Structured Review

MJ Research thermocycler
Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional <t>thermocycler,</t> as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.
Thermocycler, supplied by MJ Research, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips"

Article Title: Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips

Journal: Scientific Reports

doi: 10.1038/s41598-018-30797-9

Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional thermocycler, as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.
Figure Legend Snippet: Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional thermocycler, as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.

Techniques Used: Agarose Gel Electrophoresis

2) Product Images from "Genotyping 1000 yeast strains by next-generation sequencing"

Article Title: Genotyping 1000 yeast strains by next-generation sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-90

Library preparation pipeline. DNA isolation is performed with a 96-head liquid handling robot. DNA fragmentation is achieved by sonication, either in glass tubes (Covaris) or PCR strips (Bandelin). SPRI bead cleanup is automated on a 96-head liquid handling robot. Three enzymatic steps for barcoded adapter ligation are performed by addition of enzyme (+ buffer), incubation, and heat inactivation in a thermocycler. After pooling of 48 barcoded libraries, samples are concentrated and size-selected using an E-gel. PCR is performed on the size-selected pool to enrich for adapter containing fragments and elongate them to full-length libraries. A final cleanup is performed in PCR strips mounted to a homemade magnetic stand.
Figure Legend Snippet: Library preparation pipeline. DNA isolation is performed with a 96-head liquid handling robot. DNA fragmentation is achieved by sonication, either in glass tubes (Covaris) or PCR strips (Bandelin). SPRI bead cleanup is automated on a 96-head liquid handling robot. Three enzymatic steps for barcoded adapter ligation are performed by addition of enzyme (+ buffer), incubation, and heat inactivation in a thermocycler. After pooling of 48 barcoded libraries, samples are concentrated and size-selected using an E-gel. PCR is performed on the size-selected pool to enrich for adapter containing fragments and elongate them to full-length libraries. A final cleanup is performed in PCR strips mounted to a homemade magnetic stand.

Techniques Used: DNA Extraction, Sonication, Polymerase Chain Reaction, Ligation, Incubation

3) Product Images from "Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips"

Article Title: Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips

Journal: Scientific Reports

doi: 10.1038/s41598-018-30797-9

Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional thermocycler, as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.
Figure Legend Snippet: Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional thermocycler, as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.

Techniques Used: Agarose Gel Electrophoresis

Related Articles

Diagnostic Assay:

Article Title: Relapsing Fever-Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent
Article Snippet: .. The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess, Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s denaturation at 94°C, 20 s each for the first annealing reaction at 63°C with a 40 s extension at 72°C and a final extension for 2 min at 72°C. .. After the first amplification with the outer set of primers, 2 μL of the amplification product was transferred to a fresh tube containing 48 μL of the reaction mixture previously described, except that 2.5 mM MgCl2 and 20 pmol of the inner primer pair was used (16S2A 5′-AGT CAA ACG GGA TGT AGC AAT AC-3′ and 16S2B 5′-GGT ATT CTT TCT GAT ATC AAC AG-3′ positions 66–720).

Article Title: " Candidatus Neoehrlichia mikurensis," Anaplasma phagocytophilum, and Lyme Disease Spirochetes in Questing European Vector Ticks and in Feeding Ticks Removed from People
Article Snippet: .. The mixture was placed in a thermocycler (PTC 200; MJ Research Biozym, Diagnostic, Hess. ..

Clone Assay:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: Paragraph title: Guide RNA Cloning Protocol ... Hybridization was performed in a thermocycler (MJ Research).

Centrifugation:

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min. .. The sequencing reaction products were precipitated by adding 40 μ L of 75% isopropanol and centrifugation R-5810 centrifuge (Eppendorf) at 3220 ×g for 30 min at room temperature.

Amplification:

Article Title: White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus
Article Snippet: .. A fragment of the rDNA containing the ITS regions 1 and 2 and the 5.8S ribosomal RNA (rRNA) gene was amplified by polymerase chain reaction (PCR) using the primer combinations ITS4 and ITS5 (White et al., ) in an automated thermocycler (Minicycler PTC-150, MJ Research, USA). .. The PCR products were used directly after purification (QIAquick PCR Purification Kit, Qiagen) for DNA sequencing on an Applied Biosystems 3730 DNA Analyzer (PE Applied Biosystems, Foster City, California, USA).

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: .. Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation. .. The PCR products were separated on a 2% agarose gel in TBE (Genmedika Biotechnology Corp., Taipei, Taiwan) by electrophoresis and stained with ethidium bromide (Sigma).

Article Title: Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes
Article Snippet: .. PCR amplification was performed in a thermocycler (PTC-150 Minicycler PCR system; MJ Research Inc., Waltham, MA, USA) using rTaq polymerase (Takara) in a 25 μL reaction mixture consisting of an initial 5 min denaturation step at 95 °C, 35 cycles of 95 °C for 45 s, 55–58 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C. .. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a positive control to confirm the presence of cDNA [ ].

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Article Snippet: The thermocycler, which was used for PCR, was a Tetrad PTC-225 Thermal Cycler (MJ research, St. Bruno (Quebec), Canada). .. PCR, which was performed on samples that were reverse transcribed to cDNA in the absence of reverse transcriptase, did not show any amplified product.

Article Title: Relapsing Fever-Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent
Article Snippet: The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess, Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s denaturation at 94°C, 20 s each for the first annealing reaction at 63°C with a 40 s extension at 72°C and a final extension for 2 min at 72°C. .. After the first amplification with the outer set of primers, 2 μL of the amplification product was transferred to a fresh tube containing 48 μL of the reaction mixture previously described, except that 2.5 mM MgCl2 and 20 pmol of the inner primer pair was used (16S2A 5′-AGT CAA ACG GGA TGT AGC AAT AC-3′ and 16S2B 5′-GGT ATT CTT TCT GAT ATC AAC AG-3′ positions 66–720).

Article Title: Genotyping 1000 yeast strains by next-generation sequencing
Article Snippet: PCR enrichment In a 50 μl reaction, 5–10 ng of the pooled libraries were amplified. .. The PCR was performed on a thermocycler (MJ Research tetrad) containing 1x Phusion Master Mix with HF Buffer (Thermo Scientific) and 0.2 μM Illumina PE 1.0 and 2.0 primers.

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: Analysis of amplified products was by electrophoresis (100 V/60 min) agarose gel at 1.5% in Tris plug, sodium acetate, EDTA pH 8.0, and stained with ethidium bromide. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

Article Title: Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites
Article Snippet: .. The extension and amplification were carried out in a thermocycler (MJ Research, Inc.) programmed for three cycles, with each cycle consisting of 5 min of denaturation at 95°C, 1 min of annealing at 50°C, and 2 min of extension at 72°C. .. The final PCR products from the two separate reactions were digested with Nde I and Bam HI, gel purified, and then ligated to pT7-7/H-IN previously cut with Nde I and Bam HI to form pE2C/IN1-288 and pE2C/IN11-288, respectively.

Article Title: Genital mycoplasma trachomatis infections in treatment na?ve HIV-1 infected adults
Article Snippet: Briefly, the 50 μl amplification reaction mixture contained 5.00 μl of 10× PCR buffer [1× PCR buffer is 10 mmol/l Tris-HCl (pH 8.8 at 25°C), 50 mmol/l KCl, and 0.1% Triton X-100], 3.0 mM MgCl2, 1.25 U of Taq polymerase (GeneiTaq, Bangalore Genei, India), 400 μmol/l (each) deoxynucleoside triphosphate mixture, 25 pmol of each primer, 16 μl of sample DNA and ultrapure sterile water. .. The PCR conditions used were 1 cycle of initial denaturation at 95°C for 10 min, followed by 35, two-step cycles of 95°C for 15 sec, 60°C for 1 min, and followed by 5 min at 72°C in a thermocycler (MJ Research, Waltham, MA).

