Structured Review

PerkinElmer dntp
Agarose gel electrophoresis of PCRs. The first tier (from the top) of the gel represents the amplicons produced from the QRDR sequencing primers. The second tier shows the amplification products produced with primers JL238 and JL239. Markers (100 bp) were loaded in the first lanes. Each lane was loaded with 20 μl of a 50-μl <t>PCR</t> mixture. The PCR conditions were as specified in the text with the exceptions that Platinum Taq DNA polymerase (Life Technologies, Gibco BRL, Grand Island, N.Y.) was the enzyme used, the concentration of each <t>dNTP</t> was 0.2 mM, and the MgCl 2 concentration was 1.5 mM. TaqMan probes and buffer, Amperase UNG, Tween 20, and gelatin were not included in these reactions.
Dntp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 97/100, based on 357 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dntp/product/PerkinElmer
Average 97 stars, based on 357 article reviews
Price from $9.99 to $1999.99
dntp - by Bioz Stars, 2020-03
97/100 stars

Images

1) Product Images from "Identification of Ciprofloxacin-Resistant Campylobacter jejuni by Use of a Fluorogenic PCR Assay"

Article Title: Identification of Ciprofloxacin-Resistant Campylobacter jejuni by Use of a Fluorogenic PCR Assay

Journal: Journal of Clinical Microbiology

doi:

Agarose gel electrophoresis of PCRs. The first tier (from the top) of the gel represents the amplicons produced from the QRDR sequencing primers. The second tier shows the amplification products produced with primers JL238 and JL239. Markers (100 bp) were loaded in the first lanes. Each lane was loaded with 20 μl of a 50-μl PCR mixture. The PCR conditions were as specified in the text with the exceptions that Platinum Taq DNA polymerase (Life Technologies, Gibco BRL, Grand Island, N.Y.) was the enzyme used, the concentration of each dNTP was 0.2 mM, and the MgCl 2 concentration was 1.5 mM. TaqMan probes and buffer, Amperase UNG, Tween 20, and gelatin were not included in these reactions.
Figure Legend Snippet: Agarose gel electrophoresis of PCRs. The first tier (from the top) of the gel represents the amplicons produced from the QRDR sequencing primers. The second tier shows the amplification products produced with primers JL238 and JL239. Markers (100 bp) were loaded in the first lanes. Each lane was loaded with 20 μl of a 50-μl PCR mixture. The PCR conditions were as specified in the text with the exceptions that Platinum Taq DNA polymerase (Life Technologies, Gibco BRL, Grand Island, N.Y.) was the enzyme used, the concentration of each dNTP was 0.2 mM, and the MgCl 2 concentration was 1.5 mM. TaqMan probes and buffer, Amperase UNG, Tween 20, and gelatin were not included in these reactions.

Techniques Used: Agarose Gel Electrophoresis, Produced, Sequencing, Amplification, Polymerase Chain Reaction, Concentration Assay

Related Articles

Clone Assay:

Article Title: Xenoduplex Analysis--A Method for Comparative Gene Mapping Using Hybrid Panels
Article Snippet: PCR was performed in 10 μl containing 120 μ m dNTP, 1–2 m m MgCl2 , 4 pmoles of each primer, 0.25 units of ampliTaq Gold (Perkin-Elmer) with a PE9600 (Perkin Elmer) or a PTC100 (M.J. Research) thermocycler. .. A touch-down program was used that included an initial denaturation step at 94°C for 5 min, followed by 45 cycles with denaturation at 94°C for 30 sec, annealing at 59–49°C for 30 sec (the first 10 cycles touch down 1°C per cycle), extension at 72°C for 45 sec, and a final extension at 72°C for 5 min. After column purification (Qiagen), the pig PCR products were sequenced directly (without cloning) by use of DyePrimer chemistry with an ABI377 instrument (Perkin Elmer).

Article Title: Mapping of panda plumage color locus on the microsatellite linkage map of the Japanese quail
Article Snippet: Microsatellite markers PCR amplifications of five microsatellite markers (Table ) were carried out on a PCR Thermal Cycler (TaKaRa Biomedicals, Shiga, Japan) in 10 μl reaction mixtures containing 14 ng of the DNA template, 0.3 μM of forward and reverse primers, 130 μM of dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.4 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA, USA). .. To confirm whether the chicken primers of ABR0544 and ADL0266 amplified the orthologous Japanese quail microsatellite region, these PCR productswere cloned into TA cloning vector pCR2.1 (Invitrogen Corp., CA, USA) and sequenced by the dye termination method using ABI 3100 DNA Sequencer (Perkin-Elmer) (Table ).

Amplification:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl. .. DNA amplification conditions included an initial 5-min denaturation step at 95°C (which also activated the Gold variant of Taq Polymerase) and 35 cycles of 30 s at 95°C, 30 s at 60°C, 40 s at 72°C, and finally, 5 min at 72°C.

Article Title: Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro
Article Snippet: .. One microliter of the cDNA was amplified in a 50-μl solution containing 10 mM Tris⋅HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 μM of each dNTP, 2.5 units of Taq DNA polymerase (Perkin–Elmer) and 0.4 μM each primer. ..

Article Title: Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma
Article Snippet: .. For PCR, 3 μl of the cDNA template were used for the first round of amplification with the external primer sets (Figure 3) in a 30-μl reaction volume with 0.1 μmol/L for each primer, 200 μmol/L each dNTP, 0.8 U Ampli Taq DNA polymerase in Buffer II containing 2.0 mmol/L MgCl2 (Perkin-Elmer). .. After a 12-minute hot start at 94°C, nine cycles of touchdown amplification were performed (progressively lowering the annealing temperature from 61°C to 55°C), followed by 40 cycles of amplification (94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 45 seconds) with a Perkin-Elmer 9700 thermal cycler.

Article Title: Ancient origin and evolution of the Indian wolf: evidence from mitochondrial DNA typing of wolves from Trans-Himalayan region and Pennisular India
Article Snippet: Paragraph title: DNA amplification and sequencing for nucleotide diversity/ DNA polymorphisms ... The PCR reactions were set using approximately 50 ng of template genomic DNA in 15-20 μl reaction volume containing: 5 pico-moles of each primer, 150 μM dNTP, 1.5 mM MgCl2 , 0.1 M KCl, 20 mM Tris-HCl, and 0.5 to 1.0 U of AmpliTaq Gold polymerase (Perkin Elmer).

