Structured Review

Roche dna polymerase
Antigenic region of the VacA gene from Helicobacter pylori amplified by <t>PCR.</t> Lane 1: <t>DNA</t> marker, Lane 2: PCR product
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1) Product Images from "Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori"

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

Journal: Iranian Journal of Basic Medical Sciences

doi:

Antigenic region of the VacA gene from Helicobacter pylori amplified by PCR. Lane 1: DNA marker, Lane 2: PCR product
Figure Legend Snippet: Antigenic region of the VacA gene from Helicobacter pylori amplified by PCR. Lane 1: DNA marker, Lane 2: PCR product

Techniques Used: Amplification, Polymerase Chain Reaction, Marker

2) Product Images from "Myocyte Enhancer Factor 2 and Class II Histone Deacetylases Control a Gender-Specific Pathway of Cardioprotection Mediated by the Estrogen Receptor"

Article Title: Myocyte Enhancer Factor 2 and Class II Histone Deacetylases Control a Gender-Specific Pathway of Cardioprotection Mediated by the Estrogen Receptor

Journal: Circulation research

doi: 10.1161/CIRCRESAHA.109.207084

Cardiac expression of ER α is regulated by MEF2 and class II HDACs. A, Cross-species conservation of genomic sequences upstream of the ER α gene is shown at the top. Sequence of putative MEF2 transcription factor-binding sites (in boxes) in the promoter of the mouse ERα gene. Numbers in brackets refer to location of the MEF2 site relative to the ATG start codon for ERα . B, EMSA for binding of MEF2C to the putative MEF2 sites of the ERα promoter using nuclear extracts from COS-1 cells transfected with a MYC-MEF2C expression plasmid and the radiolabeled MEF2 site. The MEF2-DNA complex was supershifted by anti-MYC antibody and was abolished in the presence of an excess of the unlabeled cognate DNA sequence or the prototypical MEF2 site from the muscle creatine kinase enhancer as a competitor, whereas a mutant sequence that did not bind MEF2C failed to compete for MEF2C binding. C, Luciferase reporter experiments using an upstream promoter fragment of the ERα gene indicates that MEF2 can induce transcriptional activity, which is enhanced in the presence of myocardin. Mutational analysis indicates that the MEF2 site 770 bp upstream of the transcriptional start is responsible for the MEF2 responsiveness of the ERα gene. D, Real-time PCR analysis of ER α expression in noninfected neonatal rat cardiomyocytes compared with cells infected with AdCMV- β gal or AdCMV-asHDAC5 or asHDAC9 indicates that both asHDAC5 and asHDAC9 increases ER α expression in myocytes in a dose-dependent manner. MOI indicates multiplicity of infection. Additionally, adenoviral overexpression of MEF2C (AdMEF2C) is capable of inducing ER α expression, whereas titrating in a signal resistant form of HDAC5 (AdHDAC5 S > A) reduces this induction. E, Real-time PCR analysis of ER α expression in cardiac samples of WT, HDAC5 and HDAC9 KO animals shows an in vivo upregulation of ER α expression in the absence of HDAC5 and -9 compared to WT. * P
Figure Legend Snippet: Cardiac expression of ER α is regulated by MEF2 and class II HDACs. A, Cross-species conservation of genomic sequences upstream of the ER α gene is shown at the top. Sequence of putative MEF2 transcription factor-binding sites (in boxes) in the promoter of the mouse ERα gene. Numbers in brackets refer to location of the MEF2 site relative to the ATG start codon for ERα . B, EMSA for binding of MEF2C to the putative MEF2 sites of the ERα promoter using nuclear extracts from COS-1 cells transfected with a MYC-MEF2C expression plasmid and the radiolabeled MEF2 site. The MEF2-DNA complex was supershifted by anti-MYC antibody and was abolished in the presence of an excess of the unlabeled cognate DNA sequence or the prototypical MEF2 site from the muscle creatine kinase enhancer as a competitor, whereas a mutant sequence that did not bind MEF2C failed to compete for MEF2C binding. C, Luciferase reporter experiments using an upstream promoter fragment of the ERα gene indicates that MEF2 can induce transcriptional activity, which is enhanced in the presence of myocardin. Mutational analysis indicates that the MEF2 site 770 bp upstream of the transcriptional start is responsible for the MEF2 responsiveness of the ERα gene. D, Real-time PCR analysis of ER α expression in noninfected neonatal rat cardiomyocytes compared with cells infected with AdCMV- β gal or AdCMV-asHDAC5 or asHDAC9 indicates that both asHDAC5 and asHDAC9 increases ER α expression in myocytes in a dose-dependent manner. MOI indicates multiplicity of infection. Additionally, adenoviral overexpression of MEF2C (AdMEF2C) is capable of inducing ER α expression, whereas titrating in a signal resistant form of HDAC5 (AdHDAC5 S > A) reduces this induction. E, Real-time PCR analysis of ER α expression in cardiac samples of WT, HDAC5 and HDAC9 KO animals shows an in vivo upregulation of ER α expression in the absence of HDAC5 and -9 compared to WT. * P

Techniques Used: Expressing, Genomic Sequencing, Sequencing, Binding Assay, Transfection, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Infection, Over Expression, In Vivo

3) Product Images from "The Potassium Transporter AtHAK5 Functions in K+ Deprivation-Induced High-Affinity K+ Uptake and AKT1 K+ Channel Contribution to K+ Uptake Kinetics in Arabidopsis Roots 1 Uptake Kinetics in Arabidopsis Roots 1 [w]"

Article Title: The Potassium Transporter AtHAK5 Functions in K+ Deprivation-Induced High-Affinity K+ Uptake and AKT1 K+ Channel Contribution to K+ Uptake Kinetics in Arabidopsis Roots 1 Uptake Kinetics in Arabidopsis Roots 1 [w]

Journal: Plant Physiology

doi: 10.1104/pp.104.057216

Isolation of two homozygous T-DNA insertion mutants in the AtHAK5 gene. A, The cartoon shows the positions of the two T-DNA insertion lines, which were verified by sequencing of the left border PCR products. B, RT-PCR using cDNA from roots of wild-type
Figure Legend Snippet: Isolation of two homozygous T-DNA insertion mutants in the AtHAK5 gene. A, The cartoon shows the positions of the two T-DNA insertion lines, which were verified by sequencing of the left border PCR products. B, RT-PCR using cDNA from roots of wild-type

Techniques Used: Isolation, Sequencing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

4) Product Images from "Construction of a System for the Strawberry Nursery Production towards Elimination of Latent Infection of Anthracnose Fungi by a Combination of PCR and Microtube Hybridization"

Article Title: Construction of a System for the Strawberry Nursery Production towards Elimination of Latent Infection of Anthracnose Fungi by a Combination of PCR and Microtube Hybridization

Journal: The Plant Pathology Journal

doi: 10.5423/PPJ.NT.05.2016.0132

Schematic flow of the diagnostic method developed in this study for latent infection of strawberry anthracnose fungi. The diagnostic steps consist of 4 steps, initial culturing of candidate fungi (Step 1), DNA extraction from fungal hyphae (Step 2), synthesis of PCR-amplified probes (Step 3), and microtube hybridization (MTH) and colorimetric detection (Step 4). The experimental details are described in the text.
Figure Legend Snippet: Schematic flow of the diagnostic method developed in this study for latent infection of strawberry anthracnose fungi. The diagnostic steps consist of 4 steps, initial culturing of candidate fungi (Step 1), DNA extraction from fungal hyphae (Step 2), synthesis of PCR-amplified probes (Step 3), and microtube hybridization (MTH) and colorimetric detection (Step 4). The experimental details are described in the text.