Positive Control:

Article Title: Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes
Article Snippet: PCR amplification was performed in a thermocycler (PTC-150 Minicycler PCR system; MJ Research Inc., Waltham, MA, USA) using rTaq polymerase (Takara) in a 25 μL reaction mixture consisting of an initial 5 min denaturation step at 95 °C, 35 cycles of 95 °C for 45 s, 55–58 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C. .. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a positive control to confirm the presence of cDNA [ ].

Article Title: Genital mycoplasma trachomatis infections in treatment na?ve HIV-1 infected adults
Article Snippet: The template of M. genitalium DNA used as positive control was gifted by Dr M. Jurstrand, Clinical Research Centre, University Hospital, SE-70185, Orebro, Sweden. .. The PCR conditions used were 1 cycle of initial denaturation at 95°C for 10 min, followed by 35, two-step cycles of 95°C for 15 sec, 60°C for 1 min, and followed by 5 min at 72°C in a thermocycler (MJ Research, Waltham, MA).

Construct:

Article Title: Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites
Article Snippet: Paragraph title: DNA constructs. ... The extension and amplification were carried out in a thermocycler (MJ Research, Inc.) programmed for three cycles, with each cycle consisting of 5 min of denaturation at 95°C, 1 min of annealing at 50°C, and 2 min of extension at 72°C.

Real-time Polymerase Chain Reaction:

Article Title: Interleukin-6 mediated upregulation of CYP1B1 and CYP2E1 in colorectal cancer involves DNA methylation, miR27b and STAT3
Article Snippet: Paragraph title: Reverse transcription and quantitative PCR ... Each sample was incubated with 0.5 mM dNTPs, 1 × first strand buffer, 8 μ M dithiothreitol and 100 units of Superscript II reverse transcriptase (Invitrogen, Paisley, UK) for 10 min at 25 °C, 90 min at 42 °C and 15 min at 70 °C on a thermocycler (Peltier Thermal Cycler PTC-200, MJ Research, Waltham, MA, USA).

Incubation:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: BclI (New England BioLabs) was subsequently added to the digestion reaction and incubated for a minimum of two hours at 50°C. .. Hybridization was performed in a thermocycler (MJ Research).

Article Title: Interleukin-6 mediated upregulation of CYP1B1 and CYP2E1 in colorectal cancer involves DNA methylation, miR27b and STAT3
Article Snippet: .. Each sample was incubated with 0.5 mM dNTPs, 1 × first strand buffer, 8 μ M dithiothreitol and 100 units of Superscript II reverse transcriptase (Invitrogen, Paisley, UK) for 10 min at 25 °C, 90 min at 42 °C and 15 min at 70 °C on a thermocycler (Peltier Thermal Cycler PTC-200, MJ Research, Waltham, MA, USA). .. An miRNA reverse transcription kit was used for miRNA expression according to the manufacturer's protocol (Taqman, Applied Biosystems, Life Technologies, Paisley, UK).

Article Title: Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes
Article Snippet: Total RNA was treated with DNase I (Takara, Dalian, China) for 15 min at room temperature and incubated at 70 °C for 5 min before first-strand cDNA synthesis. .. PCR amplification was performed in a thermocycler (PTC-150 Minicycler PCR system; MJ Research Inc., Waltham, MA, USA) using rTaq polymerase (Takara) in a 25 μL reaction mixture consisting of an initial 5 min denaturation step at 95 °C, 35 cycles of 95 °C for 45 s, 55–58 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C.

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min. .. The sequencing reaction products were precipitated by adding 40 μ L of 75% isopropanol and centrifugation R-5810 centrifuge (Eppendorf) at 3220 ×g for 30 min at room temperature.

Stripping Membranes:

Article Title: Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases
Article Snippet: .. Prior to reverse transcription and template switching the strip was thawed by placing it in a thermocycler (MJ Research PTC-200 now a part of Bio-Rad; Hercules, CA, USA) at 20°C for 5 min. ..

Expressing:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: A stock of digested sgRNA expression plasmid was used for cloning custom 20mer guide sequences (see below). .. Hybridization was performed in a thermocycler (MJ Research).

Article Title: Interleukin-6 mediated upregulation of CYP1B1 and CYP2E1 in colorectal cancer involves DNA methylation, miR27b and STAT3
Article Snippet: Each sample was incubated with 0.5 mM dNTPs, 1 × first strand buffer, 8 μ M dithiothreitol and 100 units of Superscript II reverse transcriptase (Invitrogen, Paisley, UK) for 10 min at 25 °C, 90 min at 42 °C and 15 min at 70 °C on a thermocycler (Peltier Thermal Cycler PTC-200, MJ Research, Waltham, MA, USA). .. An miRNA reverse transcription kit was used for miRNA expression according to the manufacturer's protocol (Taqman, Applied Biosystems, Life Technologies, Paisley, UK).

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: Paragraph title: Expression of monocytic chemokines, MIP-1 and MCP-1, by PCV2-inoculated MDM ... Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation.

Article Title: Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes
Article Snippet: Paragraph title: 4.3.2. Identification of SSC-Specific Gene Expression by Reverse Transcription-PCR (RT-PCR) ... PCR amplification was performed in a thermocycler (PTC-150 Minicycler PCR system; MJ Research Inc., Waltham, MA, USA) using rTaq polymerase (Takara) in a 25 μL reaction mixture consisting of an initial 5 min denaturation step at 95 °C, 35 cycles of 95 °C for 45 s, 55–58 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C.

Transformation Assay:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: Hybridization was performed in a thermocycler (MJ Research). .. The ligation reaction was transformed into Z-competent E. coli (Zymo Research), and plasmid DNA from the resulting colonies was isolated and sequenced using a T3 primer to confirm TRP1 guide sequence insertion into the sgRNA expression cassette.

Chloramphenicol Acetyltransferase Assay:

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: The primer sets used included porcine MIP-1 (sense: 5′-GCT CAG TTC AGT TCC AAG TC-3′, antisense: 5′-ACC ATG AAG CTC TGC GTG AC-3′) [ ], porcine MCP-1 (sense: 5′-TCA CCA GCA GCA AGT GTC-3′, antisense: 5′-CTG AGA TTC ACA GAG GA-3′) [ ], and internal controls including porcine G3PDH (sense: 5′-ACC TCC ACT ACA TGG TCT ACA TGT TC-3′, antisense: 5′-CAT TGA TGA CAA GCT TCC CAT TC-3′), and β-actin (sense: 5′-CAT CAC CAT CGG CAA CGA-3′, antisense: 5′-GCG TAG AGG TCC TTC CTG ATG T-3′) [ ]. .. Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation.

Derivative Assay:

Article Title: Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes
Article Snippet: PCR amplification was performed in a thermocycler (PTC-150 Minicycler PCR system; MJ Research Inc., Waltham, MA, USA) using rTaq polymerase (Takara) in a 25 μL reaction mixture consisting of an initial 5 min denaturation step at 95 °C, 35 cycles of 95 °C for 45 s, 55–58 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C. .. To conform that the RT-PCR signals derived not from genomic DNA, for each gene tested a negative control identical to the test assay using a RNA sample was included.

Hybridization:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: .. Hybridization was performed in a thermocycler (MJ Research). .. Reactions were initially held at 95° C for six minutes, and then decreased 1° C per one minute cycle for seventy cycles to reach a final temperature of 25° C. Hybridized oligonucleotide substrates containing the 20mer guide sequence and 5' end of the sgRNA were then directly ligated into cut sgRNA expression plasmids (e.g., pT040, pML104, or pML107) typically overnight at 16° C. The hybridized the nucleotide insert was added at a 40-100 fold molar ratio to the cut vector.

Sequencing:

Article Title: White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus
Article Snippet: A fragment of the rDNA containing the ITS regions 1 and 2 and the 5.8S ribosomal RNA (rRNA) gene was amplified by polymerase chain reaction (PCR) using the primer combinations ITS4 and ITS5 (White et al., ) in an automated thermocycler (Minicycler PTC-150, MJ Research, USA). .. BLAST (basic local alignment search tool) was used to perform similarity search of the sequence from the isolate obtained in this study with those from GenBank.