Article Title: O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification of Vibrio cholerae O1, O139, and Non-O1/Non-O139
Article Snippet: .. Touchdown-Multiplex PCR Amplification Initially, two-template combinations of the V. cholerae O1 and O139 were tested under the simultaneous locus-specific amplification conditions [ , ] using a 96-well MyCycler Thermal Cycler (BIORAD, USA) or PCR gradient thermal cycler (ThermoHybraid Px2, Ashford, UK), in which two combined factors such as concentrations of primer sets and gDNA templates were empirically determined in a 25 μ L PCR mixture consisting of 1x PCR buffer (50 mM KCl, 1.5 mM MgCl2 , and 10 mM Tris-HCl pH 9.0), 200 μ M each dNTP (dATP, dTTP, dCTP, and dGTP), 1 U of AmpliTaq (Perkin Elmer, USA), or Taq DNA polymerase (Promega, USA). .. The amplification conditions were performed on predenaturation at 95°C for 5 min and followed by 30 cycles of denaturation at 94°C for 1 min and extension at 72°C for 1 min, except that primer annealing temperature decrements (70 → 50°C) were used each for 1 min.

Article Title: Polarized Expression of CD74 by Gastric Epithelial Cells
Article Snippet: Next, 5 μ RNA was reverse-transcribed using 0.1 μ of oligo dT primer, 1 mM of each dNTP, and 50 U MuLV reverse transcriptase (Perkin-Elmer; Branchburg, NJ), at 42C for 15 min as previously described ( ). .. The resulting cDNA was then amplified by PCR in a reaction mixture consisting of 1 mM MgCl2 , 50 mM KCl, 0.2 mM of each dNTP, 0.4 μ of each primer, and 2.5 U Amplitaq DNA polymerase (Perkin Elmer).

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers. .. Amplification products were purified by isopropanol precipitation and sequenced with standard sequencing methods.

Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity
Article Snippet: One-fourth of the cDNA samples were used for PCR amplification of MEA , of the samples was used to amplify ACT11 , and 0.5 ng of genomic L er DNA for the controls. .. PCR was performed in 1× PCR buffer (Perkin Elmer) containing 2 m m MgCl2 , 0.2 m m of each dNTP, 1 unit of Taq DNA polymerase (Perkin-Elmer/Cetus), TaqStart antibody (Clontech) in a molar ratio of 28:1 relative to Taq DNA polymerase, and 20 pmoles of each gene specific primer for 30 cycles at an annealing temperature of 58°C.

Article Title: Xenoduplex Analysis--A Method for Comparative Gene Mapping Using Hybrid Panels
Article Snippet: Paragraph title: PCR Amplification ... PCR was performed in 10 μl containing 120 μ m dNTP, 1–2 m m MgCl2 , 4 pmoles of each primer, 0.25 units of ampliTaq Gold (Perkin-Elmer) with a PE9600 (Perkin Elmer) or a PTC100 (M.J. Research) thermocycler.

Article Title: Mapping of panda plumage color locus on the microsatellite linkage map of the Japanese quail
Article Snippet: Microsatellite markers PCR amplifications of five microsatellite markers (Table ) were carried out on a PCR Thermal Cycler (TaKaRa Biomedicals, Shiga, Japan) in 10 μl reaction mixtures containing 14 ng of the DNA template, 0.3 μM of forward and reverse primers, 130 μM of dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.4 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA, USA). .. After an initial incubation at 95°C for 9 min, amplification reactions were performed for 30 cycles each with denaturing at 95°C for 30 sec, annealing for 1 min at 50 to 60°C depending on the optimized annealing temperature of the primer used (Table ), and extension at 72°C for 1 min.

Article Title: Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing
Article Snippet: For DGGE, specific regions of the COL1A1 and COL2A1 genes were amplified by PCR with primers designed on the basis of published sequences ( – ) to generate PCR products that were 211–528 bp with a 40-bp GC-clamp added to the 5′-end of one of the PCR primers ( , , ). .. Typically, PCR amplifications were carried out in a reaction volume of 40 μl containing 50–100 ng of genomic DNA, 200 μM of each dNTP, 0.25 μM of each primer, and 1 unit of Taq polymerase (AmpliTaq Gold; Perkin–Elmer).

Article Title: Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome
Article Snippet: .. The amplification was performed in a 50-μL reaction mixture containing 1.25 mM MgCl2 , 1 × PCR buffer, 0.2 mM of each dNTP, 1 μM of each primer, and 2.5 U of Taq DNA polymerase (PerkinElmer Inc., Orlando, Florida, USA/Applied Biosystems, Foster City, California, USA). .. The 2 reactions were run in a thermocycler (GeneAmp PCR System 9600; PerkinElmer/Cetus, Norwalk, Connecticut, USA) under the same conditions: 35 cycles of denaturation at 95°C for 1 min, primer annealing at 65°C for 1 min, and extension at 72°C for 1 min.

Synthesized:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: Primers for the human pannexin synthesized by Life Technologies were as follows: forward 5′-CCCAATTGTGGAGCAGTACTTG-3′ (963–984) and reverse 5′-AGACACTTGTATGAC TTGACCTCAC-3′ (1403–1427). .. To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl.

TA Cloning:

Article Title: Mapping of panda plumage color locus on the microsatellite linkage map of the Japanese quail
Article Snippet: Microsatellite markers PCR amplifications of five microsatellite markers (Table ) were carried out on a PCR Thermal Cycler (TaKaRa Biomedicals, Shiga, Japan) in 10 μl reaction mixtures containing 14 ng of the DNA template, 0.3 μM of forward and reverse primers, 130 μM of dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.4 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA, USA). .. To confirm whether the chicken primers of ABR0544 and ADL0266 amplified the orthologous Japanese quail microsatellite region, these PCR productswere cloned into TA cloning vector pCR2.1 (Invitrogen Corp., CA, USA) and sequenced by the dye termination method using ABI 3100 DNA Sequencer (Perkin-Elmer) (Table ).

Random Hexamer Labeling:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: After a DNase I (Life Technologies) treatment to eliminate genomic DNA, 2 μg of total RNA was reverse transcribed into cDNA at 42°C using random hexamer primers (Perkin Elmer) and MuLV reverse transcriptase (Perkin Elmer) in a 20-μl final volume, followed by PCR. .. To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl.

Expressing:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: Paragraph title: Analysis of PanX1 expression (RT-PCR) ... To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl.

Article Title: Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro
Article Snippet: One microliter of the cDNA was amplified in a 50-μl solution containing 10 mM Tris⋅HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 μM of each dNTP, 2.5 units of Taq DNA polymerase (Perkin–Elmer) and 0.4 μM each primer. .. PCR consisted of 1 cycle at 95°C for 3 min, 62°C for 1 min, and 72°C for 1 min and subsequently 26 cycles for GAPDH or 26 to 33 cycles for IGF-II, depending on the endogenous levels of expression, at 95°C for 35 sec, 62°C for 40 sec, and 72°C for 40 sec.

Touchdown PCR:

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA. .. PCR cycling conditions (Touchdown PCR) were as follows: 5 min at 95°C, two cycles of 60 s denaturation at 94°C, 60 s at 66°C annealing temperature, a 120 s extension at 72°C, two cycles of 60 s at 94°C, 60 s at 64, 62, 60, 58, and 56°C, 120 s at 72°C, 30 cycles of 60 s at 94°C, 60 s at 54°C, 120 s at 72°C, and a final extension step of 7 min at 72°C.

