Structured Review

Biometra thermocycler
Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The <t>thermocycler</t> (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
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1) Product Images from "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism"

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

Journal: BMC Microbiology

doi: 10.1186/1471-2180-4-21

Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
Figure Legend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

Related Articles

Clone Assay:

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Once the four single stranded DNA (ssDNA) sequences were synthesized by the manufacturer, they were assembled as a single double-stranded DNA (dsDNA) fragment to be subsequently cloned into the PGEM-T plasmid (PGEM-T Easy Vector System I kit, Promega France SARL, Charbonnieres-les-Bains, France). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

Article Title: Molecular characterization of activated sludge from a seawater-processing wastewater treatment plant
Article Snippet: Reactions were carried out in an automated thermocycler (Biometra) with the following cycle: an initial denaturation step at 94°C for 5 min, followed by 30 cycles of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C, and a final extension step of 10 min at 72°C. .. PCR products were cloned with the TOPO TA cloning kit (Invitrogen) according to the manufacturer's instructions.

Amplification:

Article Title: Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity
Article Snippet: RNA was transcribed to complementary DNA (cDNA) with random primers (Promega, Madison, WI, USA), and SuperScript II Reverse Transcriptase (Invitrogen GmbH, Karlsruhe, Germany) on a thermocycler (Biometra, Göttingen, Germany). cDNA was subjected to qRT-PCR using the Power SYBRGreen PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA, USA). .. Amplification reaction data were analyzed by the complementary Mx3000P analysis software.

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: PCR amplification of each of the 4 single stranded DNA sequences by using primers consisting of the stabilization and the restriction site sequences (italics in ). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

Article Title: Contribution of BRCA1 5382insC mutation in triple negative breast cancer in Tunisia
Article Snippet: Exon 20 was amplified in 20 μl with 100 ng DNA, 1× reaction buffer, 200 μM dNTPs, 1.5 mM MgCl2 , 0.8 μM primers (designed by Centre Jean Perrin, sequences available on request) and 1 unit Taq polymerase (except primers, all other reagents from Promega, France). .. PCR was performed in an thermocycler (Biometra, Germany) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of (94 °C 20 s, 54 °C 20 s, 72 °C 20 s).

Article Title: RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors
Article Snippet: To screen for the RHD 1227A allele in all samples with RhC(+), the forward primer for RHD 1227A allele: 5′-GATGACCAAGTTTTCTGGAAA-3′, and the reverse primer for RHD 1227A: 5′-GTTCTGTCACCCGCATGTCAG-3′ were used in amplified a 348 bp product. .. Forty cycles were programmed on the thermocycler (TGradient, Biometra) as follows: denaturation at 94°C for 5 minutes, then 35 cycles of 30 seconds at 94°C, 40 seconds at 68°C, and 30 seconds at 72°C.

Article Title: Oxidative DNA damage in peripheral leukocytes and its association with expression and polymorphisms of hOGG1: A study of adolescents in a high risk region for hepatocellular carcinoma in China
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Article Title: Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR
Article Snippet: The two reverse primers (Peri1004R and Peri1403R) were paired with the eukaryote-specific primer EukA ( ) to amplify longer fragments of 18S rDNA, and short fragments were amplified with two forward primers (Peri974F and Peri979F) ( ). .. The PCR program was carried out on a thermocycler (Biometra, Göttingen, Germany), consisting of 94°C for 4 min followed by 35 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 2 min, with an additional 7 min at 72°C.

Article Title: Molecular characterization of activated sludge from a seawater-processing wastewater treatment plant
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Article Title: Mitochondrial and nuclear ribosomal DNA dataset supports that Paramphistomum leydeni (Trematoda: Digenea) is a distinct rumen fluke species
Article Snippet: The full mt genome of P. leydeni was amplified in 4 overlapping long fragments between cox 3 and atp 6 (approximately 3.5 kb), between atp 6 to cox 1 (approximately 4 kb), between cox 1 to rrn S (approximately 2.6 kb) and between rrn L to cox 3 (approximately 5.5 kb) (Table ). .. PCR reactions were conducted in a total volume of 50 μl using 4 mM MgCl2 , 0.4 mM each of dNTPs, 5 μl 10× LATaq buffer, 5 mM of each primer, 0.5 μl LA Taq DNA polymerase (Takara, Dalian, China) and 2 μl DNA templates in a thermocycler (Biometra, Göttingen, Germany).

Synthesized:

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Once the four single stranded DNA (ssDNA) sequences were synthesized by the manufacturer, they were assembled as a single double-stranded DNA (dsDNA) fragment to be subsequently cloned into the PGEM-T plasmid (PGEM-T Easy Vector System I kit, Promega France SARL, Charbonnieres-les-Bains, France). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

Article Title: Chronic Effects of Fusarium Mycotoxins in Rations with or without Increased Concentrate Proportion on the Insulin Sensitivity in Lactating Dairy Cows
Article Snippet: The mixture was incubated at 25 °C for 10 min., at 42 °C for 60 min. and at 99 °C for 5 min on a thermocycler (TProfessional Standard 96, Biometra GmbH, Göttingen, Germany). .. The synthesized complementary DNA (cDNA) was diluted with water at 1:1 or 1:3 and stored at −20 °C until analysis.

TA Cloning:

Article Title: Molecular characterization of activated sludge from a seawater-processing wastewater treatment plant
Article Snippet: Reactions were carried out in an automated thermocycler (Biometra) with the following cycle: an initial denaturation step at 94°C for 5 min, followed by 30 cycles of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C, and a final extension step of 10 min at 72°C. .. PCR products were cloned with the TOPO TA cloning kit (Invitrogen) according to the manufacturer's instructions.

Quantitative RT-PCR:

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Article Snippet: Paragraph title: QRT-PCR and RT-PCR ... For RT-PCR, the reaction was carried out with Taq DNA polymerase (D1806, Sigma) using a thermocycler (Biometra).