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: Hybridization was performed in a thermocycler (MJ Research). .. Reactions were initially held at 95° C for six minutes, and then decreased 1° C per one minute cycle for seventy cycles to reach a final temperature of 25° C. Hybridized oligonucleotide substrates containing the 20mer guide sequence and 5' end of the sgRNA were then directly ligated into cut sgRNA expression plasmids (e.g., pT040, pML104, or pML107) typically overnight at 16° C. The hybridized the nucleotide insert was added at a 40-100 fold molar ratio to the cut vector.

Article Title: Relapsing Fever-Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent
Article Snippet: Borrelia genospecies were characterized by amplifying and sequencing a 600-nucleotide fragment of the gene encoding the 16S rRNA. .. The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess, Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s denaturation at 94°C, 20 s each for the first annealing reaction at 63°C with a 40 s extension at 72°C and a final extension for 2 min at 72°C.

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: The positive PCR products were purified with the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI, USA—Cat. no. A7170) and subjected to sequencing reaction by chain termination with dideoxynucleotides marked with fluorophores. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

Article Title: Genital mycoplasma trachomatis infections in treatment na?ve HIV-1 infected adults
Article Snippet: The PCR conditions used were 1 cycle of initial denaturation at 95°C for 10 min, followed by 35, two-step cycles of 95°C for 15 sec, 60°C for 1 min, and followed by 5 min at 72°C in a thermocycler (MJ Research, Waltham, MA). .. PCR for C. trachomatis: C. trachomatis DNA was detected by PCR targeting a sequence of the cryptic plasmid using primers KL-1 and KL-2 .

Ligation:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: Hybridization was performed in a thermocycler (MJ Research). .. The ligation reaction was transformed into Z-competent E. coli (Zymo Research), and plasmid DNA from the resulting colonies was isolated and sequenced using a T3 primer to confirm TRP1 guide sequence insertion into the sgRNA expression cassette.

Cell Culture:

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Article Snippet: Polymerase Chain Reaction (PCR) RNA was isolated from freshly isolated equine RPE and cultured RPE of passage-4 using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. .. The thermocycler, which was used for PCR, was a Tetrad PTC-225 Thermal Cycler (MJ research, St. Bruno (Quebec), Canada).

Generated:

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min. .. The generated sequences were analyzed by the program Bioedit v.7.0.9 [ ] to generate a unique sequence from the bidirectional sequence data.

Article Title: Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites
Article Snippet: The fusion genes were obtained by an overlapping PCR method that generated a 5′ and 3′ fragment with a shared region of homology ( ). .. The extension and amplification were carried out in a thermocycler (MJ Research, Inc.) programmed for three cycles, with each cycle consisting of 5 min of denaturation at 95°C, 1 min of annealing at 50°C, and 2 min of extension at 72°C.

DNA Sequencing:

Article Title: White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus
Article Snippet: A fragment of the rDNA containing the ITS regions 1 and 2 and the 5.8S ribosomal RNA (rRNA) gene was amplified by polymerase chain reaction (PCR) using the primer combinations ITS4 and ITS5 (White et al., ) in an automated thermocycler (Minicycler PTC-150, MJ Research, USA). .. The PCR products were used directly after purification (QIAquick PCR Purification Kit, Qiagen) for DNA sequencing on an Applied Biosystems 3730 DNA Analyzer (PE Applied Biosystems, Foster City, California, USA).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: Expression of monocytic chemokines, MIP-1 and MCP-1, by PCV2-inoculated MDM The expression levels of MIP-1 and MCP-1 mRNA by PCV2-inoculated MDM were measured semiquantitatively by RT-PCR as described previously [ , ]. .. Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation.

Article Title: Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes
Article Snippet: Paragraph title: 4.3.2. Identification of SSC-Specific Gene Expression by Reverse Transcription-PCR (RT-PCR) ... PCR amplification was performed in a thermocycler (PTC-150 Minicycler PCR system; MJ Research Inc., Waltham, MA, USA) using rTaq polymerase (Takara) in a 25 μL reaction mixture consisting of an initial 5 min denaturation step at 95 °C, 35 cycles of 95 °C for 45 s, 55–58 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C.

Cellular Antioxidant Activity Assay:

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: The primer sets used included porcine MIP-1 (sense: 5′-GCT CAG TTC AGT TCC AAG TC-3′, antisense: 5′-ACC ATG AAG CTC TGC GTG AC-3′) [ ], porcine MCP-1 (sense: 5′-TCA CCA GCA GCA AGT GTC-3′, antisense: 5′-CTG AGA TTC ACA GAG GA-3′) [ ], and internal controls including porcine G3PDH (sense: 5′-ACC TCC ACT ACA TGG TCT ACA TGT TC-3′, antisense: 5′-CAT TGA TGA CAA GCT TCC CAT TC-3′), and β-actin (sense: 5′-CAT CAC CAT CGG CAA CGA-3′, antisense: 5′-GCG TAG AGG TCC TTC CTG ATG T-3′) [ ]. .. Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation.

Article Title: Relapsing Fever-Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent
Article Snippet: The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess, Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s denaturation at 94°C, 20 s each for the first annealing reaction at 63°C with a 40 s extension at 72°C and a final extension for 2 min at 72°C. .. After the first amplification with the outer set of primers, 2 μL of the amplification product was transferred to a fresh tube containing 48 μL of the reaction mixture previously described, except that 2.5 mM MgCl2 and 20 pmol of the inner primer pair was used (16S2A 5′-AGT CAA ACG GGA TGT AGC AAT AC-3′ and 16S2B 5′-GGT ATT CTT TCT GAT ATC AAC AG-3′ positions 66–720).

Nucleic Acid Electrophoresis:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: Digested plasmids were purified by gel electrophoresis and isolated from gel fragments using the Zymoclean Gel DNA recovery kit (Zymo Research). .. Hybridization was performed in a thermocycler (MJ Research).

Article Title: Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips
Article Snippet: .. The optimum temperature was evaluated by conventionally amplifying the NTC and target at three different temperatures, 65 °C, 68 °C, and 70 °C for 30 minutes using a thermocycler (MJ Research, Waltham, MA, USA), followed by refrigeration at 4 °C in preparation for gel electrophoresis confirmation. .. Multiple paper types were tested for optimizing the paper microfluidic RT-LAMP assay.

Article Title: Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips
Article Snippet: .. Temperature optimization The optimum temperature was evaluated by conventionally amplifying the NTC and target at three different temperatures, 65 °C, 68 °C, and 70 °C for 30 minutes using a thermocycler (MJ Research, Waltham, MA, USA), followed by refrigeration at 4 °C in preparation for gel electrophoresis confirmation. .. Paper type optimization Multiple paper types were tested for optimizing the paper microfluidic RT-LAMP assay.

Isolation:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: Digested plasmids were purified by gel electrophoresis and isolated from gel fragments using the Zymoclean Gel DNA recovery kit (Zymo Research). .. Hybridization was performed in a thermocycler (MJ Research).

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Article Snippet: Polymerase Chain Reaction (PCR) RNA was isolated from freshly isolated equine RPE and cultured RPE of passage-4 using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. .. The thermocycler, which was used for PCR, was a Tetrad PTC-225 Thermal Cycler (MJ research, St. Bruno (Quebec), Canada).

Negative Control:

Article Title: Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes
Article Snippet: PCR amplification was performed in a thermocycler (PTC-150 Minicycler PCR system; MJ Research Inc., Waltham, MA, USA) using rTaq polymerase (Takara) in a 25 μL reaction mixture consisting of an initial 5 min denaturation step at 95 °C, 35 cycles of 95 °C for 45 s, 55–58 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C. .. To conform that the RT-PCR signals derived not from genomic DNA, for each gene tested a negative control identical to the test assay using a RNA sample was included.