Modification:

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: Methylation analysis The assay involved sodium bisulphite modification followed by sequence and pyrosequencing to determine the methylation status of GNG7 promoter region. .. PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA.

Generated:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: The expected DNA length of the PCR product generated by these primers was 465 bp (NM_015368, National Center for Biotechnology Information database). .. To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl.

Polymerase Chain Reaction:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: .. To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl. .. DNA amplification conditions included an initial 5-min denaturation step at 95°C (which also activated the Gold variant of Taq Polymerase) and 35 cycles of 30 s at 95°C, 30 s at 60°C, 40 s at 72°C, and finally, 5 min at 72°C.

Article Title: Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro
Article Snippet: Paragraph title: PCR Amplification. ... One microliter of the cDNA was amplified in a 50-μl solution containing 10 mM Tris⋅HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 μM of each dNTP, 2.5 units of Taq DNA polymerase (Perkin–Elmer) and 0.4 μM each primer.

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: .. PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA. .. PCR cycling conditions (Touchdown PCR) were as follows: 5 min at 95°C, two cycles of 60 s denaturation at 94°C, 60 s at 66°C annealing temperature, a 120 s extension at 72°C, two cycles of 60 s at 94°C, 60 s at 64, 62, 60, 58, and 56°C, 120 s at 72°C, 30 cycles of 60 s at 94°C, 60 s at 54°C, 120 s at 72°C, and a final extension step of 7 min at 72°C.

Article Title: Identification of Ciprofloxacin-Resistant Campylobacter jejuni by Use of a Fluorogenic PCR Assay
Article Snippet: .. The TaqMan PCR concentrations were as follows: 1× TaqMan buffer (Perkin-Elmer), 0.2 mM (each) dNTP (0.4 mM dUTP), 0.5 pmol of each primer/μl, 200 nM TaqMan probe, 0.05 U of Amplitaq Gold polymerase (Perkin-Elmer)/μl, 0.01 U of Amperase UNG (Perkin-Elmer)/μl, 4.5 mM MgCl2 , 0.05% gelatin, 0.01% Tween 20. .. The PCR thermocycling parameters were as follows.

Article Title: Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma
Article Snippet: .. For PCR, 3 μl of the cDNA template were used for the first round of amplification with the external primer sets (Figure 3) in a 30-μl reaction volume with 0.1 μmol/L for each primer, 200 μmol/L each dNTP, 0.8 U Ampli Taq DNA polymerase in Buffer II containing 2.0 mmol/L MgCl2 (Perkin-Elmer). .. After a 12-minute hot start at 94°C, nine cycles of touchdown amplification were performed (progressively lowering the annealing temperature from 61°C to 55°C), followed by 40 cycles of amplification (94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 45 seconds) with a Perkin-Elmer 9700 thermal cycler.

Article Title: Polymerase Chain Reaction in the Diagnosis of Uveitis
Article Snippet: .. Polymerase chain reaction was performed in a total volume of 10 µL containing 1 µL of microdissected DNA, 4.0 pmol each of 32 P-labeled sense primer and unlabeled antisense primer, 4.0 nmol each of dNTP, 1X GeneAmp buffer, 1.0 unit of AmpliTaq Gold Polymerase (Perkin Elmer, Hayward, CA), and a final concentration of 2.0 mM MgCl2 . .. The cycling parameter with the CDR3 primer pair included an initial incubation at 94°C for 9 minutes; 40 cycles of 94°C for 45 seconds, 56°C for 45 seconds and 72°C for 1 minute; and a final incubation at 70°C for 10 minutes.

Article Title: Ancient origin and evolution of the Indian wolf: evidence from mitochondrial DNA typing of wolves from Trans-Himalayan region and Pennisular India
Article Snippet: .. The PCR reactions were set using approximately 50 ng of template genomic DNA in 15-20 μl reaction volume containing: 5 pico-moles of each primer, 150 μM dNTP, 1.5 mM MgCl2 , 0.1 M KCl, 20 mM Tris-HCl, and 0.5 to 1.0 U of AmpliTaq Gold polymerase (Perkin Elmer). .. In general, PCR profile comprised an initial denaturation of 10 min at 95°C, followed by 35 cycles of: 94°C for 1 min., 50-55°C for 1 min., 72°C for 2 min.; and 72°C for 5 min.

Article Title: O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification of Vibrio cholerae O1, O139, and Non-O1/Non-O139
Article Snippet: .. Touchdown-Multiplex PCR Amplification Initially, two-template combinations of the V. cholerae O1 and O139 were tested under the simultaneous locus-specific amplification conditions [ , ] using a 96-well MyCycler Thermal Cycler (BIORAD, USA) or PCR gradient thermal cycler (ThermoHybraid Px2, Ashford, UK), in which two combined factors such as concentrations of primer sets and gDNA templates were empirically determined in a 25 μ L PCR mixture consisting of 1x PCR buffer (50 mM KCl, 1.5 mM MgCl2 , and 10 mM Tris-HCl pH 9.0), 200 μ M each dNTP (dATP, dTTP, dCTP, and dGTP), 1 U of AmpliTaq (Perkin Elmer, USA), or Taq DNA polymerase (Promega, USA). .. The amplification conditions were performed on predenaturation at 95°C for 5 min and followed by 30 cycles of denaturation at 94°C for 1 min and extension at 72°C for 1 min, except that primer annealing temperature decrements (70 → 50°C) were used each for 1 min.

Article Title: Polarized Expression of CD74 by Gastric Epithelial Cells
Article Snippet: Next, 5 μ RNA was reverse-transcribed using 0.1 μ of oligo dT primer, 1 mM of each dNTP, and 50 U MuLV reverse transcriptase (Perkin-Elmer; Branchburg, NJ), at 42C for 15 min as previously described ( ). .. The resulting cDNA was then amplified by PCR in a reaction mixture consisting of 1 mM MgCl2 , 50 mM KCl, 0.2 mM of each dNTP, 0.4 μ of each primer, and 2.5 U Amplitaq DNA polymerase (Perkin Elmer).

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: Five μl of RNA was added to 45 μl of RT-PCR mixture comprising components for reverse transcription and PCR steps (Access RT-PCR System; Promega Co., Madison, WI). .. Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers.

Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity
Article Snippet: .. PCR was performed in 1× PCR buffer (Perkin Elmer) containing 2 m m MgCl2 , 0.2 m m of each dNTP, 1 unit of Taq DNA polymerase (Perkin-Elmer/Cetus), TaqStart antibody (Clontech) in a molar ratio of 28:1 relative to Taq DNA polymerase, and 20 pmoles of each gene specific primer for 30 cycles at an annealing temperature of 58°C. .. The primers used for amplification of ACT11 were as described ( ).

Article Title: Mapping of panda plumage color locus on the microsatellite linkage map of the Japanese quail
Article Snippet: .. Microsatellite markers PCR amplifications of five microsatellite markers (Table ) were carried out on a PCR Thermal Cycler (TaKaRa Biomedicals, Shiga, Japan) in 10 μl reaction mixtures containing 14 ng of the DNA template, 0.3 μM of forward and reverse primers, 130 μM of dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.4 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA, USA). .. After an initial incubation at 95°C for 9 min, amplification reactions were performed for 30 cycles each with denaturing at 95°C for 30 sec, annealing for 1 min at 50 to 60°C depending on the optimized annealing temperature of the primer used (Table ), and extension at 72°C for 1 min.

Article Title: Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing
Article Snippet: .. Typically, PCR amplifications were carried out in a reaction volume of 40 μl containing 50–100 ng of genomic DNA, 200 μM of each dNTP, 0.25 μM of each primer, and 1 unit of Taq polymerase (AmpliTaq Gold; Perkin–Elmer). .. The PCR conditions were an initial denaturation at 95°C for 10 min, followed by 95°C for 40 sec, 56–60°C for 40 sec, and 72°C for 40 sec for 30–35 cycles, and a final extension 72°C for 10 min in an automated thermocycler (either GeneAmp 9600, Perkin–Elmer or PTC 225 DNA Engine Tetrad, MJ-Research, Inc., Watertown, MA).

Article Title: Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome
Article Snippet: .. The amplification was performed in a 50-μL reaction mixture containing 1.25 mM MgCl2 , 1 × PCR buffer, 0.2 mM of each dNTP, 1 μM of each primer, and 2.5 U of Taq DNA polymerase (PerkinElmer Inc., Orlando, Florida, USA/Applied Biosystems, Foster City, California, USA). .. The 2 reactions were run in a thermocycler (GeneAmp PCR System 9600; PerkinElmer/Cetus, Norwalk, Connecticut, USA) under the same conditions: 35 cycles of denaturation at 95°C for 1 min, primer annealing at 65°C for 1 min, and extension at 72°C for 1 min.

DNA Sequencing:

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA. .. To verify the methylation status, DNA bands of each transcript were excised from the gel, purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) and sequenced on the Applied Biosystems Prism 377 DNA sequencing system.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: Paragraph title: Analysis of PanX1 expression (RT-PCR) ... To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl.

Article Title: Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma
Article Snippet: Paragraph title: Nested RT-PCR for RET/PTC1 and RET/PTC3 Rearrangements ... For PCR, 3 μl of the cDNA template were used for the first round of amplification with the external primer sets (Figure 3) in a 30-μl reaction volume with 0.1 μmol/L for each primer, 200 μmol/L each dNTP, 0.8 U Ampli Taq DNA polymerase in Buffer II containing 2.0 mmol/L MgCl2 (Perkin-Elmer).

Article Title: Polarized Expression of CD74 by Gastric Epithelial Cells
Article Snippet: Paragraph title: RT-PCR ... Next, 5 μ RNA was reverse-transcribed using 0.1 μ of oligo dT primer, 1 mM of each dNTP, and 50 U MuLV reverse transcriptase (Perkin-Elmer; Branchburg, NJ), at 42C for 15 min as previously described ( ).

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: Paragraph title: RNA extraction and nested RT-PCR from clinical samples. ... Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers.

Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity
Article Snippet: Paragraph title: RT–PCR analysis ... PCR was performed in 1× PCR buffer (Perkin Elmer) containing 2 m m MgCl2 , 0.2 m m of each dNTP, 1 unit of Taq DNA polymerase (Perkin-Elmer/Cetus), TaqStart antibody (Clontech) in a molar ratio of 28:1 relative to Taq DNA polymerase, and 20 pmoles of each gene specific primer for 30 cycles at an annealing temperature of 58°C.

Denaturing Gradient Gel Electrophoresis:

Article Title: Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing
Article Snippet: For DGGE, specific regions of the COL1A1 and COL2A1 genes were amplified by PCR with primers designed on the basis of published sequences ( – ) to generate PCR products that were 211–528 bp with a 40-bp GC-clamp added to the 5′-end of one of the PCR primers ( , , ). .. Typically, PCR amplifications were carried out in a reaction volume of 40 μl containing 50–100 ng of genomic DNA, 200 μM of each dNTP, 0.25 μM of each primer, and 1 unit of Taq polymerase (AmpliTaq Gold; Perkin–Elmer).

Cellular Antioxidant Activity Assay:

Article Title: Polymerase Chain Reaction in the Diagnosis of Uveitis
Article Snippet: The PCR primer pairs were as follows: CDR3 sense (5′-CCG GRA ARR GTC TGG AGT GG-3′) and antisense (5′-ATC CTG AGG AGA CGG TGA CC-3′); FR3A sense (′-ACA CGG CYS TGT ATT ACT GT-3′) and antisense (5′-GGA TGG TAC CAA GCT TTG AGG AGA CGG TGA CCA-3′); and FR2A sense (5′-TGG RTC CGM CAG CAG SCV YCN GG-3′) and antisense (5′-ACC TGA GGA GAC GGT GAC C-3′). .. Polymerase chain reaction was performed in a total volume of 10 µL containing 1 µL of microdissected DNA, 4.0 pmol each of 32 P-labeled sense primer and unlabeled antisense primer, 4.0 nmol each of dNTP, 1X GeneAmp buffer, 1.0 unit of AmpliTaq Gold Polymerase (Perkin Elmer, Hayward, CA), and a final concentration of 2.0 mM MgCl2 .

Methylation:

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: Paragraph title: Methylation analysis ... PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA.

Isolation:

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: .. Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers. .. Nested PCR assays were subjected to an initial cycle of 94°C for 2 min and thirty cycles (94°C for 1 min, 50°C for 1 min and 72°C for 1 min) followed by a final incubation at 72°C for 5 min. Amplifications were carried out in a PTC-200 (Peltier Thermal Cycler, MJ Research, Inc., MA) utilizing thin-walled reaction tubes.

Microelectrode Array:

Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity
Article Snippet: One-fourth of the cDNA samples were used for PCR amplification of MEA , of the samples was used to amplify ACT11 , and 0.5 ng of genomic L er DNA for the controls. .. PCR was performed in 1× PCR buffer (Perkin Elmer) containing 2 m m MgCl2 , 0.2 m m of each dNTP, 1 unit of Taq DNA polymerase (Perkin-Elmer/Cetus), TaqStart antibody (Clontech) in a molar ratio of 28:1 relative to Taq DNA polymerase, and 20 pmoles of each gene specific primer for 30 cycles at an annealing temperature of 58°C.

Size-exclusion Chromatography:

Article Title: Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro
Article Snippet: One microliter of the cDNA was amplified in a 50-μl solution containing 10 mM Tris⋅HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 μM of each dNTP, 2.5 units of Taq DNA polymerase (Perkin–Elmer) and 0.4 μM each primer. .. PCR consisted of 1 cycle at 95°C for 3 min, 62°C for 1 min, and 72°C for 1 min and subsequently 26 cycles for GAPDH or 26 to 33 cycles for IGF-II, depending on the endogenous levels of expression, at 95°C for 35 sec, 62°C for 40 sec, and 72°C for 40 sec.

