emerald Amp GT PCR Master Mix (Clontech, USA, cat #RR310A).

    Techniques: Polymerase Chain Reaction, Positive Control, Molecular Weight, Mutagenesis

    Genotyping of Arabidopsis thaliana plants from SALK_100012 line with (a) Phusion High-Fidelity DNA polymerase; (b) Ex-Taq DNA polymerase; (c) Taq DNA polymerase; (d) Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–8 - individual plants from SALK_100012 line. Molecular weight DNA standards are shown on each side. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.

    Journal: BioNanoScience

    Article Title: Selection of efficient Taq DNA polymerase to optimize T-DNA genotyping method for rapid detection of mutant Arabidopsis thaliana plants

    doi: 10.1007/s12668-016-0253-6

    Figure Lengend Snippet: Genotyping of Arabidopsis thaliana plants from SALK_100012 line with (a) Phusion High-Fidelity DNA polymerase; (b) Ex-Taq DNA polymerase; (c) Taq DNA polymerase; (d) Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–8 - individual plants from SALK_100012 line. Molecular weight DNA standards are shown on each side. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.

    Article Snippet: The following DNA polymerases were tested: Phusion High-Fidelity DNA polymerase (ThermoFisher Scientific, USA, cat #F530S), Ex-Taq DNA polymerase (Clontech, USA, cat #RR001A), Taq DNA polymerase (SibEnzyme, Russia, cat #E331), Emerald Amp GT PCR Master Mix (Clontech, USA, cat #RR310A).

    Techniques: Polymerase Chain Reaction, Positive Control, Molecular Weight, Mutagenesis

    cDNA sequence of mRNA de novo transcribed from TNFSF9 in HepG2 clone 9-1 (A) HepG2 clone 9-1 cells were incubated with 500 ng/ml LPS for the indicated times. cDNA was synthesized from total RNA, and TNFSF9 expression was quantified by real-time PCR as described in “Materials and Methods”. (B) HepG2 cells were incubated with 500 ng/ml LPS in the presence of 5-ethynyl uridine (EU) for 16 hrs. EU-labeled RNA was isolated as described in “Materials and Methods”. TNFSF9 cDNA was amplified by PCR and the PCR products were isolated and sequenced. This experiment is representative of four independent experiments.

    Journal: Molecules and Cells

    Article Title: Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript

    doi: 10.14348/molcells.2018.0209

    Figure Lengend Snippet: cDNA sequence of mRNA de novo transcribed from TNFSF9 in HepG2 clone 9-1 (A) HepG2 clone 9-1 cells were incubated with 500 ng/ml LPS for the indicated times. cDNA was synthesized from total RNA, and TNFSF9 expression was quantified by real-time PCR as described in “Materials and Methods”. (B) HepG2 cells were incubated with 500 ng/ml LPS in the presence of 5-ethynyl uridine (EU) for 16 hrs. EU-labeled RNA was isolated as described in “Materials and Methods”. TNFSF9 cDNA was amplified by PCR and the PCR products were isolated and sequenced. This experiment is representative of four independent experiments.

    Article Snippet: Real-Time PCR amplification of TNFSF9 cDNA Real-time PCR analysis (MiniOpticon, Bio Rad, USA) was performed using PCR Master Mix (Takara SYBR® PreMix Ex Taq II) to quantify expression of TNFSF9 mRNA (normalized to β-actin expression).

    Techniques: Sequencing, Incubation, Synthesized, Expressing, Real-time Polymerase Chain Reaction, Labeling, Isolation, Amplification, Polymerase Chain Reaction

    DNA sequence of genomic TNFSF9 gene edited by CRISPR-Cas9 The TNFSF9 genes in genomic DNAs from wild type and mutated HepG2 cells were amplified by PCR. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the genomic TNFSF9 sequence. On the right are shown the DNA sequences around the region of the mutated triplet in the wildtype and mutant clones. This is one representative of five independent experiments.

    Journal: Molecules and Cells

    Article Title: Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript

    doi: 10.14348/molcells.2018.0209

    Figure Lengend Snippet: DNA sequence of genomic TNFSF9 gene edited by CRISPR-Cas9 The TNFSF9 genes in genomic DNAs from wild type and mutated HepG2 cells were amplified by PCR. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the genomic TNFSF9 sequence. On the right are shown the DNA sequences around the region of the mutated triplet in the wildtype and mutant clones. This is one representative of five independent experiments.

    Article Snippet: Real-Time PCR amplification of TNFSF9 cDNA Real-time PCR analysis (MiniOpticon, Bio Rad, USA) was performed using PCR Master Mix (Takara SYBR® PreMix Ex Taq II) to quantify expression of TNFSF9 mRNA (normalized to β-actin expression).