Size-exclusion Chromatography:

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min. .. The sequencing reaction products were precipitated by adding 40 μ L of 75% isopropanol and centrifugation R-5810 centrifuge (Eppendorf) at 3220 ×g for 30 min at room temperature.

Article Title: Genital mycoplasma trachomatis infections in treatment na?ve HIV-1 infected adults
Article Snippet: .. The PCR conditions used were 1 cycle of initial denaturation at 95°C for 10 min, followed by 35, two-step cycles of 95°C for 15 sec, 60°C for 1 min, and followed by 5 min at 72°C in a thermocycler (MJ Research, Waltham, MA). ..

Purification:

Article Title: White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus
Article Snippet: A fragment of the rDNA containing the ITS regions 1 and 2 and the 5.8S ribosomal RNA (rRNA) gene was amplified by polymerase chain reaction (PCR) using the primer combinations ITS4 and ITS5 (White et al., ) in an automated thermocycler (Minicycler PTC-150, MJ Research, USA). .. The PCR products were used directly after purification (QIAquick PCR Purification Kit, Qiagen) for DNA sequencing on an Applied Biosystems 3730 DNA Analyzer (PE Applied Biosystems, Foster City, California, USA).

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: Digested plasmids were purified by gel electrophoresis and isolated from gel fragments using the Zymoclean Gel DNA recovery kit (Zymo Research). .. Hybridization was performed in a thermocycler (MJ Research).

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: The positive PCR products were purified with the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI, USA—Cat. no. A7170) and subjected to sequencing reaction by chain termination with dideoxynucleotides marked with fluorophores. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

Article Title: Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites
Article Snippet: The extension and amplification were carried out in a thermocycler (MJ Research, Inc.) programmed for three cycles, with each cycle consisting of 5 min of denaturation at 95°C, 1 min of annealing at 50°C, and 2 min of extension at 72°C. .. The final PCR products from the two separate reactions were digested with Nde I and Bam HI, gel purified, and then ligated to pT7-7/H-IN previously cut with Nde I and Bam HI to form pE2C/IN1-288 and pE2C/IN11-288, respectively.

Polymerase Chain Reaction:

Article Title: White-spot disease of Chinese soft-shelled turtles (Trionyx sinens) caused by Paecilomyces lilacinus
Article Snippet: .. A fragment of the rDNA containing the ITS regions 1 and 2 and the 5.8S ribosomal RNA (rRNA) gene was amplified by polymerase chain reaction (PCR) using the primer combinations ITS4 and ITS5 (White et al., ) in an automated thermocycler (Minicycler PTC-150, MJ Research, USA). .. The PCR products were used directly after purification (QIAquick PCR Purification Kit, Qiagen) for DNA sequencing on an Applied Biosystems 3730 DNA Analyzer (PE Applied Biosystems, Foster City, California, USA).

Article Title: Interleukin-6 mediated upregulation of CYP1B1 and CYP2E1 in colorectal cancer involves DNA methylation, miR27b and STAT3
Article Snippet: Each sample was incubated with 0.5 mM dNTPs, 1 × first strand buffer, 8 μ M dithiothreitol and 100 units of Superscript II reverse transcriptase (Invitrogen, Paisley, UK) for 10 min at 25 °C, 90 min at 42 °C and 15 min at 70 °C on a thermocycler (Peltier Thermal Cycler PTC-200, MJ Research, Waltham, MA, USA). .. Quantitative PCR was performed using pre-designed gene expression assays and FAST PCR master mix (Taqman, Applied Biosystems, Life Technologies), and measured in a StepOnePlus fast real-time PCR system (Applied Biosystems, Life Technologies) according to the manufacturer's protocol.

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: The total RNA of MDM after 0 and 24 h of mock- or PCV2-inoculation was extracted using TRIzol reagent (Life Technologies, Paisley, Scotland, UK), reverse-transcribed to cDNA, and amplified by PCR. .. Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation.

Article Title: Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes
Article Snippet: .. PCR amplification was performed in a thermocycler (PTC-150 Minicycler PCR system; MJ Research Inc., Waltham, MA, USA) using rTaq polymerase (Takara) in a 25 μL reaction mixture consisting of an initial 5 min denaturation step at 95 °C, 35 cycles of 95 °C for 45 s, 55–58 °C for 30 s, 72 °C for 1 min, and a final extension for 10 min at 72 °C. .. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a positive control to confirm the presence of cDNA [ ].

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Article Snippet: .. The thermocycler, which was used for PCR, was a Tetrad PTC-225 Thermal Cycler (MJ research, St. Bruno (Quebec), Canada). .. PCR, which was performed on samples that were reverse transcribed to cDNA in the absence of reverse transcriptase, did not show any amplified product.

Article Title: Relapsing Fever-Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent
Article Snippet: Aliquots of DNA suspensions (2 μL) were diluted to 50 μL by using 200 μM of each deoxynucleoside triphosphate, 1.5 mM MgCl2 , 0.5 U Taq polymerase (Qiagen QmbH) as well as 15 pmol of the outer primer pair and PCR-buffer supplied with the Taq polymerase. .. The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess, Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s denaturation at 94°C, 20 s each for the first annealing reaction at 63°C with a 40 s extension at 72°C and a final extension for 2 min at 72°C.

Article Title: Genotyping 1000 yeast strains by next-generation sequencing
Article Snippet: .. The PCR was performed on a thermocycler (MJ Research tetrad) containing 1x Phusion Master Mix with HF Buffer (Thermo Scientific) and 0.2 μM Illumina PE 1.0 and 2.0 primers. .. The low primer concentration reduced the formation of primer dimers often observed at standard primer concentrations (1.25 μM).

Article Title: " Candidatus Neoehrlichia mikurensis," Anaplasma phagocytophilum, and Lyme Disease Spirochetes in Questing European Vector Ticks and in Feeding Ticks Removed from People
Article Snippet: Neoehrlichia mikurensis” and A. phagocytophilum in a random subsample of these questing ticks, as well as in ticks removed from patients, we amplified a fragment of the 16S rRNA gene for each of the first two pathogens by nested PCR and a fragment of the p44 gene for A. phagocytophilum by conventional PCR (for primers and references see ). .. The mixture was placed in a thermocycler (PTC 200; MJ Research Biozym, Diagnostic, Hess.

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: The reaction was performed with 5.68 μ L of the PCR product, 2 μ L reagent Big Dye 3.1 (Applied Biosystems), 2 μ L of dilution buffer (0.2 M Tris-HCl pH 9.0, 5 μ L MgCl2 ), and 0.32 μ L of a primer used in PCR (final concentration 0.32 μ L) to a total volume of 10 μ L. Each sample was sequenced in both directions using forward and reverse primers. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

Article Title: Fusion Proteins Consisting of Human Immunodeficiency Virus Type 1 Integrase and the Designed Polydactyl Zinc Finger Protein E2C Direct Integration of Viral DNA into Specific Sites
Article Snippet: The two PCR products containing a common overlapping region were annealed together, extended, and amplified in the presence of 0.2 mM deoxyribonucleoside triphosphates (United States Biochemicals), 2.5 U of Pfu polymerase ( Pfu Turbo; Stratagene), and E2CF1 and INR as the forward and reverse primers, respectively. .. The extension and amplification were carried out in a thermocycler (MJ Research, Inc.) programmed for three cycles, with each cycle consisting of 5 min of denaturation at 95°C, 1 min of annealing at 50°C, and 2 min of extension at 72°C.

Article Title: Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases
Article Snippet: 5 μl of the CCM were deposited in a well of an 8-well PCR strip kept on ice. .. Prior to reverse transcription and template switching the strip was thawed by placing it in a thermocycler (MJ Research PTC-200 now a part of Bio-Rad; Hercules, CA, USA) at 20°C for 5 min.

Article Title: Genital mycoplasma trachomatis infections in treatment na?ve HIV-1 infected adults
Article Snippet: .. The PCR conditions used were 1 cycle of initial denaturation at 95°C for 10 min, followed by 35, two-step cycles of 95°C for 15 sec, 60°C for 1 min, and followed by 5 min at 72°C in a thermocycler (MJ Research, Waltham, MA). ..

Nested PCR:

Article Title: Relapsing Fever-Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent
Article Snippet: To increase the sensitivity for spirochetal DNA detection in ticks, we used nested PCR. .. The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess, Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s denaturation at 94°C, 20 s each for the first annealing reaction at 63°C with a 40 s extension at 72°C and a final extension for 2 min at 72°C.

Article Title: " Candidatus Neoehrlichia mikurensis," Anaplasma phagocytophilum, and Lyme Disease Spirochetes in Questing European Vector Ticks and in Feeding Ticks Removed from People
Article Snippet: Neoehrlichia mikurensis” and A. phagocytophilum in a random subsample of these questing ticks, as well as in ticks removed from patients, we amplified a fragment of the 16S rRNA gene for each of the first two pathogens by nested PCR and a fragment of the p44 gene for A. phagocytophilum by conventional PCR (for primers and references see ). .. The mixture was placed in a thermocycler (PTC 200; MJ Research Biozym, Diagnostic, Hess.

Activated Clotting Time Assay:

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: The primer sets used included porcine MIP-1 (sense: 5′-GCT CAG TTC AGT TCC AAG TC-3′, antisense: 5′-ACC ATG AAG CTC TGC GTG AC-3′) [ ], porcine MCP-1 (sense: 5′-TCA CCA GCA GCA AGT GTC-3′, antisense: 5′-CTG AGA TTC ACA GAG GA-3′) [ ], and internal controls including porcine G3PDH (sense: 5′-ACC TCC ACT ACA TGG TCT ACA TGT TC-3′, antisense: 5′-CAT TGA TGA CAA GCT TCC CAT TC-3′), and β-actin (sense: 5′-CAT CAC CAT CGG CAA CGA-3′, antisense: 5′-GCG TAG AGG TCC TTC CTG ATG T-3′) [ ]. .. Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation.

Plasmid Preparation:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: A stock of digested sgRNA expression plasmid was used for cloning custom 20mer guide sequences (see below). .. Hybridization was performed in a thermocycler (MJ Research).

Article Title: Genital mycoplasma trachomatis infections in treatment na?ve HIV-1 infected adults
Article Snippet: The PCR conditions used were 1 cycle of initial denaturation at 95°C for 10 min, followed by 35, two-step cycles of 95°C for 15 sec, 60°C for 1 min, and followed by 5 min at 72°C in a thermocycler (MJ Research, Waltham, MA). .. PCR for C. trachomatis: C. trachomatis DNA was detected by PCR targeting a sequence of the cryptic plasmid using primers KL-1 and KL-2 .

Electrophoresis:

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation. .. The PCR products were separated on a 2% agarose gel in TBE (Genmedika Biotechnology Corp., Taipei, Taiwan) by electrophoresis and stained with ethidium bromide (Sigma).

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Article Snippet: The thermocycler, which was used for PCR, was a Tetrad PTC-225 Thermal Cycler (MJ research, St. Bruno (Quebec), Canada). .. Amplified products were separated by 2% agarose gel electrophoresis, stained with Serva DNA Stain G (Serva Electrophoresis GmbH, Heidelberg, Germany) and visualized with UV illumination (Herolab UVT-40M, Wiesloch, Germany) ( ).

Article Title: Relapsing Fever-Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent
Article Snippet: The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess, Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s denaturation at 94°C, 20 s each for the first annealing reaction at 63°C with a 40 s extension at 72°C and a final extension for 2 min at 72°C. .. This mixture was subjected to 35 amplification cycles by using the cycle conditions described previously, except that the annealing reaction was performed at 56°C and the extension reaction lasted 30 s. DNA was extracted, reaction vials were prepared for amplification, templates were added, and products underwent electrophoresis in separate rooms.

Article Title: " Candidatus Neoehrlichia mikurensis," Anaplasma phagocytophilum, and Lyme Disease Spirochetes in Questing European Vector Ticks and in Feeding Ticks Removed from People
Article Snippet: The mixture was placed in a thermocycler (PTC 200; MJ Research Biozym, Diagnostic, Hess. .. PCR products were detected by electrophoresis in a 1.5% agarose gel stained with ethidium bromide.

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: Analysis of amplified products was by electrophoresis (100 V/60 min) agarose gel at 1.5% in Tris plug, sodium acetate, EDTA pH 8.0, and stained with ethidium bromide. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

Multiplex Assay:

Article Title: Genital mycoplasma trachomatis infections in treatment na?ve HIV-1 infected adults
Article Snippet: Multiplex PCR for Ureaplasma spp. and M. hominis : Multiplex PCR targeting the urease gene of Ureaplasma spp. and 16S rDNA of M. hominis was used to detect the presence of DNA of these two organisms using the protocol by Stellrecht et al . .. The PCR conditions used were 1 cycle of initial denaturation at 95°C for 10 min, followed by 35, two-step cycles of 95°C for 15 sec, 60°C for 1 min, and followed by 5 min at 72°C in a thermocycler (MJ Research, Waltham, MA).

Agarose Gel Electrophoresis:

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation. .. The PCR products were separated on a 2% agarose gel in TBE (Genmedika Biotechnology Corp., Taipei, Taiwan) by electrophoresis and stained with ethidium bromide (Sigma).

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Article Snippet: The thermocycler, which was used for PCR, was a Tetrad PTC-225 Thermal Cycler (MJ research, St. Bruno (Quebec), Canada). .. Amplified products were separated by 2% agarose gel electrophoresis, stained with Serva DNA Stain G (Serva Electrophoresis GmbH, Heidelberg, Germany) and visualized with UV illumination (Herolab UVT-40M, Wiesloch, Germany) ( ).

Article Title: " Candidatus Neoehrlichia mikurensis," Anaplasma phagocytophilum, and Lyme Disease Spirochetes in Questing European Vector Ticks and in Feeding Ticks Removed from People
Article Snippet: The mixture was placed in a thermocycler (PTC 200; MJ Research Biozym, Diagnostic, Hess. .. PCR products were detected by electrophoresis in a 1.5% agarose gel stained with ethidium bromide.

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: Analysis of amplified products was by electrophoresis (100 V/60 min) agarose gel at 1.5% in Tris plug, sodium acetate, EDTA pH 8.0, and stained with ethidium bromide. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

Concentration Assay:

Article Title: New Vectors for Simple and Streamlined CRISPR-Cas9 Genome Editing in Saccharomyces cerevisiae
Article Snippet: For TRP1 gene targeting, oligonucleotides OWY251 and OWY252 were hybridized at a concentration of 3 μM in a solution of 1× T4 DNA ligase Buffer (50mM Tris-HCl, 10mM MgCl2, 1mM ATP, 10mM DTT, pH 7.5). .. Hybridization was performed in a thermocycler (MJ Research).

Article Title: Genotyping 1000 yeast strains by next-generation sequencing
Article Snippet: The PCR was performed on a thermocycler (MJ Research tetrad) containing 1x Phusion Master Mix with HF Buffer (Thermo Scientific) and 0.2 μM Illumina PE 1.0 and 2.0 primers. .. The low primer concentration reduced the formation of primer dimers often observed at standard primer concentrations (1.25 μM).