SYBR Green Assay:

Article Title: Gibberellin deficiency is responsible for shy-flowering nature of Epipremnum aureum
Article Snippet: For RT-PCR, the reaction was carried out with Taq DNA polymerase (D1806, Sigma) using a thermocycler (Biometra). .. For qRT-PCR, the Power SYBR Green PCR Master mix (4367659, Applied Biosystems) was used.

Incubation:

Article Title: RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors
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Article Title: Chronic Effects of Fusarium Mycotoxins in Rations with or without Increased Concentrate Proportion on the Insulin Sensitivity in Lactating Dairy Cows
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Article Title: Tolerance to Shock: An Exploration of Mechanism
Article Snippet: Oligodeoxythymidine primer (1 μl) was added to 4 μg RNA and DEPC water to make a total volume of 10 μl, and this was placed in the thermocycler (Biometra TRIO Thermoblock, Tampa, FL) at 70°C for 10 minutes, with a pause at 4°C. .. These Eppendorf tubes were then incubated in the thermocycler for 15 minutes at 25°C, 50 minutes at 42°C, and 15 minutes at 70°C, with a pause at 4°C.

In Silico:

Article Title: Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR
Article Snippet: Primer specificity was checked by submitting the sequences to probeCheck ( ) and evaluated against the GenBank database by in silico analyses using BLAST ( ). .. The PCR program was carried out on a thermocycler (Biometra, Göttingen, Germany), consisting of 94°C for 4 min followed by 35 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 2 min, with an additional 7 min at 72°C.

Modification:

Article Title: Oxidative DNA damage in peripheral leukocytes and its association with expression and polymorphisms of hOGG1: A study of adolescents in a high risk region for hepatocellular carcinoma in China
Article Snippet: PCR-SSCP analysis of hOGG1 Cys326Ser SNP was modified from a technique described by Kohno et al[ ]. .. PCR reaction on a thermocycler (Biometra TGradient, Göttingen, Germany) began with pre-incubation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and elongation at 72 °C for 30 s. PCR products were diluted with 4 volumes of loading buffer (0.5 × TBE, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM methylmercury hydroxide), heat-denatured at 85 °C for 5 min and rapidly cooled on ice before loading.

Cell Culture:

Article Title: Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity
Article Snippet: RNA isolation and quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) RNA from frozen monocyte pellets, cultured THP-1 monocytes/macrophages or from mouse adipose tissue was isolated using an RNAeasy Mini or Micro kit from Qiagen (Hilden, Germany). .. RNA was transcribed to complementary DNA (cDNA) with random primers (Promega, Madison, WI, USA), and SuperScript II Reverse Transcriptase (Invitrogen GmbH, Karlsruhe, Germany) on a thermocycler (Biometra, Göttingen, Germany). cDNA was subjected to qRT-PCR using the Power SYBRGreen PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA, USA).

Generated:

Article Title: Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development
Article Snippet: Droplets were generated using QX200 Droplet Generator (Bio-Rad Laboratories, Munich, Germany). .. PCR was performed in a thermocycler (Biometra, Göttingen, Germany) and cycling conditions were as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 30 sec and 60°C for 1 min.

Polymerase Chain Reaction:

Article Title: Two Novel Type II Restriction-Modification Systems Occupying Genomically Equivalent Locations on the Chromosomes of Listeria monocytogenes Strains
Article Snippet: .. PCR was carried out with Ex Taq polymerase (TaKaRa, Madison, WI) in a thermocycler (Biometra, Goettingen, Germany). .. The PCR was initiated at 95°C for 5 min and was followed by 31 cycles (each 95°C 1 min, 52°C 1 min, and 72°C 1 min), with a final extension at 72°C for 10 min. Primers employed in this study were purchased from Eurofins (Huntsville, AL) and are listed in .

Article Title: Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity
Article Snippet: .. RNA was transcribed to complementary DNA (cDNA) with random primers (Promega, Madison, WI, USA), and SuperScript II Reverse Transcriptase (Invitrogen GmbH, Karlsruhe, Germany) on a thermocycler (Biometra, Göttingen, Germany). cDNA was subjected to qRT-PCR using the Power SYBRGreen PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA, USA). ..

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

Article Title: Contribution of BRCA1 5382insC mutation in triple negative breast cancer in Tunisia
Article Snippet: .. PCR was performed in an thermocycler (Biometra, Germany) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of (94 °C 20 s, 54 °C 20 s, 72 °C 20 s). ..

Article Title: Mitochondrial DNA-depleted A549 cells are resistant to bleomycin
Article Snippet: .. PCR was performed using the GeneAmp XL PCR Kit (Roche, Applied Biosystems, Branchburg, NJ) on a thermocycler (T Gradient, Biometra, Goettingen, Germany). ..

Article Title: Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development
Article Snippet: .. PCR was performed in a thermocycler (Biometra, Göttingen, Germany) and cycling conditions were as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 30 sec and 60°C for 1 min. .. Final droplet stabilization was performed at 98°C for 10 min. Droplets were analyzed using the QX200 Droplet Reader (Bio-Rad Laboratories, Munich, Germany).

Article Title: Gibberellin deficiency is responsible for shy-flowering nature of Epipremnum aureum
Article Snippet: For RT-PCR, the reaction was carried out with Taq DNA polymerase (D1806, Sigma) using a thermocycler (Biometra). .. Each 25 μl PCR reaction contained cDNA made from the original 20 ng of RNA together with 1x PCR reaction buffer, 300 nM of each primer, 2 mM MgCl2 , 0.2 mM dNTP and 1.25U of Taq DNA polymerase.

Article Title: RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors
Article Snippet: The PCR reactions were performed at a total volume 10 μ L. It contained 1 μ L of genomic DNA, 0.5 U DNA polymerase, 200 μ M dNTPs, primers, and 2.5 mM MgCl2 in the buffer provided by the manufacturer. .. Forty cycles were programmed on the thermocycler (TGradient, Biometra) as follows: denaturation at 94°C for 5 minutes, then 35 cycles of 30 seconds at 94°C, 40 seconds at 68°C, and 30 seconds at 72°C.

Article Title: Metagenomic potential for and diversity of N‐cycle driving microorganisms in the Bothnian Sea sediment. Metagenomic potential for and diversity of N‐cycle driving microorganisms in the Bothnian Sea sediment
Article Snippet: .. PCR was performed in a thermocycler (Professional thermal cycler, Biometra, Jena) with following parameters: initial denaturation for 4 min at 94°C, followed by 30 cycles of denaturation for 1 min at 94°C, primer annealing for 1 min at 43–56°C (parallel PCR reactions were performed at different annealing temperatures), elongation for 2 min at 72°C, and final elongation for 10 min at 72°C. ..

Article Title: Oxidative DNA damage in peripheral leukocytes and its association with expression and polymorphisms of hOGG1: A study of adolescents in a high risk region for hepatocellular carcinoma in China
Article Snippet: .. PCR reaction on a thermocycler (Biometra TGradient, Göttingen, Germany) began with pre-incubation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and elongation at 72 °C for 30 s. PCR products were diluted with 4 volumes of loading buffer (0.5 × TBE, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM methylmercury hydroxide), heat-denatured at 85 °C for 5 min and rapidly cooled on ice before loading. ..

Article Title: Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR
Article Snippet: .. The PCR program was carried out on a thermocycler (Biometra, Göttingen, Germany), consisting of 94°C for 4 min followed by 35 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 2 min, with an additional 7 min at 72°C. .. Amplicons were checked and separated by electrophoresis in 1% agarose gel containing Gelview (BioTeke, Beijing, China) in 0.5×TAE buffer and visualized under UV light.

Article Title: Chronic Effects of Fusarium Mycotoxins in Rations with or without Increased Concentrate Proportion on the Insulin Sensitivity in Lactating Dairy Cows
Article Snippet: Samples containing 1 µg of DNase-treated RNA were added to the reaction-mix containing 2.5 unit MULV-reverse transcriptase (Applied Biosystems, Waltham, MA, USA), one unit of RNase inhibitor (Applied Biosystems, Waltham, MA, USA), 1 mM dNTPs (BioRad Laboratories, Inc., Hercules, CA, USA), 2.5 µM random hexamers (Applied Biosystems, Waltham, MA, USA), 5 mM MgCl2 (Invitrogen, Carlsbad, CA, USA), PCR buffer with 50 mM KCl and 10 mM Tris-HCl (pH 8.3; Applied Biosystems, Waltham, MA, USA), and water. .. The mixture was incubated at 25 °C for 10 min., at 42 °C for 60 min. and at 99 °C for 5 min on a thermocycler (TProfessional Standard 96, Biometra GmbH, Göttingen, Germany).

Article Title: Molecular characterization of activated sludge from a seawater-processing wastewater treatment plant
Article Snippet: Reactions were carried out in an automated thermocycler (Biometra) with the following cycle: an initial denaturation step at 94°C for 5 min, followed by 30 cycles of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C, and a final extension step of 10 min at 72°C. .. The cycle was as follows: 5 min at 94°C, 38 cycles consisting of primer annealing at 52°C for 1 min, DNA elongation at 72°C for 90 s and denaturation at 94°C for 1 min, and a final cycle of 52°C for 1 min and 72°C for 6 min. PCR mixtures contained 1–10 ng of template DNA, each deoxynucleoside triphosphate at a concentration of 200 µM, 1.5 mM MgCl2 , each primer at a concentration of 0.3 µM, 2.5 U Taq DNA polymerase (Invitrogen) and PCR buffer supplied by the manufacturer.

Article Title: Mitochondrial and nuclear ribosomal DNA dataset supports that Paramphistomum leydeni (Trematoda: Digenea) is a distinct rumen fluke species
Article Snippet: .. PCR reactions were conducted in a total volume of 50 μl using 4 mM MgCl2 , 0.4 mM each of dNTPs, 5 μl 10× LATaq buffer, 5 mM of each primer, 0.5 μl LA Taq DNA polymerase (Takara, Dalian, China) and 2 μl DNA templates in a thermocycler (Biometra, Göttingen, Germany). .. The PCR cycling conditions began with an initial denaturation at 92°C for 2 min, then 12 cycles of denaturation at 92°C for 20 s, annealing at 55–62°C for 30 s and extension at 60°C for 3–5 min, followed by 92°C denaturation for 2 min, plus 28 cycles of 92°C for 20 s (denaturation), 55–62°C for 30 s (annealing) and 66°C for 3–5 min, with 10 min of the final extension at 66°C.

DNA Sequencing:

Article Title: Contribution of BRCA1 5382insC mutation in triple negative breast cancer in Tunisia
Article Snippet: BRCA1 5382inC mutation detection As for BRCA1 5382inC mutation detection, the exon 20 coding region and exon–intron boundaries of BRCA1 was analyzed using direct DNA sequencing. .. PCR was performed in an thermocycler (Biometra, Germany) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of (94 °C 20 s, 54 °C 20 s, 72 °C 20 s).

Sequencing:

Article Title: Contribution of BRCA1 5382insC mutation in triple negative breast cancer in Tunisia
Article Snippet: PCR was performed in an thermocycler (Biometra, Germany) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of (94 °C 20 s, 54 °C 20 s, 72 °C 20 s). .. Both DNA strands were sequenced using BigDye Deoxy terminator cycle sequencing kit (BD V3.1, Applied Biosystems).

Article Title: RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors
Article Snippet: Paragraph title: 2.4. Specific Sequence Primer-Polymerase Chain Reaction (SSP-PCR) Analysis for RHD 1227A Allele ... Forty cycles were programmed on the thermocycler (TGradient, Biometra) as follows: denaturation at 94°C for 5 minutes, then 35 cycles of 30 seconds at 94°C, 40 seconds at 68°C, and 30 seconds at 72°C.

Article Title: Oxidative DNA damage in peripheral leukocytes and its association with expression and polymorphisms of hOGG1: A study of adolescents in a high risk region for hepatocellular carcinoma in China
Article Snippet: PCR reaction on a thermocycler (Biometra TGradient, Göttingen, Germany) began with pre-incubation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and elongation at 72 °C for 30 s. PCR products were diluted with 4 volumes of loading buffer (0.5 × TBE, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM methylmercury hydroxide), heat-denatured at 85 °C for 5 min and rapidly cooled on ice before loading. .. Genotype of each band-pattern was confirmed by a subsequent sequencing in a commercial laboratory (Research Biolabs Pte., Ltd, Singapore) using BigDyeTM Terminator kits on a ABI PRISM® 377-96 DNA Sequencer.

Article Title: Mitochondrial and nuclear ribosomal DNA dataset supports that Paramphistomum leydeni (Trematoda: Digenea) is a distinct rumen fluke species
Article Snippet: Paragraph title: Long-range PCR-based sequencing of mt genome ... PCR reactions were conducted in a total volume of 50 μl using 4 mM MgCl2 , 0.4 mM each of dNTPs, 5 μl 10× LATaq buffer, 5 mM of each primer, 0.5 μl LA Taq DNA polymerase (Takara, Dalian, China) and 2 μl DNA templates in a thermocycler (Biometra, Göttingen, Germany).

DNA Extraction:

Article Title: Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR
Article Snippet: Paragraph title: DNA extraction, primer design and PCR ... The PCR program was carried out on a thermocycler (Biometra, Göttingen, Germany), consisting of 94°C for 4 min followed by 35 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 2 min, with an additional 7 min at 72°C.

Fluorescence:

Article Title: Mitochondrial DNA-depleted A549 cells are resistant to bleomycin
Article Snippet: Purified DNA and PCR products were quantified by PicoGreen dye (Molecular Probes/Life Technologies, Grand Island, NY) and read on a fluorescence plate reader (model FL600, Bio-Tek) with 485-nm excitation and 535-nm emission. λ DNA/ Hin dIII (Invitrogen) was used to generate a standard curve. .. PCR was performed using the GeneAmp XL PCR Kit (Roche, Applied Biosystems, Branchburg, NJ) on a thermocycler (T Gradient, Biometra, Goettingen, Germany).

Mutagenesis:

Article Title: Contribution of BRCA1 5382insC mutation in triple negative breast cancer in Tunisia
Article Snippet: Paragraph title: BRCA1 5382inC mutation detection ... PCR was performed in an thermocycler (Biometra, Germany) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of (94 °C 20 s, 54 °C 20 s, 72 °C 20 s).

Isolation:

Article Title: Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity
Article Snippet: Paragraph title: RNA isolation and quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) ... RNA was transcribed to complementary DNA (cDNA) with random primers (Promega, Madison, WI, USA), and SuperScript II Reverse Transcriptase (Invitrogen GmbH, Karlsruhe, Germany) on a thermocycler (Biometra, Göttingen, Germany). cDNA was subjected to qRT-PCR using the Power SYBRGreen PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA, USA).

Size-exclusion Chromatography:

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. .. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination.

Article Title: Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development
Article Snippet: .. PCR was performed in a thermocycler (Biometra, Göttingen, Germany) and cycling conditions were as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 30 sec and 60°C for 1 min. .. Final droplet stabilization was performed at 98°C for 10 min. Droplets were analyzed using the QX200 Droplet Reader (Bio-Rad Laboratories, Munich, Germany).

Purification:

Article Title: Contribution of BRCA1 5382insC mutation in triple negative breast cancer in Tunisia
Article Snippet: PCR was performed in an thermocycler (Biometra, Germany) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of (94 °C 20 s, 54 °C 20 s, 72 °C 20 s). .. The product was cut from the gel and purified using QIAquick gel extraction kit (Qiagen, CA).

Article Title: Mitochondrial DNA-depleted A549 cells are resistant to bleomycin
Article Snippet: Purified DNA and PCR products were quantified by PicoGreen dye (Molecular Probes/Life Technologies, Grand Island, NY) and read on a fluorescence plate reader (model FL600, Bio-Tek) with 485-nm excitation and 535-nm emission. λ DNA/ Hin dIII (Invitrogen) was used to generate a standard curve. .. PCR was performed using the GeneAmp XL PCR Kit (Roche, Applied Biosystems, Branchburg, NJ) on a thermocycler (T Gradient, Biometra, Goettingen, Germany).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Gibberellin deficiency is responsible for shy-flowering nature of Epipremnum aureum
Article Snippet: .. For RT-PCR, the reaction was carried out with Taq DNA polymerase (D1806, Sigma) using a thermocycler (Biometra). .. Each 25 μl PCR reaction contained cDNA made from the original 20 ng of RNA together with 1x PCR reaction buffer, 300 nM of each primer, 2 mM MgCl2 , 0.2 mM dNTP and 1.25U of Taq DNA polymerase.

Silver Staining:

Article Title: Oxidative DNA damage in peripheral leukocytes and its association with expression and polymorphisms of hOGG1: A study of adolescents in a high risk region for hepatocellular carcinoma in China
Article Snippet: PCR reaction on a thermocycler (Biometra TGradient, Göttingen, Germany) began with pre-incubation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and elongation at 72 °C for 30 s. PCR products were diluted with 4 volumes of loading buffer (0.5 × TBE, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM methylmercury hydroxide), heat-denatured at 85 °C for 5 min and rapidly cooled on ice before loading. .. Silver staining followed Forsberg et al[ ].

Plasmid Preparation:

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: Once the four single stranded DNA (ssDNA) sequences were synthesized by the manufacturer, they were assembled as a single double-stranded DNA (dsDNA) fragment to be subsequently cloned into the PGEM-T plasmid (PGEM-T Easy Vector System I kit, Promega France SARL, Charbonnieres-les-Bains, France). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

Software:

Article Title: Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity
Article Snippet: RNA was transcribed to complementary DNA (cDNA) with random primers (Promega, Madison, WI, USA), and SuperScript II Reverse Transcriptase (Invitrogen GmbH, Karlsruhe, Germany) on a thermocycler (Biometra, Göttingen, Germany). cDNA was subjected to qRT-PCR using the Power SYBRGreen PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA, USA). .. Amplification reaction data were analyzed by the complementary Mx3000P analysis software.

Real-time Polymerase Chain Reaction:

Article Title: Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity
Article Snippet: Paragraph title: RNA isolation and quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) ... RNA was transcribed to complementary DNA (cDNA) with random primers (Promega, Madison, WI, USA), and SuperScript II Reverse Transcriptase (Invitrogen GmbH, Karlsruhe, Germany) on a thermocycler (Biometra, Göttingen, Germany). cDNA was subjected to qRT-PCR using the Power SYBRGreen PCR Master Mix Reagent Kit (Applied Biosystems, Foster City, CA, USA).

Article Title: Mitochondrial DNA-depleted A549 cells are resistant to bleomycin
Article Snippet: Paragraph title: qPCR for mtDNA and nDNA. ... PCR was performed using the GeneAmp XL PCR Kit (Roche, Applied Biosystems, Branchburg, NJ) on a thermocycler (T Gradient, Biometra, Goettingen, Germany).

Article Title: Gibberellin deficiency is responsible for shy-flowering nature of Epipremnum aureum
Article Snippet: For RT-PCR, the reaction was carried out with Taq DNA polymerase (D1806, Sigma) using a thermocycler (Biometra). .. The reactions and fluorescent signal detections were performed under the 7500-Fast Real-Time PCR system (Applied Biosystems).

Negative Control:

Article Title: Mitochondrial and nuclear ribosomal DNA dataset supports that Paramphistomum leydeni (Trematoda: Digenea) is a distinct rumen fluke species
Article Snippet: PCR reactions were conducted in a total volume of 50 μl using 4 mM MgCl2 , 0.4 mM each of dNTPs, 5 μl 10× LATaq buffer, 5 mM of each primer, 0.5 μl LA Taq DNA polymerase (Takara, Dalian, China) and 2 μl DNA templates in a thermocycler (Biometra, Göttingen, Germany). .. A negative control containing nuclease-free water was included in every amplification run.

RNA Expression:

Article Title: Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development
Article Snippet: Paragraph title: Digital droplet PCR assay for RNA expression analysis ... PCR was performed in a thermocycler (Biometra, Göttingen, Germany) and cycling conditions were as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 30 sec and 60°C for 1 min.

Agarose Gel Electrophoresis:

Article Title: Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR
Article Snippet: The PCR program was carried out on a thermocycler (Biometra, Göttingen, Germany), consisting of 94°C for 4 min followed by 35 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 2 min, with an additional 7 min at 72°C. .. Amplicons were checked and separated by electrophoresis in 1% agarose gel containing Gelview (BioTeke, Beijing, China) in 0.5×TAE buffer and visualized under UV light.

Electrophoresis:

Article Title: Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR
Article Snippet: The PCR program was carried out on a thermocycler (Biometra, Göttingen, Germany), consisting of 94°C for 4 min followed by 35 cycles at 94°C for 1 min, 50°C for 1 min and 72°C for 2 min, with an additional 7 min at 72°C. .. Amplicons were checked and separated by electrophoresis in 1% agarose gel containing Gelview (BioTeke, Beijing, China) in 0.5×TAE buffer and visualized under UV light.

Concentration Assay:

Article Title: Metagenomic potential for and diversity of N‐cycle driving microorganisms in the Bothnian Sea sediment. Metagenomic potential for and diversity of N‐cycle driving microorganisms in the Bothnian Sea sediment
Article Snippet: PCR was performed in a thermocycler (Professional thermal cycler, Biometra, Jena) with following parameters: initial denaturation for 4 min at 94°C, followed by 30 cycles of denaturation for 1 min at 94°C, primer annealing for 1 min at 43–56°C (parallel PCR reactions were performed at different annealing temperatures), elongation for 2 min at 72°C, and final elongation for 10 min at 72°C. .. Due to low final concentration of PCR products, a seminested PCR was performed with hzsA_1600F Scalindua and hzsA_1829R Scalindua primers (Harhangi et al., ).

Article Title: Molecular characterization of activated sludge from a seawater-processing wastewater treatment plant
Article Snippet: Reactions were carried out in an automated thermocycler (Biometra) with the following cycle: an initial denaturation step at 94°C for 5 min, followed by 30 cycles of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C, and a final extension step of 10 min at 72°C. .. The cycle was as follows: 5 min at 94°C, 38 cycles consisting of primer annealing at 52°C for 1 min, DNA elongation at 72°C for 90 s and denaturation at 94°C for 1 min, and a final cycle of 52°C for 1 min and 72°C for 6 min. PCR mixtures contained 1–10 ng of template DNA, each deoxynucleoside triphosphate at a concentration of 200 µM, 1.5 mM MgCl2 , each primer at a concentration of 0.3 µM, 2.5 U Taq DNA polymerase (Invitrogen) and PCR buffer supplied by the manufacturer.

Gel Extraction:

Article Title: Contribution of BRCA1 5382insC mutation in triple negative breast cancer in Tunisia
Article Snippet: PCR was performed in an thermocycler (Biometra, Germany) with an initial denaturation at 94 °C for 5 min, followed by 30 cycles of (94 °C 20 s, 54 °C 20 s, 72 °C 20 s). .. The product was cut from the gel and purified using QIAquick gel extraction kit (Qiagen, CA).

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  • 97
    Biometra conventional thermocycler
    Computational simulations of microscale thermal convection and corresponding PCR results aspect ratios of a) h / d = 9 (38.2 μL, Ra = 2.66 × 10 7 ) and b) h/d = 3 (18.5 μL, Ra = 1.45 × 10 6 ) with temperatures of 53 and 96 °C imposed at the top and bottom surfaces, respectively. A representative trajectory followed by a passive tracer advected in the 3D flow field is shown for each geometry along with top- and side view projections of the path. Excursions across the reactor midplane ( h /2) are used to construct Poincaré maps for the trajectory shown (blue points) and for two additional trajectories (red and green points). At h / d = 9, the flow field transports reagents along pseudo-2D trajectories that are essentially closed loops, as evident by quasi-periodic oscillation in the corresponding plot of temperature versus time following a fluid element. A more complex chaotic flow field is generated at h/d = 3, disrupting periodicity in the thermal profile. The chaotic nature of the convective flow field at h/d = 3 greatly accelerates DNA replication via the PCR flow, as evident by strong products in gel electrophoresis after only 10 min of reaction time (M: 100 bp ladder, lanes 1–4: products from 4 parallel reactions). In contrast, the reaction must be run for at least 20 min to obtain visible products at h / d = 9, and multiple bands are evident indicating non-specific replication (M: 100 bp ladder, lanes 1–2: products from 2 parallel reactions). Similar multiple product bands also appear in a conventional <t>thermocycler</t> when different pairs of denaturing ( T d ) and annealing ( T a ) temperatures are applied to mimick thermal profiles encountered at different locations within the reactor at h / d = 9 (M: 100 bp ladder, lanes 1–6: reaction products where T d (°C)/ T a (°C) = 91.6/58.4, 92.5/57.5, 93.7/56.3, 94.9/55.1, 96/54, and 96.5/53.5, respectively).
    Conventional Thermocycler, supplied by Biometra, used in various techniques. Bioz Stars score: 97/100, based on 182 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/conventional thermocycler/product/Biometra
    Average 97 stars, based on 182 article reviews
    Price from $9.99 to $1999.99
    conventional thermocycler - by Bioz Stars, 2020-02
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    78
    Biometra professional standard thermocycler
    Optimization of different influence factors in specific PCR of A. decursiva . M: DNA Marker (containing a mix of 6 individual DNA fragments from top to bottom denoting 600, 500, 400, 300, 200, and 100 bp). A Amplification results using A. decursiva identification primer pairs ZHQH-CP3s\ZHQH-CP3a: 1, 2, and 3 indicate A. decursiva ; 4, 5, and 6 indicate P. praeruptorum ; 7 indicates negative control without DNA template. B. Annealing temperatures: numbers from 1 to 21 indicate annealing temperatures of A. decursiva with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C; numbers from 22 to 42 indicate annealing temperatures of P. praeruptorum with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C. C. Cycle numbers: numbers from 1 to 4 indicate cycle numbers of A. decursiva with 20, 25, 30, and 35 cycles; numbers from 5 to 8 indicate cycle numbers of P. praeruptorum with 20, 25, 30, and 35 cycles. D. DNA template dosages: numbers from 1 to 7 indicate DNA template dosages of A. decursiva with 144, 72, 36, 18, 9, 4.5 and 2.25 ng; numbers from 8 to 14 indicate DNA template dosages of A. decursiva with 144, 72, 36, 18, 9, 4.5 and 2.25 ng. E. Taq enzyme species: a indicates Taq DNA Polymerase, b indicates Taq Plus DNA Polymerase, c indicates Hot Start Taq DNA Polymerase, 1 indicates A. decursiva , and 2 indicates P. praeruptorum . F . PCR instruments: a indicates LightCycler ®96 System (Roche company), b indicates Professional standard <t>Thermocycler</t> (Biometra company), c indicates MJ research PTC-200 Peltier Thermal Cycler (Bio-Rad company), 1 indicates A. decursiva , and 2 indicates P. praeruptorum
    Professional Standard Thermocycler, supplied by Biometra, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    professional standard thermocycler - by Bioz Stars, 2020-02
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    99
    Biometra biometra tgradient thermocycler
    Optimization of different influence factors in specific PCR of A. decursiva . M: DNA Marker (containing a mix of 6 individual DNA fragments from top to bottom denoting 600, 500, 400, 300, 200, and 100 bp). A Amplification results using A. decursiva identification primer pairs ZHQH-CP3s\ZHQH-CP3a: 1, 2, and 3 indicate A. decursiva ; 4, 5, and 6 indicate P. praeruptorum ; 7 indicates negative control without DNA template. B. Annealing temperatures: numbers from 1 to 21 indicate annealing temperatures of A. decursiva with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C; numbers from 22 to 42 indicate annealing temperatures of P. praeruptorum with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C. C. Cycle numbers: numbers from 1 to 4 indicate cycle numbers of A. decursiva with 20, 25, 30, and 35 cycles; numbers from 5 to 8 indicate cycle numbers of P. praeruptorum with 20, 25, 30, and 35 cycles. D. DNA template dosages: numbers from 1 to 7 indicate DNA template dosages of A. decursiva with 144, 72, 36, 18, 9, 4.5 and 2.25 ng; numbers from 8 to 14 indicate DNA template dosages of A. decursiva with 144, 72, 36, 18, 9, 4.5 and 2.25 ng. E. Taq enzyme species: a indicates Taq DNA Polymerase, b indicates Taq Plus DNA Polymerase, c indicates Hot Start Taq DNA Polymerase, 1 indicates A. decursiva , and 2 indicates P. praeruptorum . F . PCR instruments: a indicates LightCycler ®96 System (Roche company), b indicates Professional standard <t>Thermocycler</t> (Biometra company), c indicates MJ research PTC-200 Peltier Thermal Cycler (Bio-Rad company), 1 indicates A. decursiva , and 2 indicates P. praeruptorum
    Biometra Tgradient Thermocycler, supplied by Biometra, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biometra tgradient thermocycler/product/Biometra
    Average 99 stars, based on 7 article reviews
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    biometra tgradient thermocycler - by Bioz Stars, 2020-02
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    Computational simulations of microscale thermal convection and corresponding PCR results aspect ratios of a) h / d = 9 (38.2 μL, Ra = 2.66 × 10 7 ) and b) h/d = 3 (18.5 μL, Ra = 1.45 × 10 6 ) with temperatures of 53 and 96 °C imposed at the top and bottom surfaces, respectively. A representative trajectory followed by a passive tracer advected in the 3D flow field is shown for each geometry along with top- and side view projections of the path. Excursions across the reactor midplane ( h /2) are used to construct Poincaré maps for the trajectory shown (blue points) and for two additional trajectories (red and green points). At h / d = 9, the flow field transports reagents along pseudo-2D trajectories that are essentially closed loops, as evident by quasi-periodic oscillation in the corresponding plot of temperature versus time following a fluid element. A more complex chaotic flow field is generated at h/d = 3, disrupting periodicity in the thermal profile. The chaotic nature of the convective flow field at h/d = 3 greatly accelerates DNA replication via the PCR flow, as evident by strong products in gel electrophoresis after only 10 min of reaction time (M: 100 bp ladder, lanes 1–4: products from 4 parallel reactions). In contrast, the reaction must be run for at least 20 min to obtain visible products at h / d = 9, and multiple bands are evident indicating non-specific replication (M: 100 bp ladder, lanes 1–2: products from 2 parallel reactions). Similar multiple product bands also appear in a conventional thermocycler when different pairs of denaturing ( T d ) and annealing ( T a ) temperatures are applied to mimick thermal profiles encountered at different locations within the reactor at h / d = 9 (M: 100 bp ladder, lanes 1–6: reaction products where T d (°C)/ T a (°C) = 91.6/58.4, 92.5/57.5, 93.7/56.3, 94.9/55.1, 96/54, and 96.5/53.5, respectively).

    Journal: Angewandte Chemie (International ed. in English)

    Article Title: Chaotically accelerated PCR by microscale Rayleigh-B?nard convection **

    doi: 10.1002/anie.201004217

    Figure Lengend Snippet: Computational simulations of microscale thermal convection and corresponding PCR results aspect ratios of a) h / d = 9 (38.2 μL, Ra = 2.66 × 10 7 ) and b) h/d = 3 (18.5 μL, Ra = 1.45 × 10 6 ) with temperatures of 53 and 96 °C imposed at the top and bottom surfaces, respectively. A representative trajectory followed by a passive tracer advected in the 3D flow field is shown for each geometry along with top- and side view projections of the path. Excursions across the reactor midplane ( h /2) are used to construct Poincaré maps for the trajectory shown (blue points) and for two additional trajectories (red and green points). At h / d = 9, the flow field transports reagents along pseudo-2D trajectories that are essentially closed loops, as evident by quasi-periodic oscillation in the corresponding plot of temperature versus time following a fluid element. A more complex chaotic flow field is generated at h/d = 3, disrupting periodicity in the thermal profile. The chaotic nature of the convective flow field at h/d = 3 greatly accelerates DNA replication via the PCR flow, as evident by strong products in gel electrophoresis after only 10 min of reaction time (M: 100 bp ladder, lanes 1–4: products from 4 parallel reactions). In contrast, the reaction must be run for at least 20 min to obtain visible products at h / d = 9, and multiple bands are evident indicating non-specific replication (M: 100 bp ladder, lanes 1–2: products from 2 parallel reactions). Similar multiple product bands also appear in a conventional thermocycler when different pairs of denaturing ( T d ) and annealing ( T a ) temperatures are applied to mimick thermal profiles encountered at different locations within the reactor at h / d = 9 (M: 100 bp ladder, lanes 1–6: reaction products where T d (°C)/ T a (°C) = 91.6/58.4, 92.5/57.5, 93.7/56.3, 94.9/55.1, 96/54, and 96.5/53.5, respectively).

    Article Snippet: The reactions shown in were performed using a conventional thermocycler (T-Gradient; Biometra) following a two temperature protocol (15 s denaturing, 30 s annealing) for 30 cycles.

    Techniques: Convection, Polymerase Chain Reaction, Flow Cytometry, Construct, Generated, Nucleic Acid Electrophoresis

    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

    Journal: BMC Microbiology

    Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

    doi: 10.1186/1471-2180-4-21

    Figure Lengend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

    Article Snippet: The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

    Optimization of different influence factors in specific PCR of A. decursiva . M: DNA Marker (containing a mix of 6 individual DNA fragments from top to bottom denoting 600, 500, 400, 300, 200, and 100 bp). A Amplification results using A. decursiva identification primer pairs ZHQH-CP3s\ZHQH-CP3a: 1, 2, and 3 indicate A. decursiva ; 4, 5, and 6 indicate P. praeruptorum ; 7 indicates negative control without DNA template. B. Annealing temperatures: numbers from 1 to 21 indicate annealing temperatures of A. decursiva with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C; numbers from 22 to 42 indicate annealing temperatures of P. praeruptorum with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C. C. Cycle numbers: numbers from 1 to 4 indicate cycle numbers of A. decursiva with 20, 25, 30, and 35 cycles; numbers from 5 to 8 indicate cycle numbers of P. praeruptorum with 20, 25, 30, and 35 cycles. D. DNA template dosages: numbers from 1 to 7 indicate DNA template dosages of A. decursiva with 144, 72, 36, 18, 9, 4.5 and 2.25 ng; numbers from 8 to 14 indicate DNA template dosages of A. decursiva with 144, 72, 36, 18, 9, 4.5 and 2.25 ng. E. Taq enzyme species: a indicates Taq DNA Polymerase, b indicates Taq Plus DNA Polymerase, c indicates Hot Start Taq DNA Polymerase, 1 indicates A. decursiva , and 2 indicates P. praeruptorum . F . PCR instruments: a indicates LightCycler ®96 System (Roche company), b indicates Professional standard Thermocycler (Biometra company), c indicates MJ research PTC-200 Peltier Thermal Cycler (Bio-Rad company), 1 indicates A. decursiva , and 2 indicates P. praeruptorum

    Journal: Pharmacognosy Magazine

    Article Title: Specific PCR Identification between Peucedanum praeruptorum and Angelica decursiva and Identification between Them and Adulterant Using DNA Barcode

    doi: 10.4103/0973-1296.197658

    Figure Lengend Snippet: Optimization of different influence factors in specific PCR of A. decursiva . M: DNA Marker (containing a mix of 6 individual DNA fragments from top to bottom denoting 600, 500, 400, 300, 200, and 100 bp). A Amplification results using A. decursiva identification primer pairs ZHQH-CP3s\ZHQH-CP3a: 1, 2, and 3 indicate A. decursiva ; 4, 5, and 6 indicate P. praeruptorum ; 7 indicates negative control without DNA template. B. Annealing temperatures: numbers from 1 to 21 indicate annealing temperatures of A. decursiva with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C; numbers from 22 to 42 indicate annealing temperatures of P. praeruptorum with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C. C. Cycle numbers: numbers from 1 to 4 indicate cycle numbers of A. decursiva with 20, 25, 30, and 35 cycles; numbers from 5 to 8 indicate cycle numbers of P. praeruptorum with 20, 25, 30, and 35 cycles. D. DNA template dosages: numbers from 1 to 7 indicate DNA template dosages of A. decursiva with 144, 72, 36, 18, 9, 4.5 and 2.25 ng; numbers from 8 to 14 indicate DNA template dosages of A. decursiva with 144, 72, 36, 18, 9, 4.5 and 2.25 ng. E. Taq enzyme species: a indicates Taq DNA Polymerase, b indicates Taq Plus DNA Polymerase, c indicates Hot Start Taq DNA Polymerase, 1 indicates A. decursiva , and 2 indicates P. praeruptorum . F . PCR instruments: a indicates LightCycler ®96 System (Roche company), b indicates Professional standard Thermocycler (Biometra company), c indicates MJ research PTC-200 Peltier Thermal Cycler (Bio-Rad company), 1 indicates A. decursiva , and 2 indicates P. praeruptorum

    Article Snippet: PCR instruments: MJ research PTC-200 Peltier Thermal Cycler (Bio-Rad company), Professional standard Thermocycler (Biometra company) and Light Cycler® 96 System (Roche company).

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

    Optimization of different influence factors in specific PCR of P. praeruptorum . M: DNA Marker (containing a mix of 6 individual DNA fragments from top to bottom denoting 600, 500, 400, 300, 200, and 100 bp). A Amplification results using P. praeruptorum identification primer pairs QH-CP19s\QH-CP19a: 1, 2, and 3 indicate A. decursiva ; 4, 5, and 6 indicate P. praeruptorum ; 7 indicates negative control without DNA template. B . Annealing temperatures: numbers from 1 to 21 indicate annealing temperatures of P. praeruptorum with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C; numbers from 22 to 42 indicate annealing temperatures of A. decursiva with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C. C. Cycle numbers: numbers from 1 to 4 indicate cycle numbers of P. praeruptorum with 20, 25, 30, and 35 cycles; numbers from 5 to 8 indicate cycle numbers of A. decursiva with 20, 25, 30, and 35 cycles. D. DNA template dosages: numbers from 1 to 7 indicate DNA template dosages of P. praeruptorum with 42, 21, 10.5, 5.25, 2.625, 1.313, and 0.657 ng; numbers from 8 to 14 indicate DNA template dosages of A. decursiva with 42, 21, 10.5, 5.25, 2.625, 1.313, and 0.657 ng. E. Taq enzyme species: a indicates Taq DNA Polymerase, b indicates Taq Plus DNA Polymerase, c indicates Hot Start Taq DNA Polymerase, 1 indicates P. praeruptorum , and 2 indicates A. decursiva . F. PCR instruments: a indicates LightCycler ®96 System (Roche company), b indicates Professional standard Thermocycler (Biometra company), c indicates MJ research PTC-200 Peltier Thermal Cycler (Bio-Rad company), 1 indicates P. praeruptorum , and 2 indicates A. decursiva .

    Journal: Pharmacognosy Magazine

    Article Title: Specific PCR Identification between Peucedanum praeruptorum and Angelica decursiva and Identification between Them and Adulterant Using DNA Barcode

    doi: 10.4103/0973-1296.197658

    Figure Lengend Snippet: Optimization of different influence factors in specific PCR of P. praeruptorum . M: DNA Marker (containing a mix of 6 individual DNA fragments from top to bottom denoting 600, 500, 400, 300, 200, and 100 bp). A Amplification results using P. praeruptorum identification primer pairs QH-CP19s\QH-CP19a: 1, 2, and 3 indicate A. decursiva ; 4, 5, and 6 indicate P. praeruptorum ; 7 indicates negative control without DNA template. B . Annealing temperatures: numbers from 1 to 21 indicate annealing temperatures of P. praeruptorum with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C; numbers from 22 to 42 indicate annealing temperatures of A. decursiva with 48, 49, 51, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68 °C. C. Cycle numbers: numbers from 1 to 4 indicate cycle numbers of P. praeruptorum with 20, 25, 30, and 35 cycles; numbers from 5 to 8 indicate cycle numbers of A. decursiva with 20, 25, 30, and 35 cycles. D. DNA template dosages: numbers from 1 to 7 indicate DNA template dosages of P. praeruptorum with 42, 21, 10.5, 5.25, 2.625, 1.313, and 0.657 ng; numbers from 8 to 14 indicate DNA template dosages of A. decursiva with 42, 21, 10.5, 5.25, 2.625, 1.313, and 0.657 ng. E. Taq enzyme species: a indicates Taq DNA Polymerase, b indicates Taq Plus DNA Polymerase, c indicates Hot Start Taq DNA Polymerase, 1 indicates P. praeruptorum , and 2 indicates A. decursiva . F. PCR instruments: a indicates LightCycler ®96 System (Roche company), b indicates Professional standard Thermocycler (Biometra company), c indicates MJ research PTC-200 Peltier Thermal Cycler (Bio-Rad company), 1 indicates P. praeruptorum , and 2 indicates A. decursiva .

    Article Snippet: PCR instruments: MJ research PTC-200 Peltier Thermal Cycler (Bio-Rad company), Professional standard Thermocycler (Biometra company) and Light Cycler® 96 System (Roche company).

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