    Techniques: Sequencing, CRISPR, Amplification, Polymerase Chain Reaction, Isolation, Mutagenesis, Clone Assay

    Heteroduplex formation by the cDNA of de novo transcripts of TNFSF9 from HepG2 clone 9-1 Total RNA and EU-labeled RNAs were isolated from clone 9-1 HepG2 cells. cDNA was synthesized and the TNFSF9 cDNA was amplified by PCR as described in “Materials and Methods”. The PCR products were annealed and incubated in the presence or absence of T7E1 endonuclease, and the products were fractionated on 1.2% agarose gels and visualized under UV. This is one representative of three independent experiments.

    Journal: Molecules and Cells

    Article Title: Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript

    doi: 10.14348/molcells.2018.0209

    Figure Lengend Snippet: Heteroduplex formation by the cDNA of de novo transcripts of TNFSF9 from HepG2 clone 9-1 Total RNA and EU-labeled RNAs were isolated from clone 9-1 HepG2 cells. cDNA was synthesized and the TNFSF9 cDNA was amplified by PCR as described in “Materials and Methods”. The PCR products were annealed and incubated in the presence or absence of T7E1 endonuclease, and the products were fractionated on 1.2% agarose gels and visualized under UV. This is one representative of three independent experiments.

    Article Snippet: Real-Time PCR amplification of TNFSF9 cDNA Real-time PCR analysis (MiniOpticon, Bio Rad, USA) was performed using PCR Master Mix (Takara SYBR® PreMix Ex Taq II) to quantify expression of TNFSF9 mRNA (normalized to β-actin expression).

    Techniques: Labeling, Isolation, Synthesized, Amplification, Polymerase Chain Reaction, Incubation

    cDNA sequence of mRNA transcribed from mutant genomic TNFSF9 TNFSF9 cDNA was synthesized and amplified by PCR from total RNA extracted from wild type and mutated HepG2 cells. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the TNFSF9 cDNA sequences. On the right are shown the cDNA sequences around the region of the mutated triplet in the wild type and mutant clones. This is one representative of four independent experiments.

    Journal: Molecules and Cells

    Article Title: Editing of Genomic TNFSF9 by CRISPR-Cas9 Can Be Followed by Re-Editing of Its Transcript

    doi: 10.14348/molcells.2018.0209

    Figure Lengend Snippet: cDNA sequence of mRNA transcribed from mutant genomic TNFSF9 TNFSF9 cDNA was synthesized and amplified by PCR from total RNA extracted from wild type and mutated HepG2 cells. The PCR products were isolated and sequenced as described in “Materials and Methods”. Left panels are chromatograms of the TNFSF9 cDNA sequences. On the right are shown the cDNA sequences around the region of the mutated triplet in the wild type and mutant clones. This is one representative of four independent experiments.

    Article Snippet: Real-Time PCR amplification of TNFSF9 cDNA Real-time PCR analysis (MiniOpticon, Bio Rad, USA) was performed using PCR Master Mix (Takara SYBR® PreMix Ex Taq II) to quantify expression of TNFSF9 mRNA (normalized to β-actin expression).

    Techniques: Sequencing, Mutagenesis, Synthesized, Amplification, Polymerase Chain Reaction, Isolation, Clone Assay

    Overexpression of PpGLK1 in u/u tomato. (A) qRT-PCR and western blot analysis confirming PpGLK1 overexpression in u/u tomato. (B) Phenotype of PpGLK1 -overexpressing u/u tomato. (C) Chlorophyll contents in u/u tomato and PpGLK1 -overexpressing u/u tomato. (D) Chloroplast development in fruit observed by TEM in PpGLK1 -overexpressing u/u tomato fruit and u/u tomato fruit. Data is presented as the means ± SD, n = 3. Different letters above bars denote statistical significance according to one-way ANOVA and Duncan's test ( P

    Journal: Frontiers in Plant Science

    Article Title: Transcriptomic and Functional Analyses Reveal That PpGLK1 Regulates Chloroplast Development in Peach (Prunus persica)

    doi: 10.3389/fpls.2018.00034

    Figure Lengend Snippet: Overexpression of PpGLK1 in u/u tomato. (A) qRT-PCR and western blot analysis confirming PpGLK1 overexpression in u/u tomato. (B) Phenotype of PpGLK1 -overexpressing u/u tomato. (C) Chlorophyll contents in u/u tomato and PpGLK1 -overexpressing u/u tomato. (D) Chloroplast development in fruit observed by TEM in PpGLK1 -overexpressing u/u tomato fruit and u/u tomato fruit. Data is presented as the means ± SD, n = 3. Different letters above bars denote statistical significance according to one-way ANOVA and Duncan's test ( P

    Article Snippet: The qRT-PCR mixture consisted of 10 μL SYBR Premix Ex Taq (TaKaRa Biotechnology, Dalian, China), 1 μL cDNA, and 1 μL each primer pair.

    Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Transmission Electron Microscopy