Structured Review

Stratagene dna polymerase
Cloning the full-length oppA2 gene. (a) Schematic of how 5′ and 3′ regions of the oppA2 gene were cloned. Gene-specific primers SP1 and SP2 (HAP F and HAP R) were used in concert with primers directed against pneumococcal sequences RP1 or RP2 and were used in a <t>PCR</t> at low annealing temperature to encourage mispriming of the pneumococcal primers. (b) Cloning the 3′ oppA2 region. Primer RP2 misprimed close to the oppA2 gene and generated a product that was detected by Southern blotting by using pDub3 insert as a probe (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products). (c) Cloning the 5′ sequence of oppA2 (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products probed with pDub3). Arrowheads indicate <t>DNA</t> bands taken for cloning. Molecular mass of GeneRuler 1-kb ladder (MBI) shown in kilobases.
Dna Polymerase, supplied by Stratagene, used in various techniques. Bioz Stars score: 89/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dna polymerase/product/Stratagene
Average 89 stars, based on 25 article reviews
Price from $9.99 to $1999.99
dna polymerase - by Bioz Stars, 2020-04
89/100 stars

Images

1) Product Images from "Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA"

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.17.5210-5219.2003

Cloning the full-length oppA2 gene. (a) Schematic of how 5′ and 3′ regions of the oppA2 gene were cloned. Gene-specific primers SP1 and SP2 (HAP F and HAP R) were used in concert with primers directed against pneumococcal sequences RP1 or RP2 and were used in a PCR at low annealing temperature to encourage mispriming of the pneumococcal primers. (b) Cloning the 3′ oppA2 region. Primer RP2 misprimed close to the oppA2 gene and generated a product that was detected by Southern blotting by using pDub3 insert as a probe (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products). (c) Cloning the 5′ sequence of oppA2 (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products probed with pDub3). Arrowheads indicate DNA bands taken for cloning. Molecular mass of GeneRuler 1-kb ladder (MBI) shown in kilobases.
Figure Legend Snippet: Cloning the full-length oppA2 gene. (a) Schematic of how 5′ and 3′ regions of the oppA2 gene were cloned. Gene-specific primers SP1 and SP2 (HAP F and HAP R) were used in concert with primers directed against pneumococcal sequences RP1 or RP2 and were used in a PCR at low annealing temperature to encourage mispriming of the pneumococcal primers. (b) Cloning the 3′ oppA2 region. Primer RP2 misprimed close to the oppA2 gene and generated a product that was detected by Southern blotting by using pDub3 insert as a probe (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products). (c) Cloning the 5′ sequence of oppA2 (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products probed with pDub3). Arrowheads indicate DNA bands taken for cloning. Molecular mass of GeneRuler 1-kb ladder (MBI) shown in kilobases.

Techniques Used: Clone Assay, Polymerase Chain Reaction, Generated, Southern Blot, Staining, Agarose Gel Electrophoresis, Sequencing

Related Articles

Clone Assay:

Article Title: Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5
Article Snippet: .. TRPM5 was amplified from mouse vomeronasal organ cDNA with plaque-forming unit DNA polymerase (Stratagene) as described ( ) and was cloned into pBluescript KS+ (Stratagene). .. The coding sequence was identical to that previously reported ( ) ( ) except for a deletion of three nucleotides, resulting in the deletion of a threonine at position 130.

Article Title: Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5?-Nuclease Assays
Article Snippet: .. The plasmid containing the SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the α- Proteobacteria ( ) (R57pCRbl), was prepared from extracted genomic DNA in the same manner as the environmental clones, except that the DNA polymerase used for amplification was TaqPlus Precision DNA polymerase mix (Stratagene) and the PCR products were directly cloned using a PCR ZeroBlunt kit (Invitrogen). .. The plasmid containing the SSU-ITS-LSU rDNA fragment of Haloferax volcanii (HvpCRbl) was produced in a similar fashion, except that the primers used were the degenerate archaeal SSU rDNA forward primer 21F (5′-TTC CGG TGG ATC CYG CCG GA-3′ [ ]) and the degenerate prokaryotic LSU rDNA reverse primer 2445R (5′-CCC YGG GGT ARC TTT TCT ST-3′ [ ]).

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA
Article Snippet: Paragraph title: Cloning the full-length transcript of oppA2 . ... An additional primer, oppA6, was then designed upstream of the ATG and was used in a PCR with primer oppA4 and a proofreading DNA polymerase, Pfu Turbo (Stratagene), to amplify the full-length gene product, oppA2 .

Article Title: Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome ▿ Genome ▿ †
Article Snippet: .. The DNA fragments for TOPO cloning were generated by PCR amplification of the respective regions from genomic DNA of wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, Pfu Turbo (Stratagene Inc., La Jolla, CA). .. All plasmid extractions were performed by using QIAprep Spin Miniprep kits (Qiagen Inc., Valencia, CA).

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: .. The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA). .. Recombinant plasmid was then extracted and chemically transformed into E. coli BL21(DE3) cells (Invitrogen).

Centrifugation:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: The nuclear pellet was then resuspended in nuclear extract buffer (20 mM Tris, pH 8.0, 400 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.5 mM PMSF, and 20% glycerol) and incubated on ice for 10 min. Centrifugation at 14 K rpm for 15 min followed, and the protein concentration in the resulting supernatant (nuclear extract) was determined by the Bio-Rad DC Protein Assay prior to storage at -80°C. .. The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene).

Article Title: Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5?-Nuclease Assays
Article Snippet: The plasmid containing the SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the α- Proteobacteria ( ) (R57pCRbl), was prepared from extracted genomic DNA in the same manner as the environmental clones, except that the DNA polymerase used for amplification was TaqPlus Precision DNA polymerase mix (Stratagene) and the PCR products were directly cloned using a PCR ZeroBlunt kit (Invitrogen). .. These plasmids were purified using a Qiagen Maxi plasmid kit and isolated from genomic DNA via CsCl buoyant equilibrium centrifugation ( ).

Amplification:

Article Title: A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13
Article Snippet: PCR amplification of genomic DNA was carried out in 25-μl reactions. .. To analyze multiple DNA samples, a mixture of the following components was set up with the volumes multiplied by the number of reactions: 21 μl of H2 O, 2.5 μl of 10× plaque-forming units DNA polymerase buffer (Stratagene), 0.25 μl of dNTP mixture (dATP, dTTP, dGTP, and dCTP at 25 μM each; Amersham Pharmacia), 0.25 μl each of p53 primer, and 0.125 μl of plaque-forming units DNA polymerase (2.5 units/μl; Stratagene).

Article Title: Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5
Article Snippet: .. TRPM5 was amplified from mouse vomeronasal organ cDNA with plaque-forming unit DNA polymerase (Stratagene) as described ( ) and was cloned into pBluescript KS+ (Stratagene). .. The coding sequence was identical to that previously reported ( ) ( ) except for a deletion of three nucleotides, resulting in the deletion of a threonine at position 130.

Article Title: Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5?-Nuclease Assays
Article Snippet: .. The plasmid containing the SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the α- Proteobacteria ( ) (R57pCRbl), was prepared from extracted genomic DNA in the same manner as the environmental clones, except that the DNA polymerase used for amplification was TaqPlus Precision DNA polymerase mix (Stratagene) and the PCR products were directly cloned using a PCR ZeroBlunt kit (Invitrogen). .. The plasmid containing the SSU-ITS-LSU rDNA fragment of Haloferax volcanii (HvpCRbl) was produced in a similar fashion, except that the primers used were the degenerate archaeal SSU rDNA forward primer 21F (5′-TTC CGG TGG ATC CYG CCG GA-3′ [ ]) and the degenerate prokaryotic LSU rDNA reverse primer 2445R (5′-CCC YGG GGT ARC TTT TCT ST-3′ [ ]).

Article Title: Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome ▿ Genome ▿ †
Article Snippet: .. The DNA fragments for TOPO cloning were generated by PCR amplification of the respective regions from genomic DNA of wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, Pfu Turbo (Stratagene Inc., La Jolla, CA). .. All plasmid extractions were performed by using QIAprep Spin Miniprep kits (Qiagen Inc., Valencia, CA).

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: .. The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA). .. Recombinant plasmid was then extracted and chemically transformed into E. coli BL21(DE3) cells (Invitrogen).

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). .. Amplification was carried out at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles in two steps: 95°C for 15 s and 60°C for 1 min. For absolute quantification of viral genomic RNA, a standard curve was generated by using a serially diluted RNA in vitro transcribed from a plasmid expressing RABV N, and the copy numbers of viral genomic RNA were normalized to 1 μg of total RNA.

Stable Transfection:

Article Title: Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5
Article Snippet: TRPM5 was amplified from mouse vomeronasal organ cDNA with plaque-forming unit DNA polymerase (Stratagene) as described ( ) and was cloned into pBluescript KS+ (Stratagene). .. Plasmid encoding TRPM5 was transiently transfected into cells [COS-7, Chinese hamster ovary (CHO)-K1, or HEK-293 M1, a cell line stably expressing the muscarinic M1 receptor ( )] by using Effectene (Qiagen, Valencia, CA) or Fugene (Roche Molecular Biochemicals) as suggested by the manufacturer.

Synthesized:

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: Cloning of cDNAs and molecular modeling Full-length Na-apr-1 wt was synthesized in codon-optimized form for expression in E. coli by GeneArt AG, using the Na-apr-1 cDNA sequence in GenBank (accession no. AJ245459). .. The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA).

Blocking Assay:

Article Title: A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13
Article Snippet: To analyze multiple DNA samples, a mixture of the following components was set up with the volumes multiplied by the number of reactions: 21 μl of H2 O, 2.5 μl of 10× plaque-forming units DNA polymerase buffer (Stratagene), 0.25 μl of dNTP mixture (dATP, dTTP, dGTP, and dCTP at 25 μM each; Amersham Pharmacia), 0.25 μl each of p53 primer, and 0.125 μl of plaque-forming units DNA polymerase (2.5 units/μl; Stratagene). .. PCR reactions were carried out in a 96-well block of an Ericomp (San Diego) EZCycler TwinBlock System with an initial denaturation step of 96°C for 5 min followed by 30 cycles of 96°C for 15 sec, 50°C for 30 sec, 72°C for 30 sec, and a final extension of 72°C for 5 min. PCR products were resolved on 2% NuSieve agarose (FMC)/1% agarose (GIBCO/BRL) gels and visualized by staining for 5 min with 0.5 μg/ml ethidium bromide.

Real-time Polymerase Chain Reaction:

Article Title: Hormone Depletion-Insensitivity of Prostate Cancer Cells Is Supported by the AR Without Binding to Classical Response Elements
Article Snippet: The reagents for RT-PCR and real-time PCR, including inventoried TaqMan probes (for Ras homolog gene family, member U, ELL associated factor 2, insulin-like growth factor 1 receptor, prostate transmembrane protein, androgen induced 1, and kallikrein-related peptidase 3), were purchased from Applied Biosystems (Branchburg, NJ). .. Custom made TaqMan probes (for AR, GAPDH, and selected genomic fragments) were ordered from Integrated DNA Technologies, Inc. Plaque-forming unit DNA polymerase was from Stratagene (La Jolla, CA).

Article Title: Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs
Article Snippet: Paragraph title: Quantitative real-time PCR (qRT-PCR) ... The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: Paragraph title: Real-time PCR. ... The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Incubation:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: The nuclear pellet was then resuspended in nuclear extract buffer (20 mM Tris, pH 8.0, 400 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.5 mM PMSF, and 20% glycerol) and incubated on ice for 10 min. Centrifugation at 14 K rpm for 15 min followed, and the protein concentration in the resulting supernatant (nuclear extract) was determined by the Bio-Rad DC Protein Assay prior to storage at -80°C. .. The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene).

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA). .. Dpn I (10 units) was applied to PCR products and incubated at 37°C for 1 h. Methylated, nonmutated parental plasmid DNA was digested and fragmented while the nascent PCR fragment was left intact.

Expressing:

Article Title: Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5
Article Snippet: TRPM5 was amplified from mouse vomeronasal organ cDNA with plaque-forming unit DNA polymerase (Stratagene) as described ( ) and was cloned into pBluescript KS+ (Stratagene). .. Plasmid encoding TRPM5 was transiently transfected into cells [COS-7, Chinese hamster ovary (CHO)-K1, or HEK-293 M1, a cell line stably expressing the muscarinic M1 receptor ( )] by using Effectene (Qiagen, Valencia, CA) or Fugene (Roche Molecular Biochemicals) as suggested by the manufacturer.

Article Title: Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs
Article Snippet: The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). .. Expression Levels of each molecule in each vaccinated group (LBNSE or LBNSE-dGM-CSF) were presented as the fold change over that detected in the blood samples from mock-immunized group.

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: .. The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA). .. Recombinant plasmid was then extracted and chemically transformed into E. coli BL21(DE3) cells (Invitrogen).

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: A real-time (RT) SYBR green PCR assay was carried out in an Mx3000P apparatus (Stratagene, La Jolla, CA) to quantify the rate of viral replication and the expression of chemokines and cytokines. .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: To confirm that the identified cis -regulatory element is essential for suppressing Timp2 expression after MEHP exposure, Dpn I-mediated site-directed mutagenesis was performed to alter its sequence. .. The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA).