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: The reaction was performed with 5.68 μ L of the PCR product, 2 μ L reagent Big Dye 3.1 (Applied Biosystems), 2 μ L of dilution buffer (0.2 M Tris-HCl pH 9.0, 5 μ L MgCl2 ), and 0.32 μ L of a primer used in PCR (final concentration 0.32 μ L) to a total volume of 10 μ L. Each sample was sequenced in both directions using forward and reverse primers. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

DNA Purification:

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: The positive PCR products were purified with the Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI, USA—Cat. no. A7170) and subjected to sequencing reaction by chain termination with dideoxynucleotides marked with fluorophores. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

CTG Assay:

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: The primer sets used included porcine MIP-1 (sense: 5′-GCT CAG TTC AGT TCC AAG TC-3′, antisense: 5′-ACC ATG AAG CTC TGC GTG AC-3′) [ ], porcine MCP-1 (sense: 5′-TCA CCA GCA GCA AGT GTC-3′, antisense: 5′-CTG AGA TTC ACA GAG GA-3′) [ ], and internal controls including porcine G3PDH (sense: 5′-ACC TCC ACT ACA TGG TCT ACA TGT TC-3′, antisense: 5′-CAT TGA TGA CAA GCT TCC CAT TC-3′), and β-actin (sense: 5′-CAT CAC CAT CGG CAA CGA-3′, antisense: 5′-GCG TAG AGG TCC TTC CTG ATG T-3′) [ ]. .. Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation.

Article Title: Relapsing Fever-Like Spirochetes Infecting European Vector Tick of Lyme Disease Agent
Article Snippet: We used the following primer sequences of the 16S rRNA gene as outer primers ( ): 16S1A 5′-CTA ACG CTG GCA GTG CGT CTT AAG C-3′ and 16S1B 5′-AGC GTC AGT CTT GAC CCA GAA GTT C-3′ (positions 36–757). .. The mixture was placed in a thermocycler (PTC 200, MJResearch, Biozym Diagnostic, Hess, Oldendorf, Germany), heated for 1 min at 94°C, and subjected to 30 cycles of 20 s denaturation at 94°C, 20 s each for the first annealing reaction at 63°C with a 40 s extension at 72°C and a final extension for 2 min at 72°C.

Staining:

Article Title: Porcine circovirus type 2 (PCV2) induces cell proliferation, fusion, and chemokine expression in swine monocytic cells in vitro
Article Snippet: Amplifications were performed with a thermocycler (MJ Research, Watertown, MA, USA) using 40 cycles (95 °C for 45 s, 62 °C for 45 s, and 72 °C for 1 min) for MIP-1 and MCP-1, and 40 cycles (95 °C for 15 s, 60 °C for 1 min, and 72 °C for 1 min) for G3PDH and β-actin, followed by additional extension at 72 °C for 5 min at the end of amplification for each preparation. .. The PCR products were separated on a 2% agarose gel in TBE (Genmedika Biotechnology Corp., Taipei, Taiwan) by electrophoresis and stained with ethidium bromide (Sigma).

Article Title: Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Article Snippet: The thermocycler, which was used for PCR, was a Tetrad PTC-225 Thermal Cycler (MJ research, St. Bruno (Quebec), Canada). .. Amplified products were separated by 2% agarose gel electrophoresis, stained with Serva DNA Stain G (Serva Electrophoresis GmbH, Heidelberg, Germany) and visualized with UV illumination (Herolab UVT-40M, Wiesloch, Germany) ( ).

Article Title: " Candidatus Neoehrlichia mikurensis," Anaplasma phagocytophilum, and Lyme Disease Spirochetes in Questing European Vector Ticks and in Feeding Ticks Removed from People
Article Snippet: The mixture was placed in a thermocycler (PTC 200; MJ Research Biozym, Diagnostic, Hess. .. PCR products were detected by electrophoresis in a 1.5% agarose gel stained with ethidium bromide.

Article Title: Detection of Bovine Leukemia Virus in Brains of Cattle with a Neurological Syndrome: Pathological and Molecular Studies
Article Snippet: Analysis of amplified products was by electrophoresis (100 V/60 min) agarose gel at 1.5% in Tris plug, sodium acetate, EDTA pH 8.0, and stained with ethidium bromide. .. The samples were incubated in a thermocycler (PTC-100, MJ Research) for 25 cycles at 95°C for 10 sec and 60°C for 4 min.

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    MJ Research mj research thermocycler
    Performing multiplex PCR, BaNC-HDC separation, and online dye intercalation on an integrated microfluidic platform. Panel (A) shows BaNC-HDC separation traces; the top trace was obtained from separating a 1-kbp-plus DNA ladder, and the bottom trace was obtained from separating the multiplex PCR products amplified in the capillary <t>thermocycler</t> (30 cycles; see Capillary PCR in the SI for details). The BaNC-HDC separations were executed under ∼200 psi. Panel (B) shows the slab–gel electrophoresis of the crude multiplex PCR products amplified by a MJ Research thermocycler (30 cycles). (“Lane 1” shows data from the DNA ladder; “Lane 2” shows data from crude PCR multiplex products.) Panel (C) shows BaNC-HDC separation of a PCR-amplified genomic region (30 cycles; see Capillary PCR in the SI for details). The top trace represents the separation of a 1-kbp-plus DNA ladder, and the bottom trace is the result of the separation of an actin sequence from a rice genomic DNA that was amplified by the capillary PCR and online-intercalated with YOYO-1. The BaNC-HDC separations were executed under ∼400 psi. Other conditions were similar to those described for Panel (A). Panel (D) shows the slab–gel electrophoresis of a conventional PCR-amplified actin sequence. (“Lane 1” shows data from the DNA ladder, and “Lane 2” shows data from the actin.) Other conditions were similar to those described for Panel (B).
    Mj Research Thermocycler, supplied by MJ Research, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    MJ Research mj research ptc 200 thermocycler
    RT-PCR results for 5'-termini of trypsin and collagenase. A. Agarose gel electrophoresis of PCR products generated using 5'-RACE primer for trypsin and step-out primer system. B. Agarose gel electrophoresis of PCR products generated using 5'-RACE primer for collagenase and step-out primer system. About 1 ng of the first strand cDNA was used as a starting material for PCR reaction. Cycling was performed in a MJ Research <t>PTC-200</t> <t>Thermocycler</t> in calculated mode: 25 cycles for trypsin and 26 cycles for collagenase were made using cycling profile: 95°C for 8 s, 65°C for 10 s, and 72°C for 2 min.
    Mj Research Ptc 200 Thermocycler, supplied by MJ Research, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mj research ptc 200 thermocycler - by Bioz Stars, 2020-02
    99/100 stars
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    78
    MJ Research conventional thermocycling
    Thermal characteristics and reproducibility of device. ( A ) Temperature color map of the heat gradient established between heaters with a maximum of 100°C on the left and a minimum of 50°C on the right. ( B ) Heat ramping of the two extreme temperature regions from 25°C to equilibrium within 10 min. ( C ) Representative <t>thermocycling</t> profile of the internal droplet temperature and surrounding oil temperature. Desired temperatures are consistently achieved even at sub-minute cycle times. The temperatures at each phase are 90.4° ± 0.2°C for denaturation, 68.4° ± 0.2°C for extension, and 60.2° ± 0.2°C for annealing. Droplet ramp rates up to 12°C/s and oil ramp rates up to 32°C/s are achieved by moving the droplet within the heat gradient. ( D ) Gel electropherogram showing the results from three successive trials (lanes 1 to 3) to amplify the 196-bp 16 S rRNA V3 amplicon from 7 pg of purified K. pneumoniae genomic DNA (equivalent to 1.4 × 10 3 genomic copies) and an NTC sample. The thermocycling speed was 48 s/cycle, and 30 cycles were conducted. The band intensities in lanes 1 to 3 have a coefficient of variation of 4.0%.
    Conventional Thermocycling, supplied by MJ Research, 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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    Image Search Results


    Performing multiplex PCR, BaNC-HDC separation, and online dye intercalation on an integrated microfluidic platform. Panel (A) shows BaNC-HDC separation traces; the top trace was obtained from separating a 1-kbp-plus DNA ladder, and the bottom trace was obtained from separating the multiplex PCR products amplified in the capillary thermocycler (30 cycles; see Capillary PCR in the SI for details). The BaNC-HDC separations were executed under ∼200 psi. Panel (B) shows the slab–gel electrophoresis of the crude multiplex PCR products amplified by a MJ Research thermocycler (30 cycles). (“Lane 1” shows data from the DNA ladder; “Lane 2” shows data from crude PCR multiplex products.) Panel (C) shows BaNC-HDC separation of a PCR-amplified genomic region (30 cycles; see Capillary PCR in the SI for details). The top trace represents the separation of a 1-kbp-plus DNA ladder, and the bottom trace is the result of the separation of an actin sequence from a rice genomic DNA that was amplified by the capillary PCR and online-intercalated with YOYO-1. The BaNC-HDC separations were executed under ∼400 psi. Other conditions were similar to those described for Panel (A). Panel (D) shows the slab–gel electrophoresis of a conventional PCR-amplified actin sequence. (“Lane 1” shows data from the DNA ladder, and “Lane 2” shows data from the actin.) Other conditions were similar to those described for Panel (B).

    Journal: Analytical Chemistry

    Article Title: Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis

    doi: 10.1021/ac504257b

    Figure Lengend Snippet: Performing multiplex PCR, BaNC-HDC separation, and online dye intercalation on an integrated microfluidic platform. Panel (A) shows BaNC-HDC separation traces; the top trace was obtained from separating a 1-kbp-plus DNA ladder, and the bottom trace was obtained from separating the multiplex PCR products amplified in the capillary thermocycler (30 cycles; see Capillary PCR in the SI for details). The BaNC-HDC separations were executed under ∼200 psi. Panel (B) shows the slab–gel electrophoresis of the crude multiplex PCR products amplified by a MJ Research thermocycler (30 cycles). (“Lane 1” shows data from the DNA ladder; “Lane 2” shows data from crude PCR multiplex products.) Panel (C) shows BaNC-HDC separation of a PCR-amplified genomic region (30 cycles; see Capillary PCR in the SI for details). The top trace represents the separation of a 1-kbp-plus DNA ladder, and the bottom trace is the result of the separation of an actin sequence from a rice genomic DNA that was amplified by the capillary PCR and online-intercalated with YOYO-1. The BaNC-HDC separations were executed under ∼400 psi. Other conditions were similar to those described for Panel (A). Panel (D) shows the slab–gel electrophoresis of a conventional PCR-amplified actin sequence. (“Lane 1” shows data from the DNA ladder, and “Lane 2” shows data from the actin.) Other conditions were similar to those described for Panel (B).

    Article Snippet: Figure B presents the slab-gel separation results; the multiplex PCR was performed using a conventional Eppendorf tube and a MJ Research thermocycler.

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis, Sequencing

    Schematic diagram of experimental setup for PCR-BaNC-HDC. Panel (A) illustrates the experimental setup. (Legend: S, sample; W, waste; C, microchip injector; PC, pressure chamber; and TC, thermocycler.) The solid dots depicted on the six-port injection valve indicate ports that are blocked. Capillaries are connected to positions 1, 2, 3, and 4 on microchip injector are separation capillary, PCR capillary, pressure capillary, and waste capillary, respectively. The separation capillary had a length of 55 cm (35 cm effective), an outer diameter (o.d.) of 150 μm, and an i.d. of 2 μm. The PCR capillary had a length of 2 m, an o.d. of 150 μm, and an i.d. of 75 μm. The pressure capillary had a length of 10 cm, an o.d. of 150 μm, and an i.d. of 20 μm. The waste capillary had a length of 30 cm, an o.d. of 150 μm, and an i.d. of 75 μm. Panels (B)–(G) depict schematic diagrams for illustrating major operating procedures; the arrows indicate the flow directions (see details in text).

    Journal: Analytical Chemistry

    Article Title: Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis

    doi: 10.1021/ac504257b

    Figure Lengend Snippet: Schematic diagram of experimental setup for PCR-BaNC-HDC. Panel (A) illustrates the experimental setup. (Legend: S, sample; W, waste; C, microchip injector; PC, pressure chamber; and TC, thermocycler.) The solid dots depicted on the six-port injection valve indicate ports that are blocked. Capillaries are connected to positions 1, 2, 3, and 4 on microchip injector are separation capillary, PCR capillary, pressure capillary, and waste capillary, respectively. The separation capillary had a length of 55 cm (35 cm effective), an outer diameter (o.d.) of 150 μm, and an i.d. of 2 μm. The PCR capillary had a length of 2 m, an o.d. of 150 μm, and an i.d. of 75 μm. The pressure capillary had a length of 10 cm, an o.d. of 150 μm, and an i.d. of 20 μm. The waste capillary had a length of 30 cm, an o.d. of 150 μm, and an i.d. of 75 μm. Panels (B)–(G) depict schematic diagrams for illustrating major operating procedures; the arrows indicate the flow directions (see details in text).

    Article Snippet: Figure B presents the slab-gel separation results; the multiplex PCR was performed using a conventional Eppendorf tube and a MJ Research thermocycler.

    Techniques: Polymerase Chain Reaction, MicroChIP Assay, Injection, Flow Cytometry

    Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional thermocycler, as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.

    Journal: Scientific Reports

    Article Title: Simpler, Faster, and Sensitive Zika Virus Assay Using Smartphone Detection of Loop-mediated Isothermal Amplification on Paper Microfluidic Chips

    doi: 10.1038/s41598-018-30797-9

    Figure Lengend Snippet: Optimization of temperature and paper type. (a) Temperature optimization among 65 °C, 68 °C and 70 °C using a conventional thermocycler, as confirmed with 3% w/v agarose gel electrophoresis. (b) Paper type optimization among NC (nitrocellulose), G4 (cellulose grade 4) and G1 (cellulose grade 1) papers, as confirmed with 3% w/v agarose gel electrophoresis.

    Article Snippet: Temperature optimization The optimum temperature was evaluated by conventionally amplifying the NTC and target at three different temperatures, 65 °C, 68 °C, and 70 °C for 30 minutes using a thermocycler (MJ Research, Waltham, MA, USA), followed by refrigeration at 4 °C in preparation for gel electrophoresis confirmation.

    Techniques: Agarose Gel Electrophoresis

    RT-PCR results for 5'-termini of trypsin and collagenase. A. Agarose gel electrophoresis of PCR products generated using 5'-RACE primer for trypsin and step-out primer system. B. Agarose gel electrophoresis of PCR products generated using 5'-RACE primer for collagenase and step-out primer system. About 1 ng of the first strand cDNA was used as a starting material for PCR reaction. Cycling was performed in a MJ Research PTC-200 Thermocycler in calculated mode: 25 cycles for trypsin and 26 cycles for collagenase were made using cycling profile: 95°C for 8 s, 65°C for 10 s, and 72°C for 2 min.

    Journal: BMC Structural Biology

    Article Title: Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes

    doi: 10.1186/1472-6807-4-2

    Figure Lengend Snippet: RT-PCR results for 5'-termini of trypsin and collagenase. A. Agarose gel electrophoresis of PCR products generated using 5'-RACE primer for trypsin and step-out primer system. B. Agarose gel electrophoresis of PCR products generated using 5'-RACE primer for collagenase and step-out primer system. About 1 ng of the first strand cDNA was used as a starting material for PCR reaction. Cycling was performed in a MJ Research PTC-200 Thermocycler in calculated mode: 25 cycles for trypsin and 26 cycles for collagenase were made using cycling profile: 95°C for 8 s, 65°C for 10 s, and 72°C for 2 min.

    Article Snippet: Cycling was performed in a MJ Research PTC-200 Thermocycler in calculated mode: 25 cycles for trypsin and 26 cycles for collagenase were made using cycling profile: 95°C for 8 s, 65°C for 10 s, and 72°C for 2 min. PCR products were cloned into pT-Adv vector (Clontech) and sequenced using M13 direct and reverse universal primers by using a Beckman SEQ-2000 automated sequencer and the FS dye terminator chemistry.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Generated

    Thermal characteristics and reproducibility of device. ( A ) Temperature color map of the heat gradient established between heaters with a maximum of 100°C on the left and a minimum of 50°C on the right. ( B ) Heat ramping of the two extreme temperature regions from 25°C to equilibrium within 10 min. ( C ) Representative thermocycling profile of the internal droplet temperature and surrounding oil temperature. Desired temperatures are consistently achieved even at sub-minute cycle times. The temperatures at each phase are 90.4° ± 0.2°C for denaturation, 68.4° ± 0.2°C for extension, and 60.2° ± 0.2°C for annealing. Droplet ramp rates up to 12°C/s and oil ramp rates up to 32°C/s are achieved by moving the droplet within the heat gradient. ( D ) Gel electropherogram showing the results from three successive trials (lanes 1 to 3) to amplify the 196-bp 16 S rRNA V3 amplicon from 7 pg of purified K. pneumoniae genomic DNA (equivalent to 1.4 × 10 3 genomic copies) and an NTC sample. The thermocycling speed was 48 s/cycle, and 30 cycles were conducted. The band intensities in lanes 1 to 3 have a coefficient of variation of 4.0%.

    Journal: Science Advances

    Article Title: Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

    doi: 10.1126/sciadv.1400061

    Figure Lengend Snippet: Thermal characteristics and reproducibility of device. ( A ) Temperature color map of the heat gradient established between heaters with a maximum of 100°C on the left and a minimum of 50°C on the right. ( B ) Heat ramping of the two extreme temperature regions from 25°C to equilibrium within 10 min. ( C ) Representative thermocycling profile of the internal droplet temperature and surrounding oil temperature. Desired temperatures are consistently achieved even at sub-minute cycle times. The temperatures at each phase are 90.4° ± 0.2°C for denaturation, 68.4° ± 0.2°C for extension, and 60.2° ± 0.2°C for annealing. Droplet ramp rates up to 12°C/s and oil ramp rates up to 32°C/s are achieved by moving the droplet within the heat gradient. ( D ) Gel electropherogram showing the results from three successive trials (lanes 1 to 3) to amplify the 196-bp 16 S rRNA V3 amplicon from 7 pg of purified K. pneumoniae genomic DNA (equivalent to 1.4 × 10 3 genomic copies) and an NTC sample. The thermocycling speed was 48 s/cycle, and 30 cycles were conducted. The band intensities in lanes 1 to 3 have a coefficient of variation of 4.0%.

    Article Snippet: Conventional thermocycling Conventional thermocycling was conducted using the MJ Research MiniCycler.

    Techniques: Amplification, Purification

    Specificity, limit of detection, and speed of DOT thermocycling. ( A ) Gel electropherogram showing the differentiation of vancomycin-resistant E. faecium (VRE) and vancomycin-sensitive E. faecalis (VSE) by multiplexed amplification of the 377-bp vanA amplicon directly from bacterial culture. Simultaneous thermocycling was achieved by mounting three droplets on different thermocouples on the same motor arm. Lane 1, 1-kb Plus DNA Ladder; lane 2, VRE; lane 3, VSE; lane 4, NTC; lane 5, 1-kb Plus DNA Ladder. ( B ) Gel electropherogram showing the limit of detection at the sub-picogram level by amplification of the 196-bp 16 S rRNA V3 amplicon from 0.7 pg of K. pneumoniae genomic DNA (equivalent to 1.4 × 10 2 genomic copies) at a speed of 48 s/cycle. Lane 1, 1-kb Plus DNA Ladder; lane 2, 0.7-pg genomic DNA. ( C ) Gel electropherogram showing rapid amplification of the 16 S rRNA V3 amplicon and vanA amplicon in the presence of tissue contaminants in 30 cycles. Lane 1, vanA amplified at 40 s/cycle (20 min) from 7 × 10 5 CFU VRE inoculated to valve tissue (V3 amplified from 7 × 10 5 CFU VRE inoculated to valve tissue); lane 2, at 40 s/cycle (20 min); lane 3, at 32 s/cycle (16 min); lane 4, at 28 s/cycle (14 min); lane 5, 1-kb Plus DNA Ladder (see Fig. 2D for V3 NTC results).

    Journal: Science Advances

    Article Title: Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

    doi: 10.1126/sciadv.1400061

    Figure Lengend Snippet: Specificity, limit of detection, and speed of DOT thermocycling. ( A ) Gel electropherogram showing the differentiation of vancomycin-resistant E. faecium (VRE) and vancomycin-sensitive E. faecalis (VSE) by multiplexed amplification of the 377-bp vanA amplicon directly from bacterial culture. Simultaneous thermocycling was achieved by mounting three droplets on different thermocouples on the same motor arm. Lane 1, 1-kb Plus DNA Ladder; lane 2, VRE; lane 3, VSE; lane 4, NTC; lane 5, 1-kb Plus DNA Ladder. ( B ) Gel electropherogram showing the limit of detection at the sub-picogram level by amplification of the 196-bp 16 S rRNA V3 amplicon from 0.7 pg of K. pneumoniae genomic DNA (equivalent to 1.4 × 10 2 genomic copies) at a speed of 48 s/cycle. Lane 1, 1-kb Plus DNA Ladder; lane 2, 0.7-pg genomic DNA. ( C ) Gel electropherogram showing rapid amplification of the 16 S rRNA V3 amplicon and vanA amplicon in the presence of tissue contaminants in 30 cycles. Lane 1, vanA amplified at 40 s/cycle (20 min) from 7 × 10 5 CFU VRE inoculated to valve tissue (V3 amplified from 7 × 10 5 CFU VRE inoculated to valve tissue); lane 2, at 40 s/cycle (20 min); lane 3, at 32 s/cycle (16 min); lane 4, at 28 s/cycle (14 min); lane 5, 1-kb Plus DNA Ladder (see Fig. 2D for V3 NTC results).

    Article Snippet: Conventional thermocycling Conventional thermocycling was conducted using the MJ Research MiniCycler.

    Techniques: Amplification

    Decrease in droplet height against cycle number. ( A ) Real-time detection of 16 S rRNA amplification during early cycles by DOTS qPCR at a thermocycling speed of 48 s per cycle. Percent decrease in droplet height is plotted against C n for amplifications from 750, 75, 7.5, and 0.75 pg of genomic DNA (1.5 × 10 5 , 1.5 × 10 4 , 1.5 × 10 3 , and 1.5 × 10 2 genomic copies, respectively) and NTC. Error bars represent overall device noise. A 4.8% threshold for detection is also plotted. The threshold was chosen to optimize the R 2 value of the linear regression shown in Fig. 8 . ( B ) Smartphone camera images of the DOT submerged in oil. Images were taken every 5 thermal cycles and used to determine the droplet height.

    Journal: Science Advances

    Article Title: Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

    doi: 10.1126/sciadv.1400061

    Figure Lengend Snippet: Decrease in droplet height against cycle number. ( A ) Real-time detection of 16 S rRNA amplification during early cycles by DOTS qPCR at a thermocycling speed of 48 s per cycle. Percent decrease in droplet height is plotted against C n for amplifications from 750, 75, 7.5, and 0.75 pg of genomic DNA (1.5 × 10 5 , 1.5 × 10 4 , 1.5 × 10 3 , and 1.5 × 10 2 genomic copies, respectively) and NTC. Error bars represent overall device noise. A 4.8% threshold for detection is also plotted. The threshold was chosen to optimize the R 2 value of the linear regression shown in Fig. 8 . ( B ) Smartphone camera images of the DOT submerged in oil. Images were taken every 5 thermal cycles and used to determine the droplet height.

    Article Snippet: Conventional thermocycling Conventional thermocycling was conducted using the MJ Research MiniCycler.

    Techniques: Amplification, Real-time Polymerase Chain Reaction