Techniques Used: Flow Cytometry, Diagnostic Assay, Infection, DNA Extraction, Polymerase Chain Reaction, Amplification, Hybridization

5) Product Images from "p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts"

Article Title: p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0066892

AP-1 binds directly to the PAK3 promoter at (+52/+60) both in vivo and in vitro . A: Electrophoretic Mobility Shift Assay (EMSA) showing protein binding to the (+52/+60) PAK3 promoter region. Lane 1 shows the presence of a DNA/protein complex when the (+52/+60) PAK3 promoter radioactively-labelled oligomer was incubated with protein cell lysate of Rat1a-J4 cells treated with doxycycline. This DNA/protein complex could be competed for with unlabelled (+52/+60) wild-type (WT) oligomers (lane 2), but not with a mutated (+52/+60) sequence oligomer (lane 3). The second panel shows supershifted complexes using anti-cJun (lane 4), -JunB (lane 5) and –JunD (lane 6) antibodies. B: Chromatin Immunoprecipitation Assay (ChIP) showing cJun binding to the (+52/+60) PAK3 promoter region. Rat1a-J4 cells were treated with doxycycline for 48 hrs, after which DNA and protein complexes were cross-linked and pulled down with an anti-cJun antibody. RT-PCR amplification off the pulled-down chromatin showed cJun binding to the (+52/+60) PAK3 promoter region in the presence of doxycycline.
Figure Legend Snippet: AP-1 binds directly to the PAK3 promoter at (+52/+60) both in vivo and in vitro . A: Electrophoretic Mobility Shift Assay (EMSA) showing protein binding to the (+52/+60) PAK3 promoter region. Lane 1 shows the presence of a DNA/protein complex when the (+52/+60) PAK3 promoter radioactively-labelled oligomer was incubated with protein cell lysate of Rat1a-J4 cells treated with doxycycline. This DNA/protein complex could be competed for with unlabelled (+52/+60) wild-type (WT) oligomers (lane 2), but not with a mutated (+52/+60) sequence oligomer (lane 3). The second panel shows supershifted complexes using anti-cJun (lane 4), -JunB (lane 5) and –JunD (lane 6) antibodies. B: Chromatin Immunoprecipitation Assay (ChIP) showing cJun binding to the (+52/+60) PAK3 promoter region. Rat1a-J4 cells were treated with doxycycline for 48 hrs, after which DNA and protein complexes were cross-linked and pulled down with an anti-cJun antibody. RT-PCR amplification off the pulled-down chromatin showed cJun binding to the (+52/+60) PAK3 promoter region in the presence of doxycycline.

Techniques Used: In Vivo, In Vitro, Electrophoretic Mobility Shift Assay, Protein Binding, Incubation, Sequencing, Chromatin Immunoprecipitation, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

6) Product Images from "Characterization of a New CAMP Factor Carried by an Integrative and Conjugative Element in Streptococcus agalactiae and Spreading in Streptococci"

Article Title: Characterization of a New CAMP Factor Carried by an Integrative and Conjugative Element in Streptococcus agalactiae and Spreading in Streptococci

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048918

Analysis of the expression of the CAMP factor II (SAL_2074) gene. RT-PCR were performed using, as templates, RNA extracted from stationary-phase cultures of strain 515 (1, and negative control without RTase, 4); strain NEM316 (2 and negative control without RTase, 5); and transconjugant NEM316 (ICE_ 515_tRNA Lys ) (3 and negative control without RTase, 6). The same results were obtained in exponential phase. A positive control using genomic DNA of strain 515 was included (7) as well as a negative control with water (8). DNA molecular weight marker is marker number VI (Roche Applied Science).
Figure Legend Snippet: Analysis of the expression of the CAMP factor II (SAL_2074) gene. RT-PCR were performed using, as templates, RNA extracted from stationary-phase cultures of strain 515 (1, and negative control without RTase, 4); strain NEM316 (2 and negative control without RTase, 5); and transconjugant NEM316 (ICE_ 515_tRNA Lys ) (3 and negative control without RTase, 6). The same results were obtained in exponential phase. A positive control using genomic DNA of strain 515 was included (7) as well as a negative control with water (8). DNA molecular weight marker is marker number VI (Roche Applied Science).

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Molecular Weight, Marker

7) Product Images from "Complexity of CNC Transcription Factors As Revealed by Gene Targeting of the Nrf3 Locus"

Article Title: Complexity of CNC Transcription Factors As Revealed by Gene Targeting of the Nrf3 Locus

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.8.3286-3294.2004

Generation of Nrf3-deficient mice. (A) Replacement of two exons of the Nrf3 gene, comprising the sequences coding for the bZIP domain, by the neomycin-cytosine deaminase cassette of the targeting vector. Nrf3 gene exons are represented by open boxes, and the exon encoding the Nrf3 bZIP domain is shown. The targeting vector contains 7.5 and 3.2 kb of Nrf3 gene homologous genomic sequences on the 5′ and 3′ sides of the neomycin-cytosine deaminase cassette, respectively. The probe used for Southern analysis and the expected fragments from EcoRI/SpeI digests of wild-type and knockout genomic DNA are indicated. tk, thymidine kinase; neo, neomycin; cda, cytosine deaminase. (B) Generation of Nrf3 −/− mice. Southern blot analysis of Nrf3 +/− mouse matings yielding offspring with homozygous wild-type (wt) and heterozygous and homozygous mutant (null) genotypes. ko, knockout.
Figure Legend Snippet: Generation of Nrf3-deficient mice. (A) Replacement of two exons of the Nrf3 gene, comprising the sequences coding for the bZIP domain, by the neomycin-cytosine deaminase cassette of the targeting vector. Nrf3 gene exons are represented by open boxes, and the exon encoding the Nrf3 bZIP domain is shown. The targeting vector contains 7.5 and 3.2 kb of Nrf3 gene homologous genomic sequences on the 5′ and 3′ sides of the neomycin-cytosine deaminase cassette, respectively. The probe used for Southern analysis and the expected fragments from EcoRI/SpeI digests of wild-type and knockout genomic DNA are indicated. tk, thymidine kinase; neo, neomycin; cda, cytosine deaminase. (B) Generation of Nrf3 −/− mice. Southern blot analysis of Nrf3 +/− mouse matings yielding offspring with homozygous wild-type (wt) and heterozygous and homozygous mutant (null) genotypes. ko, knockout.