Article Title: Polarized Expression of CD74 by Gastric Epithelial Cells
Article Snippet: Next, 5 μ RNA was reverse-transcribed using 0.1 μ of oligo dT primer, 1 mM of each dNTP, and 50 U MuLV reverse transcriptase (Perkin-Elmer; Branchburg, NJ), at 42C for 15 min as previously described ( ). .. PCR for Ii was carried out in a programmed thermal cycler (Perkin Elmer Cetus; Emeryville, CA) at 30 cycles of 95C for 15 sec and 72C for 45 sec.

Article Title: Xenoduplex Analysis--A Method for Comparative Gene Mapping Using Hybrid Panels
Article Snippet: PCR was performed in 10 μl containing 120 μ m dNTP, 1–2 m m MgCl2 , 4 pmoles of each primer, 0.25 units of ampliTaq Gold (Perkin-Elmer) with a PE9600 (Perkin Elmer) or a PTC100 (M.J. Research) thermocycler. .. A touch-down program was used that included an initial denaturation step at 94°C for 5 min, followed by 45 cycles with denaturation at 94°C for 30 sec, annealing at 59–49°C for 30 sec (the first 10 cycles touch down 1°C per cycle), extension at 72°C for 45 sec, and a final extension at 72°C for 5 min. After column purification (Qiagen), the pig PCR products were sequenced directly (without cloning) by use of DyePrimer chemistry with an ABI377 instrument (Perkin Elmer).

Article Title: Mapping of panda plumage color locus on the microsatellite linkage map of the Japanese quail
Article Snippet: Microsatellite markers PCR amplifications of five microsatellite markers (Table ) were carried out on a PCR Thermal Cycler (TaKaRa Biomedicals, Shiga, Japan) in 10 μl reaction mixtures containing 14 ng of the DNA template, 0.3 μM of forward and reverse primers, 130 μM of dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.4 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA, USA). .. After an initial incubation at 95°C for 9 min, amplification reactions were performed for 30 cycles each with denaturing at 95°C for 30 sec, annealing for 1 min at 50 to 60°C depending on the optimized annealing temperature of the primer used (Table ), and extension at 72°C for 1 min.

Article Title: Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing
Article Snippet: Typically, PCR amplifications were carried out in a reaction volume of 40 μl containing 50–100 ng of genomic DNA, 200 μM of each dNTP, 0.25 μM of each primer, and 1 unit of Taq polymerase (AmpliTaq Gold; Perkin–Elmer). .. The PCR conditions were an initial denaturation at 95°C for 10 min, followed by 95°C for 40 sec, 56–60°C for 40 sec, and 72°C for 40 sec for 30–35 cycles, and a final extension 72°C for 10 min in an automated thermocycler (either GeneAmp 9600, Perkin–Elmer or PTC 225 DNA Engine Tetrad, MJ-Research, Inc., Watertown, MA).

Purification:

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA. .. To verify the methylation status, DNA bands of each transcript were excised from the gel, purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) and sequenced on the Applied Biosystems Prism 377 DNA sequencing system.

Article Title: Ancient origin and evolution of the Indian wolf: evidence from mitochondrial DNA typing of wolves from Trans-Himalayan region and Pennisular India
Article Snippet: The PCR reactions were set using approximately 50 ng of template genomic DNA in 15-20 μl reaction volume containing: 5 pico-moles of each primer, 150 μM dNTP, 1.5 mM MgCl2 , 0.1 M KCl, 20 mM Tris-HCl, and 0.5 to 1.0 U of AmpliTaq Gold polymerase (Perkin Elmer). .. Subsequently, the purified PCR products (~100 ng) were sequenced for both strands using the Big dye terminator ready reaction kit (Perkin Elmer).

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers. .. Amplification products were purified by isopropanol precipitation and sequenced with standard sequencing methods.

Article Title: Xenoduplex Analysis--A Method for Comparative Gene Mapping Using Hybrid Panels
Article Snippet: PCR was performed in 10 μl containing 120 μ m dNTP, 1–2 m m MgCl2 , 4 pmoles of each primer, 0.25 units of ampliTaq Gold (Perkin-Elmer) with a PE9600 (Perkin Elmer) or a PTC100 (M.J. Research) thermocycler. .. A touch-down program was used that included an initial denaturation step at 94°C for 5 min, followed by 45 cycles with denaturation at 94°C for 30 sec, annealing at 59–49°C for 30 sec (the first 10 cycles touch down 1°C per cycle), extension at 72°C for 45 sec, and a final extension at 72°C for 5 min. After column purification (Qiagen), the pig PCR products were sequenced directly (without cloning) by use of DyePrimer chemistry with an ABI377 instrument (Perkin Elmer).

Sequencing:

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: DNA methylation patterns in the promoter region of the GNG7 gene were ( ) determined by sequencing bisulphite-treated DNA. .. PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA.

Article Title: Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma
Article Snippet: The primer sequence and location are shown in Figure 3 . .. For PCR, 3 μl of the cDNA template were used for the first round of amplification with the external primer sets (Figure 3) in a 30-μl reaction volume with 0.1 μmol/L for each primer, 200 μmol/L each dNTP, 0.8 U Ampli Taq DNA polymerase in Buffer II containing 2.0 mmol/L MgCl2 (Perkin-Elmer).

Article Title: Ancient origin and evolution of the Indian wolf: evidence from mitochondrial DNA typing of wolves from Trans-Himalayan region and Pennisular India
Article Snippet: Paragraph title: DNA amplification and sequencing for nucleotide diversity/ DNA polymorphisms ... The PCR reactions were set using approximately 50 ng of template genomic DNA in 15-20 μl reaction volume containing: 5 pico-moles of each primer, 150 μM dNTP, 1.5 mM MgCl2 , 0.1 M KCl, 20 mM Tris-HCl, and 0.5 to 1.0 U of AmpliTaq Gold polymerase (Perkin Elmer).

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers. .. Amplification products were purified by isopropanol precipitation and sequenced with standard sequencing methods.

Article Title: Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing
Article Snippet: Here the target sequence was an exon plus at least 20 bp of 5′-flanking and 6 bp of 3′-flanking sequences. .. Typically, PCR amplifications were carried out in a reaction volume of 40 μl containing 50–100 ng of genomic DNA, 200 μM of each dNTP, 0.25 μM of each primer, and 1 unit of Taq polymerase (AmpliTaq Gold; Perkin–Elmer).

Nested PCR:

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: .. Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers. .. Nested PCR assays were subjected to an initial cycle of 94°C for 2 min and thirty cycles (94°C for 1 min, 50°C for 1 min and 72°C for 1 min) followed by a final incubation at 72°C for 5 min. Amplifications were carried out in a PTC-200 (Peltier Thermal Cycler, MJ Research, Inc., MA) utilizing thin-walled reaction tubes.

Activated Clotting Time Assay:

Article Title: Polymerase Chain Reaction in the Diagnosis of Uveitis
Article Snippet: The PCR primer pairs were as follows: CDR3 sense (5′-CCG GRA ARR GTC TGG AGT GG-3′) and antisense (5′-ATC CTG AGG AGA CGG TGA CC-3′); FR3A sense (′-ACA CGG CYS TGT ATT ACT GT-3′) and antisense (5′-GGA TGG TAC CAA GCT TTG AGG AGA CGG TGA CCA-3′); and FR2A sense (5′-TGG RTC CGM CAG CAG SCV YCN GG-3′) and antisense (5′-ACC TGA GGA GAC GGT GAC C-3′). .. Polymerase chain reaction was performed in a total volume of 10 µL containing 1 µL of microdissected DNA, 4.0 pmol each of 32 P-labeled sense primer and unlabeled antisense primer, 4.0 nmol each of dNTP, 1X GeneAmp buffer, 1.0 unit of AmpliTaq Gold Polymerase (Perkin Elmer, Hayward, CA), and a final concentration of 2.0 mM MgCl2 .

Plasmid Preparation:

Article Title: Mapping of panda plumage color locus on the microsatellite linkage map of the Japanese quail
Article Snippet: Microsatellite markers PCR amplifications of five microsatellite markers (Table ) were carried out on a PCR Thermal Cycler (TaKaRa Biomedicals, Shiga, Japan) in 10 μl reaction mixtures containing 14 ng of the DNA template, 0.3 μM of forward and reverse primers, 130 μM of dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.4 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA, USA). .. To confirm whether the chicken primers of ABR0544 and ADL0266 amplified the orthologous Japanese quail microsatellite region, these PCR productswere cloned into TA cloning vector pCR2.1 (Invitrogen Corp., CA, USA) and sequenced by the dye termination method using ABI 3100 DNA Sequencer (Perkin-Elmer) (Table ).

Software:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: The PCR primers used to amplify pannexin cDNAs were designed with Gene Runner 3.05 (Hastings Software). .. To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl.

RNA Extraction:

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: Paragraph title: RNA extraction and nested RT-PCR from clinical samples. ... Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers.

Agarose Gel Electrophoresis:

Article Title: Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma
Article Snippet: For PCR, 3 μl of the cDNA template were used for the first round of amplification with the external primer sets (Figure 3) in a 30-μl reaction volume with 0.1 μmol/L for each primer, 200 μmol/L each dNTP, 0.8 U Ampli Taq DNA polymerase in Buffer II containing 2.0 mmol/L MgCl2 (Perkin-Elmer). .. The nested RT-PCR products for RET/PTC1 and RET/PTC3 were analyzed on a 3% agarose gel and hybridized with a probe covering the tyrosine-kinase domain of RET.

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers. .. Upon completion of nested PCR assays, 5 μl of each reaction mixture was analyzed by 2% agarose gel electrophoresis.

Incubation:

Article Title: Identification of Ciprofloxacin-Resistant Campylobacter jejuni by Use of a Fluorogenic PCR Assay
Article Snippet: The TaqMan PCR concentrations were as follows: 1× TaqMan buffer (Perkin-Elmer), 0.2 mM (each) dNTP (0.4 mM dUTP), 0.5 pmol of each primer/μl, 200 nM TaqMan probe, 0.05 U of Amplitaq Gold polymerase (Perkin-Elmer)/μl, 0.01 U of Amperase UNG (Perkin-Elmer)/μl, 4.5 mM MgCl2 , 0.05% gelatin, 0.01% Tween 20. .. Prior to the initial denaturation, all TaqMan reaction mixtures were incubated at 50°C for 2 min in the presence of Amperase UNG in an effort to prevent PCR product carryover.

Article Title: Polymerase Chain Reaction in the Diagnosis of Uveitis
Article Snippet: Polymerase chain reaction was performed in a total volume of 10 µL containing 1 µL of microdissected DNA, 4.0 pmol each of 32 P-labeled sense primer and unlabeled antisense primer, 4.0 nmol each of dNTP, 1X GeneAmp buffer, 1.0 unit of AmpliTaq Gold Polymerase (Perkin Elmer, Hayward, CA), and a final concentration of 2.0 mM MgCl2 . .. The cycling parameter with the CDR3 primer pair included an initial incubation at 94°C for 9 minutes; 40 cycles of 94°C for 45 seconds, 56°C for 45 seconds and 72°C for 1 minute; and a final incubation at 70°C for 10 minutes.

Article Title: Molecular Identification of Mumps Virus Genotypes from Clinical Samples: Standardized Method of Analysis
Article Snippet: .. Samples, isolated virus, and controls were subjected to an initial cycle for reverse transcription at 42°C for 45 min, forty cycles (94°C for 1 min, 50°C for 1 min and 68°C for 1 min), and a final incubation at 68°C for 5 min. Nested PCR amplifications were performed using a reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl2 , 2.5 units of Taq polymerase (Amplitaq; Perkin-Elmer Cetus, Norwalk) and 10 pmol of Mumps-SH-2A and Mumps-SH-2S degenerate primers. .. Nested PCR assays were subjected to an initial cycle of 94°C for 2 min and thirty cycles (94°C for 1 min, 50°C for 1 min and 72°C for 1 min) followed by a final incubation at 72°C for 5 min. Amplifications were carried out in a PTC-200 (Peltier Thermal Cycler, MJ Research, Inc., MA) utilizing thin-walled reaction tubes.

Article Title: Mapping of panda plumage color locus on the microsatellite linkage map of the Japanese quail
Article Snippet: Microsatellite markers PCR amplifications of five microsatellite markers (Table ) were carried out on a PCR Thermal Cycler (TaKaRa Biomedicals, Shiga, Japan) in 10 μl reaction mixtures containing 14 ng of the DNA template, 0.3 μM of forward and reverse primers, 130 μM of dNTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 and 0.4 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA, USA). .. After an initial incubation at 95°C for 9 min, amplification reactions were performed for 30 cycles each with denaturing at 95°C for 30 sec, annealing for 1 min at 50 to 60°C depending on the optimized annealing temperature of the primer used (Table ), and extension at 72°C for 1 min.

DNA Methylation Assay:

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: DNA methylation patterns in the promoter region of the GNG7 gene were ( ) determined by sequencing bisulphite-treated DNA. .. PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA.

Concentration Assay:

Article Title: Polymerase Chain Reaction in the Diagnosis of Uveitis
Article Snippet: .. Polymerase chain reaction was performed in a total volume of 10 µL containing 1 µL of microdissected DNA, 4.0 pmol each of 32 P-labeled sense primer and unlabeled antisense primer, 4.0 nmol each of dNTP, 1X GeneAmp buffer, 1.0 unit of AmpliTaq Gold Polymerase (Perkin Elmer, Hayward, CA), and a final concentration of 2.0 mM MgCl2 . .. The cycling parameter with the CDR3 primer pair included an initial incubation at 94°C for 9 minutes; 40 cycles of 94°C for 45 seconds, 56°C for 45 seconds and 72°C for 1 minute; and a final incubation at 70°C for 10 minutes.

Article Title: O Serogroup-Specific Touchdown-Multiplex Polymerase Chain Reaction for Detection and Identification of Vibrio cholerae O1, O139, and Non-O1/Non-O139
Article Snippet: Touchdown-Multiplex PCR Amplification Initially, two-template combinations of the V. cholerae O1 and O139 were tested under the simultaneous locus-specific amplification conditions [ , ] using a 96-well MyCycler Thermal Cycler (BIORAD, USA) or PCR gradient thermal cycler (ThermoHybraid Px2, Ashford, UK), in which two combined factors such as concentrations of primer sets and gDNA templates were empirically determined in a 25 μ L PCR mixture consisting of 1x PCR buffer (50 mM KCl, 1.5 mM MgCl2 , and 10 mM Tris-HCl pH 9.0), 200 μ M each dNTP (dATP, dTTP, dCTP, and dGTP), 1 U of AmpliTaq (Perkin Elmer, USA), or Taq DNA polymerase (Promega, USA). .. The last extension was done at 72°C for 10 min. First, using a constant concentration of 1 μ M each primer set, reactions employed mixed O1 569B and O139 MO45 gDNAs (ng) at equal ratios of 10 : 10, 1 : 1, and 0.1 : 0.1 or at unequal ratios of 1 : 10 and 0.1 : 10 and vice versa .

CTG Assay:

Article Title: Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma
Article Snippet: The aldolase + primer was 5′-CGC AGA AGG GGT CCT GGT GA-3′ (nucleotides 18 to 37 of exon 1), the aldolase − primer was 5′-CAG CTC CTT CTT CTG CTC CG-3′ (nucleotides 175 to 194 of exon 2). .. For PCR, 3 μl of the cDNA template were used for the first round of amplification with the external primer sets (Figure 3) in a 30-μl reaction volume with 0.1 μmol/L for each primer, 200 μmol/L each dNTP, 0.8 U Ampli Taq DNA polymerase in Buffer II containing 2.0 mmol/L MgCl2 (Perkin-Elmer).

Article Title: Polymerase Chain Reaction in the Diagnosis of Uveitis
Article Snippet: The PCR primer pairs were as follows: CDR3 sense (5′-CCG GRA ARR GTC TGG AGT GG-3′) and antisense (5′-ATC CTG AGG AGA CGG TGA CC-3′); FR3A sense (′-ACA CGG CYS TGT ATT ACT GT-3′) and antisense (5′-GGA TGG TAC CAA GCT TTG AGG AGA CGG TGA CCA-3′); and FR2A sense (5′-TGG RTC CGM CAG CAG SCV YCN GG-3′) and antisense (5′-ACC TGA GGA GAC GGT GAC C-3′). .. Polymerase chain reaction was performed in a total volume of 10 µL containing 1 µL of microdissected DNA, 4.0 pmol each of 32 P-labeled sense primer and unlabeled antisense primer, 4.0 nmol each of dNTP, 1X GeneAmp buffer, 1.0 unit of AmpliTaq Gold Polymerase (Perkin Elmer, Hayward, CA), and a final concentration of 2.0 mM MgCl2 .

Gel Extraction:

Article Title: Clinical significance of the reduced expression of G protein gamma 7 (GNG7) in oesophageal cancer
Article Snippet: PCR was performed in 25-μ l reaction volumes containing 16.6 mM ammonium sulphate, 67 mM (pH 8.8) Trizma, 6.7 mM MgCl2 , 10 mM 2-mercaptoethanol, 10 mM DMSO, 1.25 mM dNTP, 1.6 μ M of each primer, 2.5 U of platinum Taq (Perkin-Elmer, Foster City, CA, USA), and 50 ng of bisulphite-treated DNA. .. To verify the methylation status, DNA bands of each transcript were excised from the gel, purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA) and sequenced on the Applied Biosystems Prism 377 DNA sequencing system.

Variant Assay:

Article Title: Functional implications of calcium permeability of the channel formed by pannexin 1
Article Snippet: To detect pannexin cDNAs, PCR was performed by adding 1 μl of the RT template to a mixture of (final concentrations): 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2 , 200 μM of each dNTP, 600 nM of sense and antisense primers, and 1 U AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl. .. DNA amplification conditions included an initial 5-min denaturation step at 95°C (which also activated the Gold variant of Taq Polymerase) and 35 cycles of 30 s at 95°C, 30 s at 60°C, 40 s at 72°C, and finally, 5 min at 72°C.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    PerkinElmer dntp
    Agarose gel electrophoresis of PCRs. The first tier (from the top) of the gel represents the amplicons produced from the QRDR sequencing primers. The second tier shows the amplification products produced with primers JL238 and JL239. Markers (100 bp) were loaded in the first lanes. Each lane was loaded with 20 μl of a 50-μl <t>PCR</t> mixture. The PCR conditions were as specified in the text with the exceptions that Platinum Taq DNA polymerase (Life Technologies, Gibco BRL, Grand Island, N.Y.) was the enzyme used, the concentration of each <t>dNTP</t> was 0.2 mM, and the MgCl 2 concentration was 1.5 mM. TaqMan probes and buffer, Amperase UNG, Tween 20, and gelatin were not included in these reactions.
    Dntp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 97/100, based on 583 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dntp/product/PerkinElmer
    Average 97 stars, based on 583 article reviews
    Price from $9.99 to $1999.99
    dntp - by Bioz Stars, 2020-03
    97/100 stars
      Buy from Supplier

    Image Search Results


    Agarose gel electrophoresis of PCRs. The first tier (from the top) of the gel represents the amplicons produced from the QRDR sequencing primers. The second tier shows the amplification products produced with primers JL238 and JL239. Markers (100 bp) were loaded in the first lanes. Each lane was loaded with 20 μl of a 50-μl PCR mixture. The PCR conditions were as specified in the text with the exceptions that Platinum Taq DNA polymerase (Life Technologies, Gibco BRL, Grand Island, N.Y.) was the enzyme used, the concentration of each dNTP was 0.2 mM, and the MgCl 2 concentration was 1.5 mM. TaqMan probes and buffer, Amperase UNG, Tween 20, and gelatin were not included in these reactions.

    Journal: Journal of Clinical Microbiology

    Article Title: Identification of Ciprofloxacin-Resistant Campylobacter jejuni by Use of a Fluorogenic PCR Assay

    doi:

    Figure Lengend Snippet: Agarose gel electrophoresis of PCRs. The first tier (from the top) of the gel represents the amplicons produced from the QRDR sequencing primers. The second tier shows the amplification products produced with primers JL238 and JL239. Markers (100 bp) were loaded in the first lanes. Each lane was loaded with 20 μl of a 50-μl PCR mixture. The PCR conditions were as specified in the text with the exceptions that Platinum Taq DNA polymerase (Life Technologies, Gibco BRL, Grand Island, N.Y.) was the enzyme used, the concentration of each dNTP was 0.2 mM, and the MgCl 2 concentration was 1.5 mM. TaqMan probes and buffer, Amperase UNG, Tween 20, and gelatin were not included in these reactions.

    Article Snippet: The TaqMan PCR concentrations were as follows: 1× TaqMan buffer (Perkin-Elmer), 0.2 mM (each) dNTP (0.4 mM dUTP), 0.5 pmol of each primer/μl, 200 nM TaqMan probe, 0.05 U of Amplitaq Gold polymerase (Perkin-Elmer)/μl, 0.01 U of Amperase UNG (Perkin-Elmer)/μl, 4.5 mM MgCl2 , 0.05% gelatin, 0.01% Tween 20.

    Techniques: Agarose Gel Electrophoresis, Produced, Sequencing, Amplification, Polymerase Chain Reaction, Concentration Assay

    Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, HIV-1 RT and MuLV RT with biased nucleotide substrate pools. ( A ) Template sequences used for the misincorporation assay with IAV Pol, T7 RNA polymerase and RTs of HIV-1 and MuLV. The IAV Pol template used is a 30-nt sequence from the 3′ end of the viral PA sequence with a ApG primer binding site (P:primer, T:template,). The first UTP or TTP incorporation site of each template was marked with “X”, and the first stop site in the (−) UTP or TTP reaction was marked with “1*” under each template sequence (the second and third stop sites were marked “2*” and “3*” for the IAV template). The RNA synthesis initiation sites were marked in blue. ( B ) ApG-initiated RNA-dependent RNA polymerization by IAV Pol: ApG primer was extended with a 30-nt RNA template and three different amounts of H3N2 IAV Pol protein (1x, 2x and 3x) with 500 µM four NTPs (“4”) and 0.16 µM α- 32 P-GTP, and the same reactions were repeated except using two biased NTP pools, minus UTP (“- U”) or minus ATP (“- A”). The sequence of the incorporated nucleotides near the three UTP stop sites (red) is shown at the side. The extended products in the (−) UTP reaction, which was used for calculating the misincorporation efficiency, was marked as “]”.Dotted lines refer to RNAs. ( C ) DNA dependent RNA polymerization of bacterial phage T7 RNA polymerase: A 47 bp ds DNA (box) encoding T7 promoter (grey box) and 29 bp sequence (white box) was used for RNA synthesis with T7 RNA polymerase at 37°C for 60 mins. “]”: Fully extended misincorporated product. ( D ) and ( E ) RNA dependent DNA polymerization reaction by HIV-1 RT (D) and MuLV RT (E). A 48 mer RNA template annealed to a 18-mer single stranded DNA primer was used for the DNA polymerization by HIV-1 and MuLV RTs at 37°C for 60 mins. 500 µM dNTPs mixed with α- 32 P-dNTPs (0.16 µM), which is the same nucleotide concentration and ratio as used in the reactions with IAV Pol and T7 RNA polymerase, was used for DNA synthesis. “]”: Fully extended misincorporated product. Dotted lines refer to RNA and solid lines refer to DNA. ( F ) and ( G ) Comparison of the misincorporation efficiency of the four polymerases. For calculation of the misincorporation efficiency, the fully extended misincorporated product in (−) U/(−) T reactions in all four polymerases was normalized to the total extended product in the lanes with all four NTPs (dNTPs). The fold differences of the calculated misincorporation percentages between three activities of IAV Pol ( F ) and between IAV Pol and three other polymerases ( G ) were determined. At least five repeats of the assay were conducted in this analysis.

    Journal: PLoS ONE

    Article Title: Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex

    doi: 10.1371/journal.pone.0010372

    Figure Lengend Snippet: Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, HIV-1 RT and MuLV RT with biased nucleotide substrate pools. ( A ) Template sequences used for the misincorporation assay with IAV Pol, T7 RNA polymerase and RTs of HIV-1 and MuLV. The IAV Pol template used is a 30-nt sequence from the 3′ end of the viral PA sequence with a ApG primer binding site (P:primer, T:template,). The first UTP or TTP incorporation site of each template was marked with “X”, and the first stop site in the (−) UTP or TTP reaction was marked with “1*” under each template sequence (the second and third stop sites were marked “2*” and “3*” for the IAV template). The RNA synthesis initiation sites were marked in blue. ( B ) ApG-initiated RNA-dependent RNA polymerization by IAV Pol: ApG primer was extended with a 30-nt RNA template and three different amounts of H3N2 IAV Pol protein (1x, 2x and 3x) with 500 µM four NTPs (“4”) and 0.16 µM α- 32 P-GTP, and the same reactions were repeated except using two biased NTP pools, minus UTP (“- U”) or minus ATP (“- A”). The sequence of the incorporated nucleotides near the three UTP stop sites (red) is shown at the side. The extended products in the (−) UTP reaction, which was used for calculating the misincorporation efficiency, was marked as “]”.Dotted lines refer to RNAs. ( C ) DNA dependent RNA polymerization of bacterial phage T7 RNA polymerase: A 47 bp ds DNA (box) encoding T7 promoter (grey box) and 29 bp sequence (white box) was used for RNA synthesis with T7 RNA polymerase at 37°C for 60 mins. “]”: Fully extended misincorporated product. ( D ) and ( E ) RNA dependent DNA polymerization reaction by HIV-1 RT (D) and MuLV RT (E). A 48 mer RNA template annealed to a 18-mer single stranded DNA primer was used for the DNA polymerization by HIV-1 and MuLV RTs at 37°C for 60 mins. 500 µM dNTPs mixed with α- 32 P-dNTPs (0.16 µM), which is the same nucleotide concentration and ratio as used in the reactions with IAV Pol and T7 RNA polymerase, was used for DNA synthesis. “]”: Fully extended misincorporated product. Dotted lines refer to RNA and solid lines refer to DNA. ( F ) and ( G ) Comparison of the misincorporation efficiency of the four polymerases. For calculation of the misincorporation efficiency, the fully extended misincorporated product in (−) U/(−) T reactions in all four polymerases was normalized to the total extended product in the lanes with all four NTPs (dNTPs). The fold differences of the calculated misincorporation percentages between three activities of IAV Pol ( F ) and between IAV Pol and three other polymerases ( G ) were determined. At least five repeats of the assay were conducted in this analysis.

    Article Snippet: For HIV and MuLV RT, a mixture of 200 nM 48-mer RNA template annealed to 100 nM 18-mer DNA primer , 500 µM dNTPs, 0.16 µM α-32 P-dNTPs (PerkinElmer, 3000 Ci/mmol) were incubated with RT for 1 hour at 37°C under conditions described previously (16).

    Techniques: Sequencing, Binding Assay, Concentration Assay, DNA Synthesis