Transformation Assay:

Article Title: Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome ▿ Genome ▿ †
Article Snippet: Entry vectors in this study were generated by the directional TOPO cloning of desired DNA fragments into pENTR/dTOPO (Invitrogen Inc., Carlsbad, CA) and transformed into One Shot Top10 chemically competent E. coli cells (Invitrogen) according to the manufacturer's instructions. .. The DNA fragments for TOPO cloning were generated by PCR amplification of the respective regions from genomic DNA of wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, Pfu Turbo (Stratagene Inc., La Jolla, CA).

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: .. The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA). .. Recombinant plasmid was then extracted and chemically transformed into E. coli BL21(DE3) cells (Invitrogen).

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA). .. The nicked plasmid containing the mutation was transformed into Escherichia coli to screen for mutant colonies, and the mutation site was verified by DNA sequencing.

Hybridization:

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA
Article Snippet: This was used in a low-stringency PCR with a specific primer, oppA4, followed by Southern blot hybridization and cloning and sequencing of positive fragments (Fig. ). .. An additional primer, oppA6, was then designed upstream of the ATG and was used in a PCR with primer oppA4 and a proofreading DNA polymerase, Pfu Turbo (Stratagene), to amplify the full-length gene product, oppA2 .

Transfection:

Article Title: Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5
Article Snippet: TRPM5 was amplified from mouse vomeronasal organ cDNA with plaque-forming unit DNA polymerase (Stratagene) as described ( ) and was cloned into pBluescript KS+ (Stratagene). .. Plasmid encoding TRPM5 was transiently transfected into cells [COS-7, Chinese hamster ovary (CHO)-K1, or HEK-293 M1, a cell line stably expressing the muscarinic M1 receptor ( )] by using Effectene (Qiagen, Valencia, CA) or Fugene (Roche Molecular Biochemicals) as suggested by the manufacturer.

Southern Blot:

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA
Article Snippet: This was used in a low-stringency PCR with a specific primer, oppA4, followed by Southern blot hybridization and cloning and sequencing of positive fragments (Fig. ). .. An additional primer, oppA6, was then designed upstream of the ATG and was used in a PCR with primer oppA4 and a proofreading DNA polymerase, Pfu Turbo (Stratagene), to amplify the full-length gene product, oppA2 .

Infection:

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: Muscle tissues at the site of immunization were removed from infected mice and flash frozen on dry ice before being stored at −80°C. .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Labeling:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: .. The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene). .. The sequence of the probes used in EMSAs are as follows (mutations are underlined): -390 wt : 5'-GCTTGATGATTTCCTGCCCTCGCC-3'; -390 mut Ets: 5;-GCTTGATGATTT TT TGCCCTCGCC-3'; -360 wt: 5'-GGCACAGCTTCCTGCCGCAGGCC-3'; -360 mut Ets: 5'-GGCACAGCTT TT TGCCGCAGGCC-3'; Runx/-360 Ets wt: 5'-GCGCTCACCACAGCTTCCTGCCGCAGG-3'; Runx/-360Ets mutRunx: 5'-GCGCTCA TT ACAGCTTCCTGCCGCAGG-3'; Runx/-360Ets mutEts: 5'-GCGCTCACCACAGCTT TT TGCCGCAGG-3'; Runx/-360Ets mutEts/Runx: 5'-GCGCTCA TT ACAGCTT TT TGCCGCAGG-3'; consensus Ets: 5'-GATCTCGAGCAGGAAGTTCGA-3'; consensus mutEts: 5'-GATCTCGAGCA A GAAGTTCGA-3'.

Generated:

Article Title: Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome ▿ Genome ▿ †
Article Snippet: .. The DNA fragments for TOPO cloning were generated by PCR amplification of the respective regions from genomic DNA of wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, Pfu Turbo (Stratagene Inc., La Jolla, CA). .. All plasmid extractions were performed by using QIAprep Spin Miniprep kits (Qiagen Inc., Valencia, CA).

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). .. Amplification was carried out at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles in two steps: 95°C for 15 s and 60°C for 1 min. For absolute quantification of viral genomic RNA, a standard curve was generated by using a serially diluted RNA in vitro transcribed from a plasmid expressing RABV N, and the copy numbers of viral genomic RNA were normalized to 1 μg of total RNA.

Polymerase Chain Reaction:

Article Title: A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13
Article Snippet: Paragraph title: PCR Analysis of Trp53 Status. ... To analyze multiple DNA samples, a mixture of the following components was set up with the volumes multiplied by the number of reactions: 21 μl of H2 O, 2.5 μl of 10× plaque-forming units DNA polymerase buffer (Stratagene), 0.25 μl of dNTP mixture (dATP, dTTP, dGTP, and dCTP at 25 μM each; Amersham Pharmacia), 0.25 μl each of p53 primer, and 0.125 μl of plaque-forming units DNA polymerase (2.5 units/μl; Stratagene).

Article Title: RssAB-FlhDC-ShlBA as a Major Pathogenesis Pathway in Serratia marcescens ▿ ▿ †
Article Snippet: .. DNA polymerase and PCR-related products were purchased from Stratagene, Takara (Japan), and Perkin Elmer. .. F-12K medium and RPMI 1640 medium without phenol red and culture-related products were purchased from Invitrogen.

Article Title: Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5?-Nuclease Assays
Article Snippet: .. The plasmid containing the SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the α- Proteobacteria ( ) (R57pCRbl), was prepared from extracted genomic DNA in the same manner as the environmental clones, except that the DNA polymerase used for amplification was TaqPlus Precision DNA polymerase mix (Stratagene) and the PCR products were directly cloned using a PCR ZeroBlunt kit (Invitrogen). .. The plasmid containing the SSU-ITS-LSU rDNA fragment of Haloferax volcanii (HvpCRbl) was produced in a similar fashion, except that the primers used were the degenerate archaeal SSU rDNA forward primer 21F (5′-TTC CGG TGG ATC CYG CCG GA-3′ [ ]) and the degenerate prokaryotic LSU rDNA reverse primer 2445R (5′-CCC YGG GGT ARC TTT TCT ST-3′ [ ]).

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA
Article Snippet: .. An additional primer, oppA6, was then designed upstream of the ATG and was used in a PCR with primer oppA4 and a proofreading DNA polymerase, Pfu Turbo (Stratagene), to amplify the full-length gene product, oppA2 . ..

Article Title: Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome ▿ Genome ▿ †
Article Snippet: .. The DNA fragments for TOPO cloning were generated by PCR amplification of the respective regions from genomic DNA of wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, Pfu Turbo (Stratagene Inc., La Jolla, CA). .. All plasmid extractions were performed by using QIAprep Spin Miniprep kits (Qiagen Inc., Valencia, CA).

Article Title: Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs
Article Snippet: Quantitative real-time PCR (qRT-PCR) To quantify the mRNA levels of surface co-stimulating molecules on DC or B cells in the peripheral blood after oral vaccination, qRT-PCR was performed in ABI Prism 7500 fast sequence detector system with Power SYBR green PCR master mix (Applied Biosystems). .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: .. The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA). .. Recombinant plasmid was then extracted and chemically transformed into E. coli BL21(DE3) cells (Invitrogen).

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: A real-time (RT) SYBR green PCR assay was carried out in an Mx3000P apparatus (Stratagene, La Jolla, CA) to quantify the rate of viral replication and the expression of chemokines and cytokines. .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: .. The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA). .. Dpn I (10 units) was applied to PCR products and incubated at 37°C for 1 h. Methylated, nonmutated parental plasmid DNA was digested and fragmented while the nascent PCR fragment was left intact.

DNA Sequencing:

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA). .. The nicked plasmid containing the mutation was transformed into Escherichia coli to screen for mutant colonies, and the mutation site was verified by DNA sequencing.

Protein Concentration:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: The nuclear pellet was then resuspended in nuclear extract buffer (20 mM Tris, pH 8.0, 400 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.5 mM PMSF, and 20% glycerol) and incubated on ice for 10 min. Centrifugation at 14 K rpm for 15 min followed, and the protein concentration in the resulting supernatant (nuclear extract) was determined by the Bio-Rad DC Protein Assay prior to storage at -80°C. .. The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Hormone Depletion-Insensitivity of Prostate Cancer Cells Is Supported by the AR Without Binding to Classical Response Elements
Article Snippet: The reagents for RT-PCR and real-time PCR, including inventoried TaqMan probes (for Ras homolog gene family, member U, ELL associated factor 2, insulin-like growth factor 1 receptor, prostate transmembrane protein, androgen induced 1, and kallikrein-related peptidase 3), were purchased from Applied Biosystems (Branchburg, NJ). .. Custom made TaqMan probes (for AR, GAPDH, and selected genomic fragments) were ordered from Integrated DNA Technologies, Inc. Plaque-forming unit DNA polymerase was from Stratagene (La Jolla, CA).

Binding Assay:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene). .. For EMSAs, 5 μg of nuclear extract was used in DNA binding reactions containing 10 mM HEPES, pH 7.6, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 5% glycerol, and 0.2 μg sheared salmon sperm DNA.

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA
Article Snippet: An additional primer, oppA6, was then designed upstream of the ATG and was used in a PCR with primer oppA4 and a proofreading DNA polymerase, Pfu Turbo (Stratagene), to amplify the full-length gene product, oppA2 . .. A signal peptide was identified in the N terminus in addition to a bacterial lipoprotein motif (LXXC) and an extracellular peptide and nickel binding protein family signature sequence( AX 7 DX 4 TX3 R X3 K ) ( ) (Fig. ).

DC Protein Assay:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: The nuclear pellet was then resuspended in nuclear extract buffer (20 mM Tris, pH 8.0, 400 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.5 mM PMSF, and 20% glycerol) and incubated on ice for 10 min. Centrifugation at 14 K rpm for 15 min followed, and the protein concentration in the resulting supernatant (nuclear extract) was determined by the Bio-Rad DC Protein Assay prior to storage at -80°C. .. The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene).

Methylation:

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: Dpn I endonuclease targeting sequence 5′-GmATC-3′ is specific for methylated and hemimethylated DNA [ , ]. .. The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA).

Mutagenesis:

Article Title: Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome ▿ Genome ▿ †
Article Snippet: Paragraph title: Mutant strain generation. ... The DNA fragments for TOPO cloning were generated by PCR amplification of the respective regions from genomic DNA of wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, Pfu Turbo (Stratagene Inc., La Jolla, CA).

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA). .. Both of the active site residues of the ORF of Na -apr-1 wt /pET41aΔGST (Asp-97 and Asp-284) were mutated to Ala by site-directed mutagenesis, using previously described methods .

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: Paragraph title: Dpn I-Mediated Site-Directed Mutagenesis ... The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA).

Isolation:

Article Title: Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5?-Nuclease Assays
Article Snippet: The plasmid containing the SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the α- Proteobacteria ( ) (R57pCRbl), was prepared from extracted genomic DNA in the same manner as the environmental clones, except that the DNA polymerase used for amplification was TaqPlus Precision DNA polymerase mix (Stratagene) and the PCR products were directly cloned using a PCR ZeroBlunt kit (Invitrogen). .. These plasmids were purified using a Qiagen Maxi plasmid kit and isolated from genomic DNA via CsCl buoyant equilibrium centrifugation ( ).

Article Title: Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs
Article Snippet: Blood samples were harvested at different time points and total RNA extracted from the isolated peripheral blood mononuclear cells (PBMCs). .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Size-exclusion Chromatography:

Article Title: A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13
Article Snippet: To analyze multiple DNA samples, a mixture of the following components was set up with the volumes multiplied by the number of reactions: 21 μl of H2 O, 2.5 μl of 10× plaque-forming units DNA polymerase buffer (Stratagene), 0.25 μl of dNTP mixture (dATP, dTTP, dGTP, and dCTP at 25 μM each; Amersham Pharmacia), 0.25 μl each of p53 primer, and 0.125 μl of plaque-forming units DNA polymerase (2.5 units/μl; Stratagene). .. PCR reactions were carried out in a 96-well block of an Ericomp (San Diego) EZCycler TwinBlock System with an initial denaturation step of 96°C for 5 min followed by 30 cycles of 96°C for 15 sec, 50°C for 30 sec, 72°C for 30 sec, and a final extension of 72°C for 5 min. PCR products were resolved on 2% NuSieve agarose (FMC)/1% agarose (GIBCO/BRL) gels and visualized by staining for 5 min with 0.5 μg/ml ethidium bromide.

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: .. The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA). .. Dpn I (10 units) was applied to PCR products and incubated at 37°C for 1 h. Methylated, nonmutated parental plasmid DNA was digested and fragmented while the nascent PCR fragment was left intact.

Electrophoretic Mobility Shift Assay:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: Paragraph title: Preparation of nuclear extracts and the electrophoretic mobility shift assay ... The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene).

Purification:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: .. The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene). .. The sequence of the probes used in EMSAs are as follows (mutations are underlined): -390 wt : 5'-GCTTGATGATTTCCTGCCCTCGCC-3'; -390 mut Ets: 5;-GCTTGATGATTT TT TGCCCTCGCC-3'; -360 wt: 5'-GGCACAGCTTCCTGCCGCAGGCC-3'; -360 mut Ets: 5'-GGCACAGCTT TT TGCCGCAGGCC-3'; Runx/-360 Ets wt: 5'-GCGCTCACCACAGCTTCCTGCCGCAGG-3'; Runx/-360Ets mutRunx: 5'-GCGCTCA TT ACAGCTTCCTGCCGCAGG-3'; Runx/-360Ets mutEts: 5'-GCGCTCACCACAGCTT TT TGCCGCAGG-3'; Runx/-360Ets mutEts/Runx: 5'-GCGCTCA TT ACAGCTT TT TGCCGCAGG-3'; consensus Ets: 5'-GATCTCGAGCAGGAAGTTCGA-3'; consensus mutEts: 5'-GATCTCGAGCA A GAAGTTCGA-3'.

Article Title: Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5?-Nuclease Assays
Article Snippet: The plasmid containing the SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the α- Proteobacteria ( ) (R57pCRbl), was prepared from extracted genomic DNA in the same manner as the environmental clones, except that the DNA polymerase used for amplification was TaqPlus Precision DNA polymerase mix (Stratagene) and the PCR products were directly cloned using a PCR ZeroBlunt kit (Invitrogen). .. These plasmids were purified using a Qiagen Maxi plasmid kit and isolated from genomic DNA via CsCl buoyant equilibrium centrifugation ( ).

Sequencing:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene). .. The sequence of the probes used in EMSAs are as follows (mutations are underlined): -390 wt : 5'-GCTTGATGATTTCCTGCCCTCGCC-3'; -390 mut Ets: 5;-GCTTGATGATTT TT TGCCCTCGCC-3'; -360 wt: 5'-GGCACAGCTTCCTGCCGCAGGCC-3'; -360 mut Ets: 5'-GGCACAGCTT TT TGCCGCAGGCC-3'; Runx/-360 Ets wt: 5'-GCGCTCACCACAGCTTCCTGCCGCAGG-3'; Runx/-360Ets mutRunx: 5'-GCGCTCA TT ACAGCTTCCTGCCGCAGG-3'; Runx/-360Ets mutEts: 5'-GCGCTCACCACAGCTT TT TGCCGCAGG-3'; Runx/-360Ets mutEts/Runx: 5'-GCGCTCA TT ACAGCTT TT TGCCGCAGG-3'; consensus Ets: 5'-GATCTCGAGCAGGAAGTTCGA-3'; consensus mutEts: 5'-GATCTCGAGCA A GAAGTTCGA-3'.

Article Title: Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5
Article Snippet: TRPM5 was amplified from mouse vomeronasal organ cDNA with plaque-forming unit DNA polymerase (Stratagene) as described ( ) and was cloned into pBluescript KS+ (Stratagene). .. The coding sequence was identical to that previously reported ( ) ( ) except for a deletion of three nucleotides, resulting in the deletion of a threonine at position 130.

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA
Article Snippet: This yielded a clone containing the missing N-terminal sequence, including the initiation codon. .. An additional primer, oppA6, was then designed upstream of the ATG and was used in a PCR with primer oppA4 and a proofreading DNA polymerase, Pfu Turbo (Stratagene), to amplify the full-length gene product, oppA2 .

Article Title: Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs
Article Snippet: Quantitative real-time PCR (qRT-PCR) To quantify the mRNA levels of surface co-stimulating molecules on DC or B cells in the peripheral blood after oral vaccination, qRT-PCR was performed in ABI Prism 7500 fast sequence detector system with Power SYBR green PCR master mix (Applied Biosystems). .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: Cloning of cDNAs and molecular modeling Full-length Na-apr-1 wt was synthesized in codon-optimized form for expression in E. coli by GeneArt AG, using the Na-apr-1 cDNA sequence in GenBank (accession no. AJ245459). .. The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA).

Quantitative RT-PCR:

Article Title: Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs
Article Snippet: .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). ..

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). .. Amplification was carried out at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles in two steps: 95°C for 15 s and 60°C for 1 min. For absolute quantification of viral genomic RNA, a standard curve was generated by using a serially diluted RNA in vitro transcribed from a plasmid expressing RABV N, and the copy numbers of viral genomic RNA were normalized to 1 μg of total RNA.

Staining:

Article Title: A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13
Article Snippet: To analyze multiple DNA samples, a mixture of the following components was set up with the volumes multiplied by the number of reactions: 21 μl of H2 O, 2.5 μl of 10× plaque-forming units DNA polymerase buffer (Stratagene), 0.25 μl of dNTP mixture (dATP, dTTP, dGTP, and dCTP at 25 μM each; Amersham Pharmacia), 0.25 μl each of p53 primer, and 0.125 μl of plaque-forming units DNA polymerase (2.5 units/μl; Stratagene). .. PCR reactions were carried out in a 96-well block of an Ericomp (San Diego) EZCycler TwinBlock System with an initial denaturation step of 96°C for 5 min followed by 30 cycles of 96°C for 15 sec, 50°C for 30 sec, 72°C for 30 sec, and a final extension of 72°C for 5 min. PCR products were resolved on 2% NuSieve agarose (FMC)/1% agarose (GIBCO/BRL) gels and visualized by staining for 5 min with 0.5 μg/ml ethidium bromide.

IA:

Article Title: Hormone Depletion-Insensitivity of Prostate Cancer Cells Is Supported by the AR Without Binding to Classical Response Elements
Article Snippet: Custom oligonucleotide primers were from Integrated DNA Technologies, Inc. (Coralville, IA). .. Custom made TaqMan probes (for AR, GAPDH, and selected genomic fragments) were ordered from Integrated DNA Technologies, Inc. Plaque-forming unit DNA polymerase was from Stratagene (La Jolla, CA).

Mouse Assay:

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: Muscle tissues at the site of immunization were removed from infected mice and flash frozen on dry ice before being stored at −80°C. .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene).

Plasmid Preparation:

Article Title: Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5
Article Snippet: TRPM5 was amplified from mouse vomeronasal organ cDNA with plaque-forming unit DNA polymerase (Stratagene) as described ( ) and was cloned into pBluescript KS+ (Stratagene). .. The coding sequence was subcloned into pEGFP-C2 (Clontech) to generate an N-terminal fusion of enhanced GFP (EGFP) and into an EGFP dual promoter vector (pHGCX).

Article Title: Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5?-Nuclease Assays
Article Snippet: .. The plasmid containing the SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the α- Proteobacteria ( ) (R57pCRbl), was prepared from extracted genomic DNA in the same manner as the environmental clones, except that the DNA polymerase used for amplification was TaqPlus Precision DNA polymerase mix (Stratagene) and the PCR products were directly cloned using a PCR ZeroBlunt kit (Invitrogen). .. The plasmid containing the SSU-ITS-LSU rDNA fragment of Haloferax volcanii (HvpCRbl) was produced in a similar fashion, except that the primers used were the degenerate archaeal SSU rDNA forward primer 21F (5′-TTC CGG TGG ATC CYG CCG GA-3′ [ ]) and the degenerate prokaryotic LSU rDNA reverse primer 2445R (5′-CCC YGG GGT ARC TTT TCT ST-3′ [ ]).

Article Title: Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome ▿ Genome ▿ †
Article Snippet: The DNA fragments for TOPO cloning were generated by PCR amplification of the respective regions from genomic DNA of wild-type D. vulgaris Hildenborough using a proofreading DNA polymerase, Pfu Turbo (Stratagene Inc., La Jolla, CA). .. All plasmid extractions were performed by using QIAprep Spin Miniprep kits (Qiagen Inc., Valencia, CA).

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: .. The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA). .. Recombinant plasmid was then extracted and chemically transformed into E. coli BL21(DE3) cells (Invitrogen).

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). .. Amplification was carried out at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles in two steps: 95°C for 15 s and 60°C for 1 min. For absolute quantification of viral genomic RNA, a standard curve was generated by using a serially diluted RNA in vitro transcribed from a plasmid expressing RABV N, and the copy numbers of viral genomic RNA were normalized to 1 μg of total RNA.

Article Title: Transcriptional Suppression of Sertoli Cell Timp2 in Rodents Following Mono-(2-ethylhexyl) Phthalate Exposure Is Regulated by CEBPA and MYC 1
Article Snippet: The pGL3-enhancer-TIMP2-P1200 plasmids were used as templates for the PCR for 12 cycles at 95°C for 30 sec, then 55°C for 1 min, and 68°C for 15 min, with 2.5 pfu of DNA polymerase (Stratagene, La Jolla, CA). .. Dpn I (10 units) was applied to PCR products and incubated at 37°C for 1 h. Methylated, nonmutated parental plasmid DNA was digested and fragmented while the nascent PCR fragment was left intact.

SYBR Green Assay:

Article Title: Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs
Article Snippet: .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). ..

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: .. The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). .. Amplification was carried out at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles in two steps: 95°C for 15 s and 60°C for 1 min. For absolute quantification of viral genomic RNA, a standard curve was generated by using a serially diluted RNA in vitro transcribed from a plasmid expressing RABV N, and the copy numbers of viral genomic RNA were normalized to 1 μg of total RNA.

Recombinant:

Article Title: An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection
Article Snippet: The mature form, excluding the proregion and stop codon—Ser-62–Phe-430 (numbering of amino acid residues is relative to the start of the proenzyme)—of the Na -APR-1wt ORF, was amplified by PCR using a proofreading DNA polymerase (Pfu Turbo; Stratagene, La Jolla, CA, USA), cloned into the Nde I and Xho I sites of the pET41a vector (thereby removing the GST fusion tag but retaining the 6×His tag and allowing for native N-terminal protein expression—the resulting plasmid after removal of the nucleotide encoding GST was denoted pET41aΔGST) and chemically transformed into E. coli TOP10 cells (Invitrogen, Carlsbad, CA, USA). .. Recombinant plasmid was then extracted and chemically transformed into E. coli BL21(DE3) cells (Invitrogen).

In Vitro:

Article Title: Rabies Virus Expressing Dendritic Cell-Activating Molecules Enhances the Innate and Adaptive Immune Response to Vaccination ▿
Article Snippet: The reverse transcriptase and DNA polymerase were utilized from a one-step Brilliant II SYBR green qRT-PCR master mix kit (Stratagene). .. Amplification was carried out at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles in two steps: 95°C for 15 s and 60°C for 1 min. For absolute quantification of viral genomic RNA, a standard curve was generated by using a serially diluted RNA in vitro transcribed from a plasmid expressing RABV N, and the copy numbers of viral genomic RNA were normalized to 1 μg of total RNA.

Knock-Out:

Article Title: A male germ cell tumor-susceptibility-determining locus, pgct1, identified on murine chromosome 13
Article Snippet: Three primers were used to distinguish between wild-type and null Trp53 alleles, a forward primer located within p53 exon 6 (5′-GTATCCCGAGTATCTGGAAGACAG-3′), a second forward primer located within the aminoglycoside 3′-phosphotransferase gene of the knockout cassette (5′-GCCTTCTATCGCCTTCTTGACG-3′), and a reverse primer located within p53 exon 7 (5′-AAGGATAGGTCGGCGGTTCATGC-3′). .. To analyze multiple DNA samples, a mixture of the following components was set up with the volumes multiplied by the number of reactions: 21 μl of H2 O, 2.5 μl of 10× plaque-forming units DNA polymerase buffer (Stratagene), 0.25 μl of dNTP mixture (dATP, dTTP, dGTP, and dCTP at 25 μM each; Amersham Pharmacia), 0.25 μl each of p53 primer, and 0.125 μl of plaque-forming units DNA polymerase (2.5 units/μl; Stratagene).

Produced:

Article Title: Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5?-Nuclease Assays
Article Snippet: The plasmid containing the SSU-ITS-LSU rDNA fragment of strain R2A57, a marine member of the α- Proteobacteria ( ) (R57pCRbl), was prepared from extracted genomic DNA in the same manner as the environmental clones, except that the DNA polymerase used for amplification was TaqPlus Precision DNA polymerase mix (Stratagene) and the PCR products were directly cloned using a PCR ZeroBlunt kit (Invitrogen). .. The plasmid containing the SSU-ITS-LSU rDNA fragment of Haloferax volcanii (HvpCRbl) was produced in a similar fashion, except that the primers used were the degenerate archaeal SSU rDNA forward primer 21F (5′-TTC CGG TGG ATC CYG CCG GA-3′ [ ]) and the degenerate prokaryotic LSU rDNA reverse primer 2445R (5′-CCC YGG GGT ARC TTT TCT ST-3′ [ ]).

Mobility Shift:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: .. The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene). .. The sequence of the probes used in EMSAs are as follows (mutations are underlined): -390 wt : 5'-GCTTGATGATTTCCTGCCCTCGCC-3'; -390 mut Ets: 5;-GCTTGATGATTT TT TGCCCTCGCC-3'; -360 wt: 5'-GGCACAGCTTCCTGCCGCAGGCC-3'; -360 mut Ets: 5'-GGCACAGCTT TT TGCCGCAGGCC-3'; Runx/-360 Ets wt: 5'-GCGCTCACCACAGCTTCCTGCCGCAGG-3'; Runx/-360Ets mutRunx: 5'-GCGCTCA TT ACAGCTTCCTGCCGCAGG-3'; Runx/-360Ets mutEts: 5'-GCGCTCACCACAGCTT TT TGCCGCAGG-3'; Runx/-360Ets mutEts/Runx: 5'-GCGCTCA TT ACAGCTT TT TGCCGCAGG-3'; consensus Ets: 5'-GATCTCGAGCAGGAAGTTCGA-3'; consensus mutEts: 5'-GATCTCGAGCA A GAAGTTCGA-3'.

Lysis:

Article Title: Regulation of the human LAT gene by the Elf-1 transcription factor
Article Snippet: The resulting lysate was centrifuged at 1500 rpm for 4 min and the nuclear pellet washed once with lysis buffer lacking NP-40. .. The 32 P labeled probes used in the electrophoretic gel mobility shift assay (EMSA) were prepared by filling in annealed oligonucleotides with the Klenow fragment of DNA polymerase in the presence of [α-32 P]dCTP and other dNTPs followed by purification using NucTrap probe purification columns (Stratagene).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Stratagene high fidelity dna polymerase
    Endogenous <t>FPC</t> is found on the centrosome in three renal epithelial cell lines. FPC is shown in red, labeled by 4883 or 803 antibodies. The centrosome is labeled by γ-tubulin in green. <t>DNA</t> is labeled by DAPI. FPC localization on the centrosome
    High Fidelity Dna Polymerase, supplied by Stratagene, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high fidelity dna polymerase/product/Stratagene
    Average 93 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    high fidelity dna polymerase - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    94
    Stratagene pfu dna polymerase
    (A) Plaque formation ability of A12-LLV2 on PK or IBRS2 cells. Cells were infected with approximately 100 <t>PFU</t> of A12-LLV2, overlaid, and stained at 36 h postinoculation. (B) Induction of IFN-α or IFN-β mRNA in PK or IBRS2 cells. Cells were infected with A12-IC, A12-LLV2, or mock infected for 6 h and used in RT-PCR as described in Materials and Methods. Aliquots from RT reactions were used in three separate PCR assays with IFN-α, IFN-β, and β-actin primers. <t>DNA</t> from uninfected PK or IBRS2 cells was used in PCR assays as target controls. IFN-α, IFN-β, and β-actin RT-PCR products are 379, 452, and 890 bp, respectively. Lanes MW, 1-kb-ladder DNA molecular weight markers.
    Pfu Dna Polymerase, supplied by Stratagene, used in various techniques. Bioz Stars score: 94/100, based on 1332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pfu dna polymerase/product/Stratagene
    Average 94 stars, based on 1332 article reviews
    Price from $9.99 to $1999.99
    pfu dna polymerase - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    Image Search Results


    Endogenous FPC is found on the centrosome in three renal epithelial cell lines. FPC is shown in red, labeled by 4883 or 803 antibodies. The centrosome is labeled by γ-tubulin in green. DNA is labeled by DAPI. FPC localization on the centrosome

    Journal: Human Molecular Genetics

    Article Title: Polycystic kidney disease protein fibrocystin localizes to the mitotic spindle and regulates spindle bipolarity

    doi: 10.1093/hmg/ddq233

    Figure Lengend Snippet: Endogenous FPC is found on the centrosome in three renal epithelial cell lines. FPC is shown in red, labeled by 4883 or 803 antibodies. The centrosome is labeled by γ-tubulin in green. DNA is labeled by DAPI. FPC localization on the centrosome

    Article Snippet: Four overlapping FPC cDNA fragments were amplified with high-fidelity DNA polymerase (PfuUltra™ High-Fidelity DNA Polymerase; Stratagene) from a human fetal kidney cDNA library.

    Techniques: Labeling

    Non-overlapping regulatory modules within angptl4 intron 3 confer liver, islet, and enterocyte-specific reporter expression. (A) Depiction of the 6 dpf zebrafish showing liver (li, green), intestine (in, blue), swim bladder (sb, grey), and muscle (mu, grey), with the fish oriented anterior (a) to the left and posterior (p) to the right. The opposite orientation reveals the exocrine pancreas (pa, yellow) and islet (is, orange). (B) Scaled schematic of the zebrafish angptl4 locus and non-coding DNA assayed for regulatory potential. Modules are color coded according to the tissues in which they confer expression. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown in parentheses next to truncation labels. (C–N) Representative images of GFP reporter expression in mosaic (column 1) and F 1 stable (column 2) animals driven by each non-coding DNA region (rows). Scale bars = 100 µm; li = liver, is = islet, in = intestine, sb = swim bladder. Colored arrowheads indicate tissue with specific reporter expression. (C–D) Full-length intron 3 (in3; 2,136 bp) is sufficient to promote expression of the reporter in the liver, islet (D, inset, scale bar = 50 µm), and intestine. (E–F) Truncation in3.1 (1,219 bp) confers expression in the liver. (G–H) Truncation in3.2 (701 bp) confers expression in both the intestine and islet (H, inset). Inset scale bar = 50 µm. (I–J) Truncation in3.3 (387 bp) confers islet expression. A transverse section (inset, J) reveals islet expression (nuclei stained with DAPI). Inset scale bar = 50 µm. (K–L) Truncation in3.4 (316 bp) confers intestinal expression. Insets in panels K and L contain transverse sections showing expression localized to the intestinal epithelium (nuclei stained with DAPI). Inset scale bar = 25 µm. The dotted lines in panels D, G, H, and I outline the pancreas. The white arrows in panels H, K, and L mark the boundary between the anterior intestine (segment 1) and mid-intestine (segment 2). (M–N) Cells expressing GFP driven by the in3.4 regulatory module colocalize with a marker (4E8, red, white arrow) of the brush border of absorptive enterocytes, but fail to co-localize with marker for secretory cells (2F11, red, asterisk). Nuclei stained with DAPI. Scale bars = 5 µm. (O) Intercross of Tg(in3.2-Mmu.Fos:tdTomato) with β-cell specific reporter line ( Tg(ins:CFP-NTR) s892 ) show colocalization of tdTomato and CFP in the islet. Scale bars = 10 µm. (P) Quantitative PCR shows that the in3.4 module and the angptl4 promoter (TATA box), but not the in3.3 module, are hypersensitive to DNase I cleavage in intestinal epithelial cells isolated from adult zebrafish. Asterisks denote P-value

    Journal: PLoS Genetics

    Article Title: Intronic Cis-Regulatory Modules Mediate Tissue-Specific and Microbial Control of angptl4/fiaf Transcription

    doi: 10.1371/journal.pgen.1002585

    Figure Lengend Snippet: Non-overlapping regulatory modules within angptl4 intron 3 confer liver, islet, and enterocyte-specific reporter expression. (A) Depiction of the 6 dpf zebrafish showing liver (li, green), intestine (in, blue), swim bladder (sb, grey), and muscle (mu, grey), with the fish oriented anterior (a) to the left and posterior (p) to the right. The opposite orientation reveals the exocrine pancreas (pa, yellow) and islet (is, orange). (B) Scaled schematic of the zebrafish angptl4 locus and non-coding DNA assayed for regulatory potential. Modules are color coded according to the tissues in which they confer expression. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown in parentheses next to truncation labels. (C–N) Representative images of GFP reporter expression in mosaic (column 1) and F 1 stable (column 2) animals driven by each non-coding DNA region (rows). Scale bars = 100 µm; li = liver, is = islet, in = intestine, sb = swim bladder. Colored arrowheads indicate tissue with specific reporter expression. (C–D) Full-length intron 3 (in3; 2,136 bp) is sufficient to promote expression of the reporter in the liver, islet (D, inset, scale bar = 50 µm), and intestine. (E–F) Truncation in3.1 (1,219 bp) confers expression in the liver. (G–H) Truncation in3.2 (701 bp) confers expression in both the intestine and islet (H, inset). Inset scale bar = 50 µm. (I–J) Truncation in3.3 (387 bp) confers islet expression. A transverse section (inset, J) reveals islet expression (nuclei stained with DAPI). Inset scale bar = 50 µm. (K–L) Truncation in3.4 (316 bp) confers intestinal expression. Insets in panels K and L contain transverse sections showing expression localized to the intestinal epithelium (nuclei stained with DAPI). Inset scale bar = 25 µm. The dotted lines in panels D, G, H, and I outline the pancreas. The white arrows in panels H, K, and L mark the boundary between the anterior intestine (segment 1) and mid-intestine (segment 2). (M–N) Cells expressing GFP driven by the in3.4 regulatory module colocalize with a marker (4E8, red, white arrow) of the brush border of absorptive enterocytes, but fail to co-localize with marker for secretory cells (2F11, red, asterisk). Nuclei stained with DAPI. Scale bars = 5 µm. (O) Intercross of Tg(in3.2-Mmu.Fos:tdTomato) with β-cell specific reporter line ( Tg(ins:CFP-NTR) s892 ) show colocalization of tdTomato and CFP in the islet. Scale bars = 10 µm. (P) Quantitative PCR shows that the in3.4 module and the angptl4 promoter (TATA box), but not the in3.3 module, are hypersensitive to DNase I cleavage in intestinal epithelial cells isolated from adult zebrafish. Asterisks denote P-value

    Article Snippet: Reporter construct cloning All PCR reactions used for cloning were performed with high-fidelity DNA polymerase (PfuTurbo, Stratagene; Phusion, Invitrogen; Platinum Taq, Invitrogen) and TOP10 chemically competent E. coli (Invitrogen).

    Techniques: Expressing, Fluorescence In Situ Hybridization, Staining, Marker, Real-time Polymerase Chain Reaction, Isolation

    Two-step polymerase chain assembly of a 612 bp EPCR gene 1 (A) and a 576 bp EPCR-2 gene (B). A) Eight microliters of each sample was analyzed on a 2% agarose gel. Lanes: (M) 1 Kb DNA Ladder (Fermentas Inc.); Lane 1: 612 bp assembly PCR product with a 4-second elongation; Lane 2: 612 bp assembly PCR product with 5-second elongation. B) Eight microliters of each sample was analyzed on a 2% agarose gel. Lanes: (M) 100 bp DNA Ladder (Fermentas Inc.); Lanes 1 and 2: 576 bp assembly PCR product with a 4-second elongation. C) Gel purified EPCR genes 1 and 2. Eight microliters of each sample was analyzed on a 2% agarose gel. Lanes: (M) 100 bp DNA Ladder (Fermentas Inc.); Lane 1: gel purified 612 bp EPCR gene 1, Lane 2: gel purified 576 bp EPCR-2 gene. D) gel purified 1548 bp TM gene

    Journal: Journal of Biotechnology

    Article Title: Rational de novo Gene Synthesis by Rapid Polymerase Chain Assembly (PCA) and Expression of Endothelial Protein-C and Thrombin Receptor Genes

    doi: 10.1016/j.jbiotec.2007.08.010

    Figure Lengend Snippet: Two-step polymerase chain assembly of a 612 bp EPCR gene 1 (A) and a 576 bp EPCR-2 gene (B). A) Eight microliters of each sample was analyzed on a 2% agarose gel. Lanes: (M) 1 Kb DNA Ladder (Fermentas Inc.); Lane 1: 612 bp assembly PCR product with a 4-second elongation; Lane 2: 612 bp assembly PCR product with 5-second elongation. B) Eight microliters of each sample was analyzed on a 2% agarose gel. Lanes: (M) 100 bp DNA Ladder (Fermentas Inc.); Lanes 1 and 2: 576 bp assembly PCR product with a 4-second elongation. C) Gel purified EPCR genes 1 and 2. Eight microliters of each sample was analyzed on a 2% agarose gel. Lanes: (M) 100 bp DNA Ladder (Fermentas Inc.); Lane 1: gel purified 612 bp EPCR gene 1, Lane 2: gel purified 576 bp EPCR-2 gene. D) gel purified 1548 bp TM gene

    Article Snippet: PCR was performed using Hot Start DNA polymerase (Stratagene).

    Techniques: Agarose Gel Electrophoresis, Polymerase Cycling Assembly, Purification

    (A) Plaque formation ability of A12-LLV2 on PK or IBRS2 cells. Cells were infected with approximately 100 PFU of A12-LLV2, overlaid, and stained at 36 h postinoculation. (B) Induction of IFN-α or IFN-β mRNA in PK or IBRS2 cells. Cells were infected with A12-IC, A12-LLV2, or mock infected for 6 h and used in RT-PCR as described in Materials and Methods. Aliquots from RT reactions were used in three separate PCR assays with IFN-α, IFN-β, and β-actin primers. DNA from uninfected PK or IBRS2 cells was used in PCR assays as target controls. IFN-α, IFN-β, and β-actin RT-PCR products are 379, 452, and 890 bp, respectively. Lanes MW, 1-kb-ladder DNA molecular weight markers.

    Journal: Journal of Virology

    Article Title: Inhibition of L-Deleted Foot-and-Mouth Disease Virus Replication by Alpha/Beta Interferon Involves Double-Stranded RNA-Dependent Protein Kinase

    doi: 10.1128/JVI.75.12.5498-5503.2001

    Figure Lengend Snippet: (A) Plaque formation ability of A12-LLV2 on PK or IBRS2 cells. Cells were infected with approximately 100 PFU of A12-LLV2, overlaid, and stained at 36 h postinoculation. (B) Induction of IFN-α or IFN-β mRNA in PK or IBRS2 cells. Cells were infected with A12-IC, A12-LLV2, or mock infected for 6 h and used in RT-PCR as described in Materials and Methods. Aliquots from RT reactions were used in three separate PCR assays with IFN-α, IFN-β, and β-actin primers. DNA from uninfected PK or IBRS2 cells was used in PCR assays as target controls. IFN-α, IFN-β, and β-actin RT-PCR products are 379, 452, and 890 bp, respectively. Lanes MW, 1-kb-ladder DNA molecular weight markers.

    Article Snippet: mRNA was harvested from PK or EBK cells infected with A12-LLV2 at 6 h postinfection (hpi) and amplified by RT-PCR using IFN-α- or -β-specific primer sets for porcine or bovine species and Pfu DNA polymerase (Stratagene, La Jolla, Calif.) for 20 to 25 cycles.

    Techniques: Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Molecular Weight