Techniques Used: Mouse Assay, Plasmid Preparation, Genomic Sequencing, Knock-Out, Southern Blot, Mutagenesis

8) Product Images from "Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery"

Article Title: Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery

Journal: Antiviral Research

doi: 10.1016/j.antiviral.2012.03.011

Dose-dependent decrease in Ad5 genome copy numbers mediated by the DNA polymerase siRNA. A549 cells were transfected with the DNA polymerase siRNA or a non-targeting control siRNA (neg. ctrl.) in decreasing concentrations as indicated, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers from triplicate infections (mean ± SD; n = 3) were determined at 48 h post-infection by qPCR, using E1A-specific primers. For each experiment, real-time qPCR quantification was performed in duplicate. * p
Figure Legend Snippet: Dose-dependent decrease in Ad5 genome copy numbers mediated by the DNA polymerase siRNA. A549 cells were transfected with the DNA polymerase siRNA or a non-targeting control siRNA (neg. ctrl.) in decreasing concentrations as indicated, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers from triplicate infections (mean ± SD; n = 3) were determined at 48 h post-infection by qPCR, using E1A-specific primers. For each experiment, real-time qPCR quantification was performed in duplicate. * p

Techniques Used: Transfection, Infection, Real-time Polymerase Chain Reaction

Effect of combinatorial silencing of Ad5 genes on virus output. A549 cells were transfected with the indicated siRNAs either alone or in combination. SiRNA combinations for E1A (A), DNA polymerase (B), pTP (C), IVa2 (D), hexon (E), and protease (F) are depicted. For all transfections, the total concentration of siRNA was 10 nM. For combinations, each siRNA was employed at a concentration of 5 nM. As a control, cells were transfected with the individual siRNAs alone at a concentration of 10 nM, or as a mix of 5 nM targeting siRNA and 5 nM non-targeting negative control siRNA. Subsequently, cells were infected with Ad5 at an MOI of 0.01 TCID 50 /cell, and cells and supernatants were harvested at 48 h post-infection. Numbers of infectious Ad5 particles of triplicate infections were determined on A549 cells by TCID 50 assays (mean ± SD; n = 3).
Figure Legend Snippet: Effect of combinatorial silencing of Ad5 genes on virus output. A549 cells were transfected with the indicated siRNAs either alone or in combination. SiRNA combinations for E1A (A), DNA polymerase (B), pTP (C), IVa2 (D), hexon (E), and protease (F) are depicted. For all transfections, the total concentration of siRNA was 10 nM. For combinations, each siRNA was employed at a concentration of 5 nM. As a control, cells were transfected with the individual siRNAs alone at a concentration of 10 nM, or as a mix of 5 nM targeting siRNA and 5 nM non-targeting negative control siRNA. Subsequently, cells were infected with Ad5 at an MOI of 0.01 TCID 50 /cell, and cells and supernatants were harvested at 48 h post-infection. Numbers of infectious Ad5 particles of triplicate infections were determined on A549 cells by TCID 50 assays (mean ± SD; n = 3).

Techniques Used: Transfection, Concentration Assay, Negative Control, Infection

Differential inhibition of Ad5 replication by DNA polymerase siRNAs binding in the immediate vicinity of, or overlapping, the Pol-si2 target sequence. (A) Region of the DNA polymerase open reading frame (indicated as DNA pol) targeted by siRNAs Pol-si2, Pol-si4, Pol-si5, and Pol-si6. The DNA sequences corresponding to the individual siRNA target sites on the target mRNAs are given below. The nucleotides corresponding to the seed regions of the respective siRNAs are shaded in grey. (B) A549 cells were transfected with the viral DNA polymerase-directed siRNAs or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers from triplicate infections (mean ± SD; n = 3) were determined at 48 h post-infection by qPCR using E1A-specific primers. *** p
Figure Legend Snippet: Differential inhibition of Ad5 replication by DNA polymerase siRNAs binding in the immediate vicinity of, or overlapping, the Pol-si2 target sequence. (A) Region of the DNA polymerase open reading frame (indicated as DNA pol) targeted by siRNAs Pol-si2, Pol-si4, Pol-si5, and Pol-si6. The DNA sequences corresponding to the individual siRNA target sites on the target mRNAs are given below. The nucleotides corresponding to the seed regions of the respective siRNAs are shaded in grey. (B) A549 cells were transfected with the viral DNA polymerase-directed siRNAs or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers from triplicate infections (mean ± SD; n = 3) were determined at 48 h post-infection by qPCR using E1A-specific primers. *** p

Techniques Used: Inhibition, Binding Assay, Sequencing, Transfection, Concentration Assay, Infection, Real-time Polymerase Chain Reaction

Impact of siRNAs on viral DNA replication and virus spreading. (A) A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers were determined at 48 h post-infection by qPCR, using E1A-specific primers. Values represent mean ± SD of three independent experiments, each performed in triplicate. For each experiment, real-time qPCR quantification was performed in duplicate. * p
Figure Legend Snippet: Impact of siRNAs on viral DNA replication and virus spreading. (A) A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers were determined at 48 h post-infection by qPCR, using E1A-specific primers. Values represent mean ± SD of three independent experiments, each performed in triplicate. For each experiment, real-time qPCR quantification was performed in duplicate. * p

Techniques Used: Transfection, Concentration Assay, Infection, Real-time Polymerase Chain Reaction

siRNAs decrease mRNA levels directly and indirectly. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Levels of direct targets (A) or indirect targets such as the E1A (B), DNA polymerase (C), pTP (D), and IVa2 (E) mRNAs were determined by RT-qPCR. Relative mRNA levels in comparison to a non-targeting siRNA are shown. Values represent mean ± SD of three independent experiments, each performed in triplicate. For each experiment, real-time qPCR quantification was performed in duplicate. * p
Figure Legend Snippet: siRNAs decrease mRNA levels directly and indirectly. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Levels of direct targets (A) or indirect targets such as the E1A (B), DNA polymerase (C), pTP (D), and IVa2 (E) mRNAs were determined by RT-qPCR. Relative mRNA levels in comparison to a non-targeting siRNA are shown. Values represent mean ± SD of three independent experiments, each performed in triplicate. For each experiment, real-time qPCR quantification was performed in duplicate. * p

Techniques Used: Transfection, Concentration Assay, Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

siRNAs decrease the numbers of infectious virus particles. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Numbers of infectious virus particles at 48 h post-infection were determined on A549 cells by TCID 50 assays. Representative data from three independent experiments, each performed in triplicate, are shown (mean ± SD; n = 3). ** p
Figure Legend Snippet: siRNAs decrease the numbers of infectious virus particles. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Numbers of infectious virus particles at 48 h post-infection were determined on A549 cells by TCID 50 assays. Representative data from three independent experiments, each performed in triplicate, are shown (mean ± SD; n = 3). ** p

Techniques Used: Transfection, Concentration Assay, Infection

SiRNAs increase the viability of infected cells. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 4 TCID 50 /cell. An MTS assay was performed at 6 days post-infection. The viability of cells from triplicate infection experiments (mean ± SD; n = 3) was calculated in relation to the viability of mock-infected cells. *** p
Figure Legend Snippet: SiRNAs increase the viability of infected cells. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 4 TCID 50 /cell. An MTS assay was performed at 6 days post-infection. The viability of cells from triplicate infection experiments (mean ± SD; n = 3) was calculated in relation to the viability of mock-infected cells. *** p

Techniques Used: Infection, Transfection, Concentration Assay, MTS Assay

9) Product Images from "Potyviral resistance derived from cultivars of Phaseolus vulgaris carrying bc‐3 is associated with the homozygotic presence of a mutated eIF4E allele"

Article Title: Potyviral resistance derived from cultivars of Phaseolus vulgaris carrying bc‐3 is associated with the homozygotic presence of a mutated eIF4E allele

Journal: Molecular Plant Pathology

doi: 10.1111/j.1364-3703.2009.00602.x

Segregation of Bean common mosaic virus (BCMV) resistance and a cleaved amplified polymorphic sequence (CAPS) marker differentiating eIF4E alleles PveIF4E 2 ( 4E 2 ) and PveIF4E 1 ( 4E 1 ). A Phaseolus vulgaris F 2 population was generated from a cross between parents (P) USCR8 ( 4E 2 /4E 2 , lane 2) and Dubbele Witte (DW) ( 4E 1 /4E 1 , lane 3). Successful crossing was verified on F 1 individuals (lane 4) and results from 17 F 2 individuals are shown (lanes 5–21).The response to BCMV was assayed by enzyme‐linked immunosorbent assay (ELISA). Plants with an ELISA A405 (absorbance at 405 nm) reading of more than 2.5 times that of the mock‐inoculated control were rated as susceptible (s); plants with an ELISA A405 reading of less than 2.5 times that of the mock‐inoculated control were rated as resistant (r). The genotype was determined by restriction enzyme Rsa I digestion of a 541‐bp polymerase chain reaction (PCR) product amplified with primers ENM‐FWe and ENM‐RVe from genomic DNA. DNA fragments were analysed by agarose gel electrophoresis with a 100‐bp ladder as marker (lane 1).
Figure Legend Snippet: Segregation of Bean common mosaic virus (BCMV) resistance and a cleaved amplified polymorphic sequence (CAPS) marker differentiating eIF4E alleles PveIF4E 2 ( 4E 2 ) and PveIF4E 1 ( 4E 1 ). A Phaseolus vulgaris F 2 population was generated from a cross between parents (P) USCR8 ( 4E 2 /4E 2 , lane 2) and Dubbele Witte (DW) ( 4E 1 /4E 1 , lane 3). Successful crossing was verified on F 1 individuals (lane 4) and results from 17 F 2 individuals are shown (lanes 5–21).The response to BCMV was assayed by enzyme‐linked immunosorbent assay (ELISA). Plants with an ELISA A405 (absorbance at 405 nm) reading of more than 2.5 times that of the mock‐inoculated control were rated as susceptible (s); plants with an ELISA A405 reading of less than 2.5 times that of the mock‐inoculated control were rated as resistant (r). The genotype was determined by restriction enzyme Rsa I digestion of a 541‐bp polymerase chain reaction (PCR) product amplified with primers ENM‐FWe and ENM‐RVe from genomic DNA. DNA fragments were analysed by agarose gel electrophoresis with a 100‐bp ladder as marker (lane 1).

Techniques Used: Amplification, Sequencing, Marker, Generated, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis

10) Product Images from "Spectrum of Activity and Mechanisms of Resistance of Various Nucleoside Derivatives against Gammaherpesviruses"

Article Title: Spectrum of Activity and Mechanisms of Resistance of Various Nucleoside Derivatives against Gammaherpesviruses

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.03957-14

Visualization of the mutations identified in HVS and MHV-68, at the homologous positions of HSV-1 TK and DNA polymerase three-dimensional (3D) structures. (A) BVDU (blue) is bound to the phosphate acceptor site of the HSV-1 TK (PDB code 1KI8). The p-loop (magenta) motif present in all the ATP-binding proteins is important for the interactions with the β and γ phosphates of the phosphate donor. The positions of the mutations identified in HVS are shown in cyan, and those identified in MHV-68 are in red. Amino acid changes at positions C372 (MHV-68) and A398 (HVS) described in this study were found under selection with BVDU. The other amino acid changes (shown in red at position P326 in HVS and positions Q401, E358, and T364 in MHV-68) were identified after selection with a novel pyrimidine nucleoside derivative (i.e., HDVD) possessing high levels of selectivity and potency against gammaherpesviruses. All the identified mutations are present in the first shell of residues surrounding the substrate (here BVDU) in the active site. (B) 3D structure of the HSV-1 DNA polymerase, showing the different domains: pre-NH 2 domain (gray), NH 2 -terminal domain (red), 3′-5′-exonuclease domain (green), polymerase palm subdomain (blue), finger subdomain (black), and thumb subdomain (orange). Amino acid changes at the homologous position (indicated in blue) are present in the NH 2 -terminal domain and the finger subdomain. All the pictures were generated using PyMol graphic system (open source; v0.99rc9).
Figure Legend Snippet: Visualization of the mutations identified in HVS and MHV-68, at the homologous positions of HSV-1 TK and DNA polymerase three-dimensional (3D) structures. (A) BVDU (blue) is bound to the phosphate acceptor site of the HSV-1 TK (PDB code 1KI8). The p-loop (magenta) motif present in all the ATP-binding proteins is important for the interactions with the β and γ phosphates of the phosphate donor. The positions of the mutations identified in HVS are shown in cyan, and those identified in MHV-68 are in red. Amino acid changes at positions C372 (MHV-68) and A398 (HVS) described in this study were found under selection with BVDU. The other amino acid changes (shown in red at position P326 in HVS and positions Q401, E358, and T364 in MHV-68) were identified after selection with a novel pyrimidine nucleoside derivative (i.e., HDVD) possessing high levels of selectivity and potency against gammaherpesviruses. All the identified mutations are present in the first shell of residues surrounding the substrate (here BVDU) in the active site. (B) 3D structure of the HSV-1 DNA polymerase, showing the different domains: pre-NH 2 domain (gray), NH 2 -terminal domain (red), 3′-5′-exonuclease domain (green), polymerase palm subdomain (blue), finger subdomain (black), and thumb subdomain (orange). Amino acid changes at the homologous position (indicated in blue) are present in the NH 2 -terminal domain and the finger subdomain. All the pictures were generated using PyMol graphic system (open source; v0.99rc9).

Techniques Used: Binding Assay, Selection, Generated

Sequence alignment of sites in HVS and MHV-68 TK and DNA polymerase in which drug resistance mutations occurred. The TK and DNA polymerase sequences of the human herpesviruses (HSV-1, HSV-2, VZV, HCMV, EBV, and KSHV) belonging to three subfamilies (α, β, and γ) were aligned. In addition, three additional gammaherpesviruses, HVS, MHV-68, and RRV, were included. The two substitutions in the DNA polymerase gene occurred in the conserved regions δ region C and VI.
Figure Legend Snippet: Sequence alignment of sites in HVS and MHV-68 TK and DNA polymerase in which drug resistance mutations occurred. The TK and DNA polymerase sequences of the human herpesviruses (HSV-1, HSV-2, VZV, HCMV, EBV, and KSHV) belonging to three subfamilies (α, β, and γ) were aligned. In addition, three additional gammaherpesviruses, HVS, MHV-68, and RRV, were included. The two substitutions in the DNA polymerase gene occurred in the conserved regions δ region C and VI.

Techniques Used: Sequencing

11) Product Images from "Genetic Typing of the Porin Protein of Neisseria gonorrhoeae from Clinical Noncultured Samples for Strain Characterization and Identification of Mixed Gonococcal Infections"

Article Title: Genetic Typing of the Porin Protein of Neisseria gonorrhoeae from Clinical Noncultured Samples for Strain Characterization and Identification of Mixed Gonococcal Infections

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.43.1.368-375.2005

por VR typing of PCR-amplified DNA from mixtures of two bacterial suspensions using checkerboard hybridization. Suspensions of PIB and PIA strains were mixed at different ratios, centrifuged, and DNA purified, and porB was PCR amplified. PCR product was
Figure Legend Snippet: por VR typing of PCR-amplified DNA from mixtures of two bacterial suspensions using checkerboard hybridization. Suspensions of PIB and PIA strains were mixed at different ratios, centrifuged, and DNA purified, and porB was PCR amplified. PCR product was

Techniques Used: Polymerase Chain Reaction, Amplification, Hybridization, Purification

12) Product Images from "Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery"

Article Title: Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery

Journal: Antiviral Research

doi: 10.1016/j.antiviral.2012.03.011

Dose-dependent decrease in Ad5 genome copy numbers mediated by the DNA polymerase siRNA. A549 cells were transfected with the DNA polymerase siRNA or a non-targeting control siRNA (neg. ctrl.) in decreasing concentrations as indicated, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers from triplicate infections (mean ± SD; n = 3) were determined at 48 h post-infection by qPCR, using E1A-specific primers. For each experiment, real-time qPCR quantification was performed in duplicate. * p
Figure Legend Snippet: Dose-dependent decrease in Ad5 genome copy numbers mediated by the DNA polymerase siRNA. A549 cells were transfected with the DNA polymerase siRNA or a non-targeting control siRNA (neg. ctrl.) in decreasing concentrations as indicated, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers from triplicate infections (mean ± SD; n = 3) were determined at 48 h post-infection by qPCR, using E1A-specific primers. For each experiment, real-time qPCR quantification was performed in duplicate. * p

Techniques Used: Transfection, Infection, Real-time Polymerase Chain Reaction

Effect of combinatorial silencing of Ad5 genes on virus output. A549 cells were transfected with the indicated siRNAs either alone or in combination. SiRNA combinations for E1A (A), DNA polymerase (B), pTP (C), IVa2 (D), hexon (E), and protease (F) are depicted. For all transfections, the total concentration of siRNA was 10 nM. For combinations, each siRNA was employed at a concentration of 5 nM. As a control, cells were transfected with the individual siRNAs alone at a concentration of 10 nM, or as a mix of 5 nM targeting siRNA and 5 nM non-targeting negative control siRNA. Subsequently, cells were infected with Ad5 at an MOI of 0.01 TCID 50 /cell, and cells and supernatants were harvested at 48 h post-infection. Numbers of infectious Ad5 particles of triplicate infections were determined on A549 cells by TCID 50 assays (mean ± SD; n = 3).
Figure Legend Snippet: Effect of combinatorial silencing of Ad5 genes on virus output. A549 cells were transfected with the indicated siRNAs either alone or in combination. SiRNA combinations for E1A (A), DNA polymerase (B), pTP (C), IVa2 (D), hexon (E), and protease (F) are depicted. For all transfections, the total concentration of siRNA was 10 nM. For combinations, each siRNA was employed at a concentration of 5 nM. As a control, cells were transfected with the individual siRNAs alone at a concentration of 10 nM, or as a mix of 5 nM targeting siRNA and 5 nM non-targeting negative control siRNA. Subsequently, cells were infected with Ad5 at an MOI of 0.01 TCID 50 /cell, and cells and supernatants were harvested at 48 h post-infection. Numbers of infectious Ad5 particles of triplicate infections were determined on A549 cells by TCID 50 assays (mean ± SD; n = 3).

Techniques Used: Transfection, Concentration Assay, Negative Control, Infection

Differential inhibition of Ad5 replication by DNA polymerase siRNAs binding in the immediate vicinity of, or overlapping, the Pol-si2 target sequence. (A) Region of the DNA polymerase open reading frame (indicated as DNA pol) targeted by siRNAs Pol-si2, Pol-si4, Pol-si5, and Pol-si6. The DNA sequences corresponding to the individual siRNA target sites on the target mRNAs are given below. The nucleotides corresponding to the seed regions of the respective siRNAs are shaded in grey. (B) A549 cells were transfected with the viral DNA polymerase-directed siRNAs or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers from triplicate infections (mean ± SD; n = 3) were determined at 48 h post-infection by qPCR using E1A-specific primers. *** p
Figure Legend Snippet: Differential inhibition of Ad5 replication by DNA polymerase siRNAs binding in the immediate vicinity of, or overlapping, the Pol-si2 target sequence. (A) Region of the DNA polymerase open reading frame (indicated as DNA pol) targeted by siRNAs Pol-si2, Pol-si4, Pol-si5, and Pol-si6. The DNA sequences corresponding to the individual siRNA target sites on the target mRNAs are given below. The nucleotides corresponding to the seed regions of the respective siRNAs are shaded in grey. (B) A549 cells were transfected with the viral DNA polymerase-directed siRNAs or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers from triplicate infections (mean ± SD; n = 3) were determined at 48 h post-infection by qPCR using E1A-specific primers. *** p

Techniques Used: Inhibition, Binding Assay, Sequencing, Transfection, Concentration Assay, Infection, Real-time Polymerase Chain Reaction

Impact of siRNAs on viral DNA replication and virus spreading. (A) A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers were determined at 48 h post-infection by qPCR, using E1A-specific primers. Values represent mean ± SD of three independent experiments, each performed in triplicate. For each experiment, real-time qPCR quantification was performed in duplicate. * p
Figure Legend Snippet: Impact of siRNAs on viral DNA replication and virus spreading. (A) A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Virus genome copy numbers were determined at 48 h post-infection by qPCR, using E1A-specific primers. Values represent mean ± SD of three independent experiments, each performed in triplicate. For each experiment, real-time qPCR quantification was performed in duplicate. * p

Techniques Used: Transfection, Concentration Assay, Infection, Real-time Polymerase Chain Reaction

siRNAs decrease mRNA levels directly and indirectly. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Levels of direct targets (A) or indirect targets such as the E1A (B), DNA polymerase (C), pTP (D), and IVa2 (E) mRNAs were determined by RT-qPCR. Relative mRNA levels in comparison to a non-targeting siRNA are shown. Values represent mean ± SD of three independent experiments, each performed in triplicate. For each experiment, real-time qPCR quantification was performed in duplicate. * p
Figure Legend Snippet: siRNAs decrease mRNA levels directly and indirectly. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Levels of direct targets (A) or indirect targets such as the E1A (B), DNA polymerase (C), pTP (D), and IVa2 (E) mRNAs were determined by RT-qPCR. Relative mRNA levels in comparison to a non-targeting siRNA are shown. Values represent mean ± SD of three independent experiments, each performed in triplicate. For each experiment, real-time qPCR quantification was performed in duplicate. * p

Techniques Used: Transfection, Concentration Assay, Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

siRNAs decrease the numbers of infectious virus particles. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Numbers of infectious virus particles at 48 h post-infection were determined on A549 cells by TCID 50 assays. Representative data from three independent experiments, each performed in triplicate, are shown (mean ± SD; n = 3). ** p
Figure Legend Snippet: siRNAs decrease the numbers of infectious virus particles. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 0.01 TCID 50 /cell. Numbers of infectious virus particles at 48 h post-infection were determined on A549 cells by TCID 50 assays. Representative data from three independent experiments, each performed in triplicate, are shown (mean ± SD; n = 3). ** p

Techniques Used: Transfection, Concentration Assay, Infection

SiRNAs increase the viability of infected cells. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 4 TCID 50 /cell. An MTS assay was performed at 6 days post-infection. The viability of cells from triplicate infection experiments (mean ± SD; n = 3) was calculated in relation to the viability of mock-infected cells. *** p
Figure Legend Snippet: SiRNAs increase the viability of infected cells. A549 cells were transfected with siRNAs directed against the E1A, DNA polymerase (Pol), pTP, IVa2, hexon (Hex), and protease (Prot) genes, or a non-targeting control siRNA (neg. ctrl.) at a concentration of 10 nM, and then infected with Ad5 at an MOI of 4 TCID 50 /cell. An MTS assay was performed at 6 days post-infection. The viability of cells from triplicate infection experiments (mean ± SD; n = 3) was calculated in relation to the viability of mock-infected cells. *** p

Techniques Used: Infection, Transfection, Concentration Assay, MTS Assay

Related Articles

Clone Assay:

Article Title: p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts
Article Snippet: .. PCR using high fidelity Expand Plus DNA Polymerase (Roche) was performed on rat genomic DNA, the products were sub-cloned into the pGEM-T Easy vector (Promega) and excised for cloning into the luciferase reported vector, pGL3 -Basic (Promega) using Mlu1 and Xho1 restriction enzymes. ..

Article Title: The Potassium Transporter AtHAK5 Functions in K+ Deprivation-Induced High-Affinity K+ Uptake and AKT1 K+ Channel Contribution to K+ Uptake Kinetics in Arabidopsis Roots 1 Uptake Kinetics in Arabidopsis Roots 1 [w]
Article Snippet: .. The 2-kb DNA fragment upstream of the AtHAK5 start codon was amplified by PCR from Col-0 genomic DNA using a proofreading DNA polymerase (Pwo, Roche, Indianapolis) and cloned into the Pst I/ Nco I sites of the binary vector pCAMBIA 1303 replacing the cauliflower mosaic virus 35S promoter. .. The 3′ Nco I site was introduced by a mismatch in the antisense primer (5′-cctcaccatccatGGtttgctgtgtt-3′), while the 5′ Pst I site was present in the native DNA sequence (5′-cttacactgctgcagcctcggctt-3′).

Luciferase:

Article Title: p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts
Article Snippet: .. PCR using high fidelity Expand Plus DNA Polymerase (Roche) was performed on rat genomic DNA, the products were sub-cloned into the pGEM-T Easy vector (Promega) and excised for cloning into the luciferase reported vector, pGL3 -Basic (Promega) using Mlu1 and Xho1 restriction enzymes. ..

Purification:

Article Title: Myocyte Enhancer Factor 2 and Class II Histone Deacetylases Control a Gender-Specific Pathway of Cardioprotection Mediated by the Estrogen Receptor
Article Snippet: .. Annealed oligonucleotides were radiolabeled with [32 P]dCTP using the Klenow fragment of DNA polymerase and purified using G50 spin columns (Roche). .. Nuclear cell extracts were isolated from COS-1 cells that were transfected with pcDNAMYC-MEF2C.

Real-time Polymerase Chain Reaction:

Article Title: Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery
Article Snippet: .. Expression levels of the E1A-12S, E1A-13S, DNA polymerase, pTP, IVa2, hexon, and protease genes were determined by TaqMan real-time quantitative PCR (qPCR), using the LightCycler 480 Probes master mix (Roche Diagnostics) and primer/probe sets specific for E1A-13S (E1A 289R-cDNA-f1 5′-GCATGTTTGTCTACAGTCCTGTGTC-3′, E1A 289R-cDNA-r1 5′-GGCGTCTCAGGATAGCAGGC-3′, and E1A 289R-cDNA-p1 5′-AGGCTCCGGTTCTGGCTCGGG-3′), E1A-12S (E1A 12S-cDNA-f1 5′-AGGATGAAGAGGGTCCTGTGTCT-3′, E1A 289R-cDNA-r1 5′-GGCGTCTCAGGATAGCAGGC-3′, and E1A 289R-cDNA-p1 5′-AGGCTCCGGTTCTGGCTCGGG-3′), DNA polymerase (Pol-cDNA-f1 5′-ATGGCCTTGGCTCAAGCTC-3′, Pol-cDNA-r1 5′-GCGTAGGTTGCTGGCGAAC-3′, and Pol-cDNA-p1 5′-CGCCTCTGCGTGAAGACGACGG-3′), pTP (pTP-cDNA-f2 5′-AAACCAACGCTCGGTGCC-3′, pTP-cDNA-r2 5′-GGACGCGGTTCCAGATGTT-3′, and pTP-cDNA-p2 5′-CGCGCGCAATCGTTGACGCT-3′), IVa2 (IVa2-cDNA-f1 5′-GGAAACCAGAGGGCGAAGA-3′, IVa2-cDNA-r1 5′-AGGGTCCTCGTCAGCGTAGTC-3′, and IVa2-cDNA-p1 5′-CTTGAGGCTGGTCCTGCTGGT-3′), hexon (Hex-cDNA-f1 5′-CAGTCACAGTCGCAAGAGGAGC-3′, Hex-cDNA-r1 5′-AGGTACTCCGAGGCGTCCTG-3′, and Hex-cDNA-p1 5′-ACCACTGCGGCATCATCGAAGGG-3′), and protease (Prot-cDNA-f1 5′-TCACAGTCGCAAGTCTTTGACG-3′, Prot-cDNA-r1 5′-GCGGCAGCTGTTGTTGATG-3′, and Prot-cDNA-p1 5′-CCGAGAAGGGCGTGCGCAGGTA-3′). ..

Generated:

Article Title: Complexity of CNC Transcription Factors As Revealed by Gene Targeting of the Nrf3 Locus
Article Snippet: .. The Nrf3 probe was generated by random priming of a 572-bp SacII/PstI fragment (corresponding to amino acids 131 to 321 of mouse Nrf3) by using [32 P]dCTP (Amersham) and the Klenow fragment of DNA polymerase I according to standard methods (Roche). ..

Amplification:

Article Title: The Potassium Transporter AtHAK5 Functions in K+ Deprivation-Induced High-Affinity K+ Uptake and AKT1 K+ Channel Contribution to K+ Uptake Kinetics in Arabidopsis Roots 1 Uptake Kinetics in Arabidopsis Roots 1 [w]
Article Snippet: .. The 2-kb DNA fragment upstream of the AtHAK5 start codon was amplified by PCR from Col-0 genomic DNA using a proofreading DNA polymerase (Pwo, Roche, Indianapolis) and cloned into the Pst I/ Nco I sites of the binary vector pCAMBIA 1303 replacing the cauliflower mosaic virus 35S promoter. .. The 3′ Nco I site was introduced by a mismatch in the antisense primer (5′-cctcaccatccatGGtttgctgtgtt-3′), while the 5′ Pst I site was present in the native DNA sequence (5′-cttacactgctgcagcctcggctt-3′).

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. The following conditions were used for amplification: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min and extension at 72°C for 1 min and further extension for 5 min at 72°C ( ).

Expressing:

Article Title: Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery
Article Snippet: .. Expression levels of the E1A-12S, E1A-13S, DNA polymerase, pTP, IVa2, hexon, and protease genes were determined by TaqMan real-time quantitative PCR (qPCR), using the LightCycler 480 Probes master mix (Roche Diagnostics) and primer/probe sets specific for E1A-13S (E1A 289R-cDNA-f1 5′-GCATGTTTGTCTACAGTCCTGTGTC-3′, E1A 289R-cDNA-r1 5′-GGCGTCTCAGGATAGCAGGC-3′, and E1A 289R-cDNA-p1 5′-AGGCTCCGGTTCTGGCTCGGG-3′), E1A-12S (E1A 12S-cDNA-f1 5′-AGGATGAAGAGGGTCCTGTGTCT-3′, E1A 289R-cDNA-r1 5′-GGCGTCTCAGGATAGCAGGC-3′, and E1A 289R-cDNA-p1 5′-AGGCTCCGGTTCTGGCTCGGG-3′), DNA polymerase (Pol-cDNA-f1 5′-ATGGCCTTGGCTCAAGCTC-3′, Pol-cDNA-r1 5′-GCGTAGGTTGCTGGCGAAC-3′, and Pol-cDNA-p1 5′-CGCCTCTGCGTGAAGACGACGG-3′), pTP (pTP-cDNA-f2 5′-AAACCAACGCTCGGTGCC-3′, pTP-cDNA-r2 5′-GGACGCGGTTCCAGATGTT-3′, and pTP-cDNA-p2 5′-CGCGCGCAATCGTTGACGCT-3′), IVa2 (IVa2-cDNA-f1 5′-GGAAACCAGAGGGCGAAGA-3′, IVa2-cDNA-r1 5′-AGGGTCCTCGTCAGCGTAGTC-3′, and IVa2-cDNA-p1 5′-CTTGAGGCTGGTCCTGCTGGT-3′), hexon (Hex-cDNA-f1 5′-CAGTCACAGTCGCAAGAGGAGC-3′, Hex-cDNA-r1 5′-AGGTACTCCGAGGCGTCCTG-3′, and Hex-cDNA-p1 5′-ACCACTGCGGCATCATCGAAGGG-3′), and protease (Prot-cDNA-f1 5′-TCACAGTCGCAAGTCTTTGACG-3′, Prot-cDNA-r1 5′-GCGGCAGCTGTTGTTGATG-3′, and Prot-cDNA-p1 5′-CCGAGAAGGGCGTGCGCAGGTA-3′). ..

Polymerase Chain Reaction:

Article Title: Construction of a System for the Strawberry Nursery Production towards Elimination of Latent Infection of Anthracnose Fungi by a Combination of PCR and Microtube Hybridization
Article Snippet: .. The reaction mixture contained 2.5 units Tth DNA polymerase (Roche Diagnostics, Tokyo, Japan), 1× PCR buffer, 1 mM dNTP (Roche Diagnostics) and 0.5 μM of each primer. ..

Article Title: p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts
Article Snippet: .. PCR using high fidelity Expand Plus DNA Polymerase (Roche) was performed on rat genomic DNA, the products were sub-cloned into the pGEM-T Easy vector (Promega) and excised for cloning into the luciferase reported vector, pGL3 -Basic (Promega) using Mlu1 and Xho1 restriction enzymes. ..

Article Title: The Potassium Transporter AtHAK5 Functions in K+ Deprivation-Induced High-Affinity K+ Uptake and AKT1 K+ Channel Contribution to K+ Uptake Kinetics in Arabidopsis Roots 1 Uptake Kinetics in Arabidopsis Roots 1 [w]
Article Snippet: .. The 2-kb DNA fragment upstream of the AtHAK5 start codon was amplified by PCR from Col-0 genomic DNA using a proofreading DNA polymerase (Pwo, Roche, Indianapolis) and cloned into the Pst I/ Nco I sites of the binary vector pCAMBIA 1303 replacing the cauliflower mosaic virus 35S promoter. .. The 3′ Nco I site was introduced by a mismatch in the antisense primer (5′-cctcaccatccatGGtttgctgtgtt-3′), while the 5′ Pst I site was present in the native DNA sequence (5′-cttacactgctgcagcctcggctt-3′).

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. The following conditions were used for amplification: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min and extension at 72°C for 1 min and further extension for 5 min at 72°C ( ).

Article Title: Characterization of a New CAMP Factor Carried by an Integrative and Conjugative Element in Streptococcus agalactiae and Spreading in Streptococci
Article Snippet: .. Conventional PCR were done using cDNA as templates for DNA polymerase (Roche Applied Science, Mannheim, Germany) as described earlier . .. Analysis of the supernatants by SDS-PAGE and mass spectrometry Supernatant of S. agalactiae NEM316 and NEM316 (ICE_515_tRNALys ) cultures grown overnight in BHI or chemically defined medium (CDM, peptide-free medium, ) were collected after centrifugation at 5000× g at 4°C for 10 min. Proteins were precipitated using a sodium deoxycholate (DOC) - trichloroacetic acid (TCA) protocol .

Plasmid Preparation:

Article Title: p21-Activated Kinase 3 (PAK3) Is an AP-1 Regulated Gene Contributing to Actin Organisation and Migration of Transformed Fibroblasts
Article Snippet: .. PCR using high fidelity Expand Plus DNA Polymerase (Roche) was performed on rat genomic DNA, the products were sub-cloned into the pGEM-T Easy vector (Promega) and excised for cloning into the luciferase reported vector, pGL3 -Basic (Promega) using Mlu1 and Xho1 restriction enzymes. ..

Article Title: The Potassium Transporter AtHAK5 Functions in K+ Deprivation-Induced High-Affinity K+ Uptake and AKT1 K+ Channel Contribution to K+ Uptake Kinetics in Arabidopsis Roots 1 Uptake Kinetics in Arabidopsis Roots 1 [w]
Article Snippet: .. The 2-kb DNA fragment upstream of the AtHAK5 start codon was amplified by PCR from Col-0 genomic DNA using a proofreading DNA polymerase (Pwo, Roche, Indianapolis) and cloned into the Pst I/ Nco I sites of the binary vector pCAMBIA 1303 replacing the cauliflower mosaic virus 35S promoter. .. The 3′ Nco I site was introduced by a mismatch in the antisense primer (5′-cctcaccatccatGGtttgctgtgtt-3′), while the 5′ Pst I site was present in the native DNA sequence (5′-cttacactgctgcagcctcggctt-3′).

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    Roche quantitative polymerization assay rna dependent dna polymerase activity
    Determination of K D -values by fluorescence anisotropy measurements. Fifteen nanomolar of a fluorescently labeled <t>DNA/DNA</t> ( A ) or <t>DNA/RNA</t> ( B ) P/T substrate was titrated with different PR–RTs at 25°C. The curves show the best fit to Equation ( 3 ) (‘Materials and methods’ section) describing the binding equilibrium with K D -values shown in Table 2 .
    Quantitative Polymerization Assay Rna Dependent Dna Polymerase Activity, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche dna polymerases
    Sequences of contaminating bacterial <t>DNA</t> in three <t>Taq</t> polymerases. Sequence alignment of three contaminants from different commercial Taq polymerases showing the presence of different strains of the same Pseudomonas species. Roche FastStart, Platinum HiFi Platinum Taq polymerases contain similar strains of a Pseudomonas species with a single base difference in the region covered by the 16S350 PCR assay.
    Dna Polymerases, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche pwo dna polymerase
    Characteristics and applications of the USER technique. ( a ) A comparison of the ability of <t>DNA</t> polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm Taq: HotMaster™ Taq DNA Polymerase, Taq: Platinum ® Taq DNA Polymerase, <t>Pwo:</t> Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo ® C x Hotstart DNA polymerase. ( b ) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus -specific USER compatible vector, pNB1u. H 2 O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. ( c ) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.
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    Determination of K D -values by fluorescence anisotropy measurements. Fifteen nanomolar of a fluorescently labeled DNA/DNA ( A ) or DNA/RNA ( B ) P/T substrate was titrated with different PR–RTs at 25°C. The curves show the best fit to Equation ( 3 ) (‘Materials and methods’ section) describing the binding equilibrium with K D -values shown in Table 2 .

    Journal: Nucleic Acids Research

    Article Title: AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP

    doi: 10.1093/nar/gkm1087

    Figure Lengend Snippet: Determination of K D -values by fluorescence anisotropy measurements. Fifteen nanomolar of a fluorescently labeled DNA/DNA ( A ) or DNA/RNA ( B ) P/T substrate was titrated with different PR–RTs at 25°C. The curves show the best fit to Equation ( 3 ) (‘Materials and methods’ section) describing the binding equilibrium with K D -values shown in Table 2 .

    Article Snippet: Quantitative polymerization assay RNA-dependent DNA polymerase activity was quantitated on a poly(rA)/oligo(dT)15 substrate (0.2 U/ml) (Roche Diagnostics GmbH, Mannheim, Germany) in a standard assay (30 μl reaction volume) as described previously ( , ) with 150 μM TTP and 41.7 Ci/ml [3 H]TTP (49.9 Ci/mmol; MP Biomedicals Inc., Irvine, CA, USA) in reaction buffer [50 mM Tris/HCl, pH 8.0, 80 mM KCl, 6 mM MgCl2 , 0.5 mM dithiothreitol (DTT), 0.05% Triton X-100].

    Techniques: Fluorescence, Labeling, Binding Assay

    Sequences of contaminating bacterial DNA in three Taq polymerases. Sequence alignment of three contaminants from different commercial Taq polymerases showing the presence of different strains of the same Pseudomonas species. Roche FastStart, Platinum HiFi Platinum Taq polymerases contain similar strains of a Pseudomonas species with a single base difference in the region covered by the 16S350 PCR assay.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Sequences of contaminating bacterial DNA in three Taq polymerases. Sequence alignment of three contaminants from different commercial Taq polymerases showing the presence of different strains of the same Pseudomonas species. Roche FastStart, Platinum HiFi Platinum Taq polymerases contain similar strains of a Pseudomonas species with a single base difference in the region covered by the 16S350 PCR assay.

    Article Snippet: Taq polymerases The following DNA polymerases from commercial vendors designed for qPCR were used in the experiments reported here: Amplitaq Gold (ABI, CA; Roche lot # J02913); Platinum Taq (Invitrogen, CA; cat # 10966–026; lot 1169610); Platinum HiFi Taq (Invitrogen, CA; cat# 11304–011; lot# 1267490); HotStar Taq (Qiagen, CA; Mat # 1007837; lot # 124125007); JumpStart Taq (Sigma, MO; cat # D-6558; lot # 71K9029).

    Techniques: Sequencing, Polymerase Chain Reaction

    Approximating the copy number of 16S rDNA in commercial Taq polymerases. Six DNA polymerases were used with primers for 16S rDNA on 7 serial 10-fold dilutions of E. coli genomic DNA (10 ng to 10 fg) with no added DNA in the 8 th sample. The least squares fit equation for each dilution series was used to assign a value to the signal from the 8 th sample, which contains Taq-associated DNA only. The efficiency of the reaction was determined from the slope of the linear fit plotting the base10 log of the DNA concentration vs. the threshold cycle. A slope of −3.322 indicates an average doubling rate of “2,” which is approximately 100% efficiency (2̂3.322∼10). The rDNA values assigned are for “E. coli equivalents.”

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Approximating the copy number of 16S rDNA in commercial Taq polymerases. Six DNA polymerases were used with primers for 16S rDNA on 7 serial 10-fold dilutions of E. coli genomic DNA (10 ng to 10 fg) with no added DNA in the 8 th sample. The least squares fit equation for each dilution series was used to assign a value to the signal from the 8 th sample, which contains Taq-associated DNA only. The efficiency of the reaction was determined from the slope of the linear fit plotting the base10 log of the DNA concentration vs. the threshold cycle. A slope of −3.322 indicates an average doubling rate of “2,” which is approximately 100% efficiency (2̂3.322∼10). The rDNA values assigned are for “E. coli equivalents.”

    Article Snippet: Taq polymerases The following DNA polymerases from commercial vendors designed for qPCR were used in the experiments reported here: Amplitaq Gold (ABI, CA; Roche lot # J02913); Platinum Taq (Invitrogen, CA; cat # 10966–026; lot 1169610); Platinum HiFi Taq (Invitrogen, CA; cat# 11304–011; lot# 1267490); HotStar Taq (Qiagen, CA; Mat # 1007837; lot # 124125007); JumpStart Taq (Sigma, MO; cat # D-6558; lot # 71K9029).

    Techniques: Concentration Assay

    Detection of Pseudomonas fluorescens at high and low Taq polymerase concentrations. Bacterial detection with 0.5 Units (A) or 0.05 Units (B) Qiagen Taq DNA polymerase with 16S350B assay on samples containing 10 3 , 10 2 , 10 1 and zero Pseudomonas fluorescens bacteria. A composite of A and B is shown in C.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Detection of Pseudomonas fluorