Structured Review

TaKaRa hot start dna polymerase
GPX3 expression and methylation in THP1 cell line before and after 5-aza-dC treatment. A: GPX3 relative expression levels. B: Electrophoresis results of <t>RQ-PCR</t> and RQ-MSP products. 1: Gene Ruler TM 100 bp <t>DNA</t> ladder; 2: 0 μM; 3: 0.1 μM;
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Images

1) Product Images from "GPX3 hypermethylation serves as an independent prognostic biomarker in non-M3 acute myeloid leukemia"

Article Title: GPX3 hypermethylation serves as an independent prognostic biomarker in non-M3 acute myeloid leukemia

Journal: American Journal of Cancer Research

doi:

GPX3 expression and methylation in THP1 cell line before and after 5-aza-dC treatment. A: GPX3 relative expression levels. B: Electrophoresis results of RQ-PCR and RQ-MSP products. 1: Gene Ruler TM 100 bp DNA ladder; 2: 0 μM; 3: 0.1 μM;
Figure Legend Snippet: GPX3 expression and methylation in THP1 cell line before and after 5-aza-dC treatment. A: GPX3 relative expression levels. B: Electrophoresis results of RQ-PCR and RQ-MSP products. 1: Gene Ruler TM 100 bp DNA ladder; 2: 0 μM; 3: 0.1 μM;

Techniques Used: Expressing, Methylation, Electrophoresis, Polymerase Chain Reaction

Electrophoresis results of RQ-PCR and RQ-MSP products in normal controls and AML patients. 1: Gene Ruler TM 100 bp DNA ladder; 2, 3: controls; 4-7: AML patients; 8: positive control; 9: negative control. A: GPX3 expression; B: GPX3 methylation; C: GPX3
Figure Legend Snippet: Electrophoresis results of RQ-PCR and RQ-MSP products in normal controls and AML patients. 1: Gene Ruler TM 100 bp DNA ladder; 2, 3: controls; 4-7: AML patients; 8: positive control; 9: negative control. A: GPX3 expression; B: GPX3 methylation; C: GPX3

Techniques Used: Electrophoresis, Polymerase Chain Reaction, Positive Control, Negative Control, Expressing, Methylation

2) Product Images from "Hypomethylation of MIR‐378 5’‐flanking region predicts poor survival in young patients with myelodysplastic syndrome, et al. Hypomethylation of MIR‐378 5’‐flanking region predicts poor survival in young patients with myelodysplastic syndrome"

Article Title: Hypomethylation of MIR‐378 5’‐flanking region predicts poor survival in young patients with myelodysplastic syndrome, et al. Hypomethylation of MIR‐378 5’‐flanking region predicts poor survival in young patients with myelodysplastic syndrome

Journal: Molecular Genetics & Genomic Medicine

doi: 10.1002/mgg3.1067

Electrophoresis results of RQ‐MSP products in MDS patients. (a) MIR‐378 methylation; (b) MIR‐378 unmethylation; (c) ALU . 1: Gene Ruler ™ 100bp DNA ladder; 2–3: normal controls; 4–10: MDS samples; 11: cloned plasmid; 12: negative control. MDS, myelodysplastic syndrome; RQ‐MSP, real‐time quantitative methylation‐specific PCR
Figure Legend Snippet: Electrophoresis results of RQ‐MSP products in MDS patients. (a) MIR‐378 methylation; (b) MIR‐378 unmethylation; (c) ALU . 1: Gene Ruler ™ 100bp DNA ladder; 2–3: normal controls; 4–10: MDS samples; 11: cloned plasmid; 12: negative control. MDS, myelodysplastic syndrome; RQ‐MSP, real‐time quantitative methylation‐specific PCR

Techniques Used: Electrophoresis, Methylation, Clone Assay, Plasmid Preparation, Negative Control, Polymerase Chain Reaction

Related Articles

Methylation Sequencing:

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: Paragraph title: Bisulfite sequencing ... Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction.

Clone Assay:

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. The purified fragments were cloned into pMD18-T Vectors (TaKaRa) and subjected to sequence analysis.

Centrifugation:

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: For semen, the sperm was collected by centrifugation, washed in SMB solution (10 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl and 2% SDS) and incubated in SMB solution supplemented with 40 mM dithiothreitol and 0.3 mg/ml PK at 56 °C for 1 h. For samples of 104 PFFs, 800 MII oocytes and 200, 100, 200, 25 and 25 pooled zona pellucida-removed embryos at the 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages, respectively, in each group, digestion was performed in M-Digestion Buffer supplemented with PK at 50 °C for 20 min. After digestion, a CT (cytosine to thymine) conversion reagent was added at 98 °C for 10 min and 64 °C for 2.5 h. Then, the samples were desalted, purified and diluted with M-Elution Buffer. .. Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction.

Amplification:

Article Title: Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes
Article Snippet: PCR reactions were carried out in 10-μl reaction mixtures containing a 0.5 μM concentration of each primer, 0.2 mM dNTPs, 0.25U Ex Taq DNA Polymerase Hot-Start Version, 1.0 μl 10×Ex Taq Buffer (Takara Bio, Shiga, Japan), and 1 μl of extracted DNA. .. The amplification conditions were 1 cycle at 96°C for 5 min of denaturation, 40 cycles of 94°C for 30 s, 55-68°C for 45 s of annealing in proportion to the Tm value of each primer, and extension at 72°C for 45 s, followed by a final extension at 72°C for 10 min. PCR products were purified by using a MultiScreenHTS PCR 96-Well Plate (Millipore, Billerica, Massachusetts, US) for sequences.

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA. .. After PCR amplification, 3 µL of products were digested with three units of restriction enzyme.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA. .. After PCR amplification, 3 μL of amplified products was digested with 3 units of restriction enzyme.

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: .. Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. Then, the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa).

Article Title: Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing
Article Snippet: PCR reactions contained 16.25 μ L Molecular Biology Grade Water (Fisher BP2819-1), 0.25 μ L TaKaRa Hot Start DNA Polymerase (Clontech TaKaRa R007A) (5 units/ μ L), 2.5 μ L 10× Buffer with MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2 ), 2.0 μ L deoxynucleotide triphosphate (2.5 mM each), 1.0 μ L Primers 515F and 806R working solutions (0.5 μ M final concentration), and 2.0 μ L of total genomic DNA. .. The DNA was denatured for at 98°C for 3 minutes, followed by 35 amplification cycles of 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 60 seconds, and a final extension step of 72°C for 10 minutes.

Article Title: Genomic Variations in Probiotic Lactobacillus plantarum P-8 in the Human and Rat Gut
Article Snippet: Paragraph title: DNA Extraction From Feces and Quantitative PCR Amplification ... The reaction mixture (20 μL) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 μM of each dNTP, 500 μg/mL bovine serum albumin (Takara, Dalian, China), a 1:75 000 dilution of SYBR Green I (Takara), 0.4 U Taq DNA polymerase Hot Start version (Takara), 0.2 μM of the specific primers and 2 μL template DNA.

Article Title: Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice 1 Affects Leaf Angle and Seed Size in Rice 1 [OPEN]
Article Snippet: PCR was performed using Hot-Start Taq DNA polymerase (DR007B; Takara Bio). .. Melting curves were read at the end of each amplification by steps of 0.3°C from 65°C to 95°C to ensure that the quantifications were derived from real PCR products and not primer dimers.

Enzyme-linked Immunosorbent Assay:

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: DNA Methylation Analysis The global levels of genomic DNA methylation were evaluated using the Global DNA Methylation ELISA Kit (Cell Biolabs) according to the manufacturer’s recommendations. .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA.

SYBR Green Assay:

Article Title: Genomic Variations in Probiotic Lactobacillus plantarum P-8 in the Human and Rat Gut
Article Snippet: .. The reaction mixture (20 μL) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 μM of each dNTP, 500 μg/mL bovine serum albumin (Takara, Dalian, China), a 1:75 000 dilution of SYBR Green I (Takara), 0.4 U Taq DNA polymerase Hot Start version (Takara), 0.2 μM of the specific primers and 2 μL template DNA. .. Isolation of P-8 From Feces and DNA Extraction From Isolated P-8 Colonies After diluting with PBS, the samples were plated and incubated on vancomycin and cycloheximide containing MRS agar under anaerobic conditions for 48 h. To confirm the identity of the P-8 colonies on MRS agar, colony PCR was performed using the strain-specific primers (also used in qPCR).

Article Title: Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice 1 Affects Leaf Angle and Seed Size in Rice 1 [OPEN]
Article Snippet: Real-time PCR analysis was performed using the CFX96 Real-Time PCR System (Bio-Rad) and SYBR Green I (S-7567; Invitrogen). .. PCR was performed using Hot-Start Taq DNA polymerase (DR007B; Takara Bio).

Incubation:

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: For semen, the sperm was collected by centrifugation, washed in SMB solution (10 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl and 2% SDS) and incubated in SMB solution supplemented with 40 mM dithiothreitol and 0.3 mg/ml PK at 56 °C for 1 h. For samples of 104 PFFs, 800 MII oocytes and 200, 100, 200, 25 and 25 pooled zona pellucida-removed embryos at the 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages, respectively, in each group, digestion was performed in M-Digestion Buffer supplemented with PK at 50 °C for 20 min. After digestion, a CT (cytosine to thymine) conversion reagent was added at 98 °C for 10 min and 64 °C for 2.5 h. Then, the samples were desalted, purified and diluted with M-Elution Buffer. .. Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction.

In Silico:

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: An in silico analysis using the UCSC Genome Bioinformatics tool ( http://genome.ucsc.edu ) was performed to identify CpG sites located upstream of the MEG3 transcription start site. .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: An in silico analysis using the UCSC Genome Bioinformatics Site ( http://genome.ucsc.edu ) was performed to identify the CpG sites associated with the transcription start site and polymerase elongation region for each gene ( ). .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA.

Modification:

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: Briefly, 1 µg of genomic DNA was subjected to bisulfite modification treatment using an EpiTect Plus kit (QIAGEN). .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: In brief, 1 μg of genomic DNA was subjected to bisulfite modification treatment using the EpiTect Plus kit (QIAGEN). .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA.

Real-time Polymerase Chain Reaction:

Article Title: Genomic Variations in Probiotic Lactobacillus plantarum P-8 in the Human and Rat Gut
Article Snippet: Paragraph title: DNA Extraction From Feces and Quantitative PCR Amplification ... The reaction mixture (20 μL) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 μM of each dNTP, 500 μg/mL bovine serum albumin (Takara, Dalian, China), a 1:75 000 dilution of SYBR Green I (Takara), 0.4 U Taq DNA polymerase Hot Start version (Takara), 0.2 μM of the specific primers and 2 μL template DNA.

Article Title: Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice 1 Affects Leaf Angle and Seed Size in Rice 1 [OPEN]
Article Snippet: Paragraph title: Reverse Transcription-PCR and Real-Time PCR ... PCR was performed using Hot-Start Taq DNA polymerase (DR007B; Takara Bio).

Article Title: A single-cell assay for telomere DNA content shows increasing telomere length heterogeneity, as well as increasing mean telomere length in human spermatozoa with advancing age
Article Snippet: A key feature of this assay is a telomere pre-amplification step, performed before quantitative polymerase chain reaction (qPCR). .. In brief, pre-PCR was performed using DNA Polymerase Hot Start Version (TAKARA).

Derivative Assay:

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: Genomic DNA from 2 lines of ESCs derived from C57BL/6J mice (Clea Japan) was extracted using the DNeasy Blood & Tissue Kit (Qiagen). .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final).

Article Title: Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice 1 Affects Leaf Angle and Seed Size in Rice 1 [OPEN]
Article Snippet: PCR was performed using Hot-Start Taq DNA polymerase (DR007B; Takara Bio). .. Melting curves were read at the end of each amplification by steps of 0.3°C from 65°C to 95°C to ensure that the quantifications were derived from real PCR products and not primer dimers.

Sequencing:

Article Title: Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes
Article Snippet: Mutation validation Validating the candidate SNVs was performed by using the standard Sanger sequencing approach. .. PCR reactions were carried out in 10-μl reaction mixtures containing a 0.5 μM concentration of each primer, 0.2 mM dNTPs, 0.25U Ex Taq DNA Polymerase Hot-Start Version, 1.0 μl 10×Ex Taq Buffer (Takara Bio, Shiga, Japan), and 1 μl of extracted DNA.

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. The purified fragments were cloned into pMD18-T Vectors (TaKaRa) and subjected to sequence analysis.

Article Title: Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing
Article Snippet: Paragraph title: Library preparation and high-throughput sequencing ... PCR reactions contained 16.25 μ L Molecular Biology Grade Water (Fisher BP2819-1), 0.25 μ L TaKaRa Hot Start DNA Polymerase (Clontech TaKaRa R007A) (5 units/ μ L), 2.5 μ L 10× Buffer with MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2 ), 2.0 μ L deoxynucleotide triphosphate (2.5 mM each), 1.0 μ L Primers 515F and 806R working solutions (0.5 μ M final concentration), and 2.0 μ L of total genomic DNA.

ChIP-sequencing:

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: Cytosine-methylated adapters (Illumina, San Diego, CA) were ligated to DNA by using the Paired-End DNA Sample Prep Kit or ChIP-Seq DNA Sample Prep Kit (Illumina). .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final).

DNA Extraction:

Article Title: Genomic Variations in Probiotic Lactobacillus plantarum P-8 in the Human and Rat Gut
Article Snippet: Paragraph title: DNA Extraction From Feces and Quantitative PCR Amplification ... The reaction mixture (20 μL) contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 μM of each dNTP, 500 μg/mL bovine serum albumin (Takara, Dalian, China), a 1:75 000 dilution of SYBR Green I (Takara), 0.4 U Taq DNA polymerase Hot Start version (Takara), 0.2 μM of the specific primers and 2 μL template DNA.

Combined Bisulfite Restriction Analysis Assay:

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA. .. After PCR amplification, 3 µL of products were digested with three units of restriction enzyme.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA. .. After PCR amplification, 3 μL of amplified products was digested with 3 units of restriction enzyme.

Methylation:

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: DNA methylation analysis Combined bisulfite restriction analysis (COBRA) was used to assess the methylation status of MEG3 -DMR. .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: COBRA was used to assess the methylation status of the specific CpG sites located in the promoter regions of ALB , SLC10A1 , CYP3A4 , and miR-122 . .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA.

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: Briefly, pooled samples were digested with Proteinase K (PK) and treated with sodium bisulfite to convert all unmethylated cytosine to uracil using an EZ DNA Methylation-Direct Kit (Zymo Research). .. Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction.

Mutagenesis:

Article Title: Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes
Article Snippet: Paragraph title: Mutation validation ... PCR reactions were carried out in 10-μl reaction mixtures containing a 0.5 μM concentration of each primer, 0.2 mM dNTPs, 0.25U Ex Taq DNA Polymerase Hot-Start Version, 1.0 μl 10×Ex Taq Buffer (Takara Bio, Shiga, Japan), and 1 μl of extracted DNA.

Isolation:

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: DNA fragments were isolated by 2–3% agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen). .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final).

Size-exclusion Chromatography:

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: .. Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. Then, the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa).

Purification:

Article Title: Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes
Article Snippet: PCR reactions were carried out in 10-μl reaction mixtures containing a 0.5 μM concentration of each primer, 0.2 mM dNTPs, 0.25U Ex Taq DNA Polymerase Hot-Start Version, 1.0 μl 10×Ex Taq Buffer (Takara Bio, Shiga, Japan), and 1 μl of extracted DNA. .. The amplification conditions were 1 cycle at 96°C for 5 min of denaturation, 40 cycles of 94°C for 30 s, 55-68°C for 45 s of annealing in proportion to the Tm value of each primer, and extension at 72°C for 45 s, followed by a final extension at 72°C for 10 min. PCR products were purified by using a MultiScreenHTS PCR 96-Well Plate (Millipore, Billerica, Massachusetts, US) for sequences.

Article Title: From Pig Breeding Environment to Subsequently Produced Pork: Comparative Analysis of Antibiotic Resistance Genes and Bacterial Community Composition
Article Snippet: .. The PCR was performed in a total reaction volume of 50 μL containing 1 μL of 50 ng/μL purified 16S rRNA genes, 5 μL of Taq reaction buffer, 0.2 mM dNTPs, 0.2 μM primers, and 1.25 units of Hot Start Taq DNA polymerase. .. A touchdown PCR was used to amplify the 16S rRNA gene V3-GC region to increase the specificity of the amplification.

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: DNA fragments were isolated by 2–3% agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen). .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final).

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: For semen, the sperm was collected by centrifugation, washed in SMB solution (10 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl and 2% SDS) and incubated in SMB solution supplemented with 40 mM dithiothreitol and 0.3 mg/ml PK at 56 °C for 1 h. For samples of 104 PFFs, 800 MII oocytes and 200, 100, 200, 25 and 25 pooled zona pellucida-removed embryos at the 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages, respectively, in each group, digestion was performed in M-Digestion Buffer supplemented with PK at 50 °C for 20 min. After digestion, a CT (cytosine to thymine) conversion reagent was added at 98 °C for 10 min and 64 °C for 2.5 h. Then, the samples were desalted, purified and diluted with M-Elution Buffer. .. Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction.

Article Title: Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing
Article Snippet: PCR reactions contained 16.25 μ L Molecular Biology Grade Water (Fisher BP2819-1), 0.25 μ L TaKaRa Hot Start DNA Polymerase (Clontech TaKaRa R007A) (5 units/ μ L), 2.5 μ L 10× Buffer with MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2 ), 2.0 μ L deoxynucleotide triphosphate (2.5 mM each), 1.0 μ L Primers 515F and 806R working solutions (0.5 μ M final concentration), and 2.0 μ L of total genomic DNA. .. Triplicate samples were pooled and purified using the MoBio UltraClean-htp 96 Well PCR Clean-Up Kit (MoBio Cat# 12596-4).

Polymerase Chain Reaction:

Article Title: Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes
Article Snippet: .. PCR reactions were carried out in 10-μl reaction mixtures containing a 0.5 μM concentration of each primer, 0.2 mM dNTPs, 0.25U Ex Taq DNA Polymerase Hot-Start Version, 1.0 μl 10×Ex Taq Buffer (Takara Bio, Shiga, Japan), and 1 μl of extracted DNA. .. The amplification conditions were 1 cycle at 96°C for 5 min of denaturation, 40 cycles of 94°C for 30 s, 55-68°C for 45 s of annealing in proportion to the Tm value of each primer, and extension at 72°C for 45 s, followed by a final extension at 72°C for 10 min. PCR products were purified by using a MultiScreenHTS PCR 96-Well Plate (Millipore, Billerica, Massachusetts, US) for sequences.

Article Title: From Pig Breeding Environment to Subsequently Produced Pork: Comparative Analysis of Antibiotic Resistance Genes and Bacterial Community Composition
Article Snippet: .. The PCR was performed in a total reaction volume of 50 μL containing 1 μL of 50 ng/μL purified 16S rRNA genes, 5 μL of Taq reaction buffer, 0.2 mM dNTPs, 0.2 μM primers, and 1.25 units of Hot Start Taq DNA polymerase. .. A touchdown PCR was used to amplify the 16S rRNA gene V3-GC region to increase the specificity of the amplification.

Article Title: Replicative mechanisms of CNV formation preferentially occur as intrachromosomal events: evidence from Potocki-Lupski duplication syndrome
Article Snippet: .. PCR amplifications were conducted with TaKaRa Hot start Taq polymerase. .. A 10 μl PCR reaction was performed with 0.1 μl of TaKaRa Hot start Taq polymerase with 1 μl of 10 × PCR buffer, 1.2 μl of dNTPs, 2 pmol of each primer and 10 ng DNA template.

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final). .. Thermocycling parameters were: initial denaturation at 94°C for 1 min, 15–25 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 30 s, followed by a final extension at 72°C for 5 min. PCR reaction products were purified using the QIAquick kit (Qiagen).

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA. .. After PCR amplification, 3 µL of products were digested with three units of restriction enzyme.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: .. Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA. .. After PCR amplification, 3 μL of amplified products was digested with 3 units of restriction enzyme.

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: .. Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. Then, the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa).

Article Title: Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing
Article Snippet: .. PCR reactions contained 16.25 μ L Molecular Biology Grade Water (Fisher BP2819-1), 0.25 μ L TaKaRa Hot Start DNA Polymerase (Clontech TaKaRa R007A) (5 units/ μ L), 2.5 μ L 10× Buffer with MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2 ), 2.0 μ L deoxynucleotide triphosphate (2.5 mM each), 1.0 μ L Primers 515F and 806R working solutions (0.5 μ M final concentration), and 2.0 μ L of total genomic DNA. .. The DNA was denatured for at 98°C for 3 minutes, followed by 35 amplification cycles of 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 60 seconds, and a final extension step of 72°C for 10 minutes.

Article Title: Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice 1 Affects Leaf Angle and Seed Size in Rice 1 [OPEN]
Article Snippet: .. PCR was performed using Hot-Start Taq DNA polymerase (DR007B; Takara Bio). .. Melting curves were read at the end of each amplification by steps of 0.3°C from 65°C to 95°C to ensure that the quantifications were derived from real PCR products and not primer dimers.

Article Title: A single-cell assay for telomere DNA content shows increasing telomere length heterogeneity, as well as increasing mean telomere length in human spermatozoa with advancing age
Article Snippet: In brief, pre-PCR was performed using DNA Polymerase Hot Start Version (TAKARA). .. The reactions were set up by aliquoting 38 μL of master mix into the PCR microtubes containing 2 μL sperm single-cell genomic DNA.

Polyacrylamide Gel Electrophoresis:

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA. .. Last, the restriction products were separated by polyacrylamide gel electrophoresis (PAGE) and visualized by ethidium bromide staining.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA. .. Finally, the restriction products were separated by 10% PAGE and visualized by ethidium bromide staining.

Staining:

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA. .. Last, the restriction products were separated by polyacrylamide gel electrophoresis (PAGE) and visualized by ethidium bromide staining.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA. .. Finally, the restriction products were separated by 10% PAGE and visualized by ethidium bromide staining.

Nested PCR:

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: .. Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. Then, the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa).

Agarose Gel Electrophoresis:

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: DNA fragments were isolated by 2–3% agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen). .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final).

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. Then, the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa).

Mouse Assay:

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: Genomic DNA from 2 lines of ESCs derived from C57BL/6J mice (Clea Japan) was extracted using the DNeasy Blood & Tissue Kit (Qiagen). .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final).

Software:

Article Title: Replicative mechanisms of CNV formation preferentially occur as intrachromosomal events: evidence from Potocki-Lupski duplication syndrome
Article Snippet: PCR amplifications were conducted with TaKaRa Hot start Taq polymerase. .. The PCR products were analyzed by capillary electrophoresis using an ABI 3130 genetic analyzer, and the STR alleles in each sample were genotyped using GeneMapper V3.2 software (Applied Biosystems, USA).

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA. .. The bands were densitometrically analyzed using the ImageJ software (v1.50i, National Institutes of Health, USA) to quantify the unmethylated (U) and methylated (M) restriction fragments.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA. .. The bands were densitometrically analyzed using the software ImageJ (v1.50; NIH, USA) to quantify the unmethylated (U) and methylated (M) restriction fragments.

Electrophoresis:

Article Title: Replicative mechanisms of CNV formation preferentially occur as intrachromosomal events: evidence from Potocki-Lupski duplication syndrome
Article Snippet: PCR amplifications were conducted with TaKaRa Hot start Taq polymerase. .. The PCR products were analyzed by capillary electrophoresis using an ABI 3130 genetic analyzer, and the STR alleles in each sample were genotyped using GeneMapper V3.2 software (Applied Biosystems, USA).

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. Then, the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa).

Sample Prep:

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: Cytosine-methylated adapters (Illumina, San Diego, CA) were ligated to DNA by using the Paired-End DNA Sample Prep Kit or ChIP-Seq DNA Sample Prep Kit (Illumina). .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final).

DNA Methylation Assay:

Article Title: MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells
Article Snippet: Paragraph title: DNA methylation analysis ... Then, COBRA PCR was performed as follows: after an initial denaturation step at 94 °C for 3 min, the following thermal cycles were repeated 40 times: 94 °C for 10 s, 55 °C for 50 s, and 72 °C for 1 min. Each COBRA PCR was performed in a total volume of 10 µL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 µL of bisulfite-treated DNA.

Article Title: Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells
Article Snippet: Paragraph title: DNA Methylation Analysis ... Then, COBRA PCR was performed as follows: after an initial denaturation step at 94°C for 3 min, the following thermal cycles were repeated 40 times: 94°C for 10 s, 55°C for 50 s, and 72°C for 1 min. Each COBRA PCR was performed in a total volume of 10 μL, which contained 0.5 units of Hot Start Taq polymerase (Takara), 10 pmol of primers, and 1 μL of bisulfite-treated DNA.

Concentration Assay:

Article Title: Exome sequencing of senescence-accelerated mice (SAM) reveals deleterious mutations in degenerative disease-causing genes
Article Snippet: .. PCR reactions were carried out in 10-μl reaction mixtures containing a 0.5 μM concentration of each primer, 0.2 mM dNTPs, 0.25U Ex Taq DNA Polymerase Hot-Start Version, 1.0 μl 10×Ex Taq Buffer (Takara Bio, Shiga, Japan), and 1 μl of extracted DNA. .. The amplification conditions were 1 cycle at 96°C for 5 min of denaturation, 40 cycles of 94°C for 30 s, 55-68°C for 45 s of annealing in proportion to the Tm value of each primer, and extension at 72°C for 45 s, followed by a final extension at 72°C for 10 min. PCR products were purified by using a MultiScreenHTS PCR 96-Well Plate (Millipore, Billerica, Massachusetts, US) for sequences.

Article Title: Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing
Article Snippet: .. PCR reactions contained 16.25 μ L Molecular Biology Grade Water (Fisher BP2819-1), 0.25 μ L TaKaRa Hot Start DNA Polymerase (Clontech TaKaRa R007A) (5 units/ μ L), 2.5 μ L 10× Buffer with MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2 ), 2.0 μ L deoxynucleotide triphosphate (2.5 mM each), 1.0 μ L Primers 515F and 806R working solutions (0.5 μ M final concentration), and 2.0 μ L of total genomic DNA. .. The DNA was denatured for at 98°C for 3 minutes, followed by 35 amplification cycles of 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 60 seconds, and a final extension step of 72°C for 10 minutes.

DNA Purification:

Article Title: Dnmt1s in donor cells is a barrier to SCNT-mediated DNA methylation reprogramming in pigs
Article Snippet: Subsequently, nested PCR was carried out to amplify the target regions of CenRep, Dnmt1, Oct4, Nanog and Sox2 using the primers described in and Hot Start Taq Polymerase (TaKaRa) with a profile of 94 °C for 5 min, 45 cycles of 94 °C for 30 sec, the optimal primer annealing temperature for 30 sec and 72 °C for 1 min, followed by 72 °C for 10 min. Products from the first amplification reaction were used in the second PCR reaction. .. Then, the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa).

High Throughput Screening Assay:

Article Title: Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing
Article Snippet: Paragraph title: Library preparation and high-throughput sequencing ... PCR reactions contained 16.25 μ L Molecular Biology Grade Water (Fisher BP2819-1), 0.25 μ L TaKaRa Hot Start DNA Polymerase (Clontech TaKaRa R007A) (5 units/ μ L), 2.5 μ L 10× Buffer with MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl2 ), 2.0 μ L deoxynucleotide triphosphate (2.5 mM each), 1.0 μ L Primers 515F and 806R working solutions (0.5 μ M final concentration), and 2.0 μ L of total genomic DNA.

Gel Extraction:

Article Title: Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks
Article Snippet: DNA fragments were isolated by 2–3% agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen). .. All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 µL 10× PCR buffer, 25 µM dNTPs, 1 µL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 µL final).

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  • 97
    TaKaRa pcr reaction mix
    <t>RT-PCR</t> confirms the ATP11B gene is alternatively spliced in rabbit endometrium. Lanes 1 and 3, Lambda <t>DNA/EcoR</t> I + Hind III markers (bp). Lane 2, the single. 939-bp product from Ishikawa cells. Lane 4, two populations of ATP11B amplicons from a single
    Pcr Reaction Mix, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr reaction mix/product/TaKaRa
    Average 97 stars, based on 52 article reviews
    Price from $9.99 to $1999.99
    pcr reaction mix - by Bioz Stars, 2020-04
    97/100 stars
      Buy from Supplier

    99
    TaKaRa hot start dna polymerase
    GPX3 expression and methylation in THP1 cell line before and after 5-aza-dC treatment. A: GPX3 relative expression levels. B: Electrophoresis results of <t>RQ-PCR</t> and RQ-MSP products. 1: Gene Ruler TM 100 bp <t>DNA</t> ladder; 2: 0 μM; 3: 0.1 μM;
    Hot Start Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hot start dna polymerase/product/TaKaRa
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hot start dna polymerase - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    86
    TaKaRa phire hot start dna polymerase
    Optimization of the UP-M-PCR. Lane A, B, C, D, E, amplicon fragments by UP (500 nmol L −1 ) and compound specific primer hpt-839, nptII-508, pat-262, bar-226 and sps-110 at a series concentrations of 500 nmol L −1 , 50 nmol L −1 , 25 nmol L −1 , 5 nmol L −1 , 0.5 nmol L −1 ; lane F1, amplicon fragments by UP at 500 nmol L −1 and all compound specific primers at 25 nmol L −1 ; lane F2, amplicon fragments by UP at 500 nmol L −1 and all compound specific primers at the optimized concentration; lane G1,G2,G3, amplicon fragments by all primers at the optimized concentration with TaKaRa Taq ™, <t>Phire™</t> Hot Start <t>DNA</t> polymerase, iProof™ High-Fidelity DNA polymerase; lane H1, amplicon fragments by all primers at the optimized concentration with Phire™ Hot Start DNA polymerase under the common amplification conditions; lane H2, amplicon fragments by all primers at the optimized concentration with Phire™ Hot Start DNA polymerase under the optimized amplification conditions; lane M, 100 bp DNA Marker.
    Phire Hot Start Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phire hot start dna polymerase/product/TaKaRa
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    phire hot start dna polymerase - by Bioz Stars, 2020-04
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    Image Search Results


    RT-PCR confirms the ATP11B gene is alternatively spliced in rabbit endometrium. Lanes 1 and 3, Lambda DNA/EcoR I + Hind III markers (bp). Lane 2, the single. 939-bp product from Ishikawa cells. Lane 4, two populations of ATP11B amplicons from a single

    Journal: Molecular and cellular endocrinology

    Article Title: Conservation of Inter -Protein Binding Sites in RUSH and RFBP, an ATP11B Isoform

    doi: 10.1016/j.mce.2008.05.007

    Figure Lengend Snippet: RT-PCR confirms the ATP11B gene is alternatively spliced in rabbit endometrium. Lanes 1 and 3, Lambda DNA/EcoR I + Hind III markers (bp). Lane 2, the single. 939-bp product from Ishikawa cells. Lane 4, two populations of ATP11B amplicons from a single

    Article Snippet: Hot start PCR reactions were performed ( ) such that each DNA sample was amplified in a 50 µl PCR reaction mix containing LA PCR buffer (1X), TaKaRa ExTaq DNA polymerase (2.5U/50 µl), TaqStart antibody (0.55 µg/50 µl), dNTPs (0.2 mM each) and primers (0.2 mM each).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Lambda DNA Preparation

    GPX3 expression and methylation in THP1 cell line before and after 5-aza-dC treatment. A: GPX3 relative expression levels. B: Electrophoresis results of RQ-PCR and RQ-MSP products. 1: Gene Ruler TM 100 bp DNA ladder; 2: 0 μM; 3: 0.1 μM;

    Journal: American Journal of Cancer Research

    Article Title: GPX3 hypermethylation serves as an independent prognostic biomarker in non-M3 acute myeloid leukemia

    doi:

    Figure Lengend Snippet: GPX3 expression and methylation in THP1 cell line before and after 5-aza-dC treatment. A: GPX3 relative expression levels. B: Electrophoresis results of RQ-PCR and RQ-MSP products. 1: Gene Ruler TM 100 bp DNA ladder; 2: 0 μM; 3: 0.1 μM;

    Article Snippet: Bisulfite sequencing PCR (BSP) reaction system contained 1 × PCR buffer (KCl 0.25 mM), dNTP Mixture 6.25 μM, primers 0.5 μM, hot start DNA polymerase 0.75 U (Takara, Tokyo, Japan), and modified DNA 20 ng.

    Techniques: Expressing, Methylation, Electrophoresis, Polymerase Chain Reaction

    Electrophoresis results of RQ-PCR and RQ-MSP products in normal controls and AML patients. 1: Gene Ruler TM 100 bp DNA ladder; 2, 3: controls; 4-7: AML patients; 8: positive control; 9: negative control. A: GPX3 expression; B: GPX3 methylation; C: GPX3

    Journal: American Journal of Cancer Research

    Article Title: GPX3 hypermethylation serves as an independent prognostic biomarker in non-M3 acute myeloid leukemia

    doi:

    Figure Lengend Snippet: Electrophoresis results of RQ-PCR and RQ-MSP products in normal controls and AML patients. 1: Gene Ruler TM 100 bp DNA ladder; 2, 3: controls; 4-7: AML patients; 8: positive control; 9: negative control. A: GPX3 expression; B: GPX3 methylation; C: GPX3

    Article Snippet: Bisulfite sequencing PCR (BSP) reaction system contained 1 × PCR buffer (KCl 0.25 mM), dNTP Mixture 6.25 μM, primers 0.5 μM, hot start DNA polymerase 0.75 U (Takara, Tokyo, Japan), and modified DNA 20 ng.

    Techniques: Electrophoresis, Polymerase Chain Reaction, Positive Control, Negative Control, Expressing, Methylation

    Electrophoresis results of RQ‐MSP products in MDS patients. (a) MIR‐378 methylation; (b) MIR‐378 unmethylation; (c) ALU . 1: Gene Ruler ™ 100bp DNA ladder; 2–3: normal controls; 4–10: MDS samples; 11: cloned plasmid; 12: negative control. MDS, myelodysplastic syndrome; RQ‐MSP, real‐time quantitative methylation‐specific PCR

    Journal: Molecular Genetics & Genomic Medicine

    Article Title: Hypomethylation of MIR‐378 5’‐flanking region predicts poor survival in young patients with myelodysplastic syndrome, et al. Hypomethylation of MIR‐378 5’‐flanking region predicts poor survival in young patients with myelodysplastic syndrome

    doi: 10.1002/mgg3.1067

    Figure Lengend Snippet: Electrophoresis results of RQ‐MSP products in MDS patients. (a) MIR‐378 methylation; (b) MIR‐378 unmethylation; (c) ALU . 1: Gene Ruler ™ 100bp DNA ladder; 2–3: normal controls; 4–10: MDS samples; 11: cloned plasmid; 12: negative control. MDS, myelodysplastic syndrome; RQ‐MSP, real‐time quantitative methylation‐specific PCR

    Article Snippet: The PCR was conducted with the reaction system containing 6.25 μM of dNTP mixture, 10× PCR buffer (0.25 mM KCl), 0.75 U of Hot start DNA polymerase (Takara), 0.5 μM of primers, and 20 ng of modified DNA on iCycler Thermal Cycler (Eppendorf).

    Techniques: Electrophoresis, Methylation, Clone Assay, Plasmid Preparation, Negative Control, Polymerase Chain Reaction

    Optimization of the UP-M-PCR. Lane A, B, C, D, E, amplicon fragments by UP (500 nmol L −1 ) and compound specific primer hpt-839, nptII-508, pat-262, bar-226 and sps-110 at a series concentrations of 500 nmol L −1 , 50 nmol L −1 , 25 nmol L −1 , 5 nmol L −1 , 0.5 nmol L −1 ; lane F1, amplicon fragments by UP at 500 nmol L −1 and all compound specific primers at 25 nmol L −1 ; lane F2, amplicon fragments by UP at 500 nmol L −1 and all compound specific primers at the optimized concentration; lane G1,G2,G3, amplicon fragments by all primers at the optimized concentration with TaKaRa Taq ™, Phire™ Hot Start DNA polymerase, iProof™ High-Fidelity DNA polymerase; lane H1, amplicon fragments by all primers at the optimized concentration with Phire™ Hot Start DNA polymerase under the common amplification conditions; lane H2, amplicon fragments by all primers at the optimized concentration with Phire™ Hot Start DNA polymerase under the optimized amplification conditions; lane M, 100 bp DNA Marker.

    Journal: PLoS ONE

    Article Title: A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

    doi: 10.1371/journal.pone.0022900

    Figure Lengend Snippet: Optimization of the UP-M-PCR. Lane A, B, C, D, E, amplicon fragments by UP (500 nmol L −1 ) and compound specific primer hpt-839, nptII-508, pat-262, bar-226 and sps-110 at a series concentrations of 500 nmol L −1 , 50 nmol L −1 , 25 nmol L −1 , 5 nmol L −1 , 0.5 nmol L −1 ; lane F1, amplicon fragments by UP at 500 nmol L −1 and all compound specific primers at 25 nmol L −1 ; lane F2, amplicon fragments by UP at 500 nmol L −1 and all compound specific primers at the optimized concentration; lane G1,G2,G3, amplicon fragments by all primers at the optimized concentration with TaKaRa Taq ™, Phire™ Hot Start DNA polymerase, iProof™ High-Fidelity DNA polymerase; lane H1, amplicon fragments by all primers at the optimized concentration with Phire™ Hot Start DNA polymerase under the common amplification conditions; lane H2, amplicon fragments by all primers at the optimized concentration with Phire™ Hot Start DNA polymerase under the optimized amplification conditions; lane M, 100 bp DNA Marker.

    Article Snippet: To test the efficiency of Taq Polymerase to be employed in PCR assays, comparative tests were made with several Taq polymerases, such as Phire™ Hot Start DNA polymerase, iProof™ High-Fidelity DNA polymerase, and TaKaRa Taq ™.

    Techniques: Polymerase Chain Reaction, Amplification, Concentration Assay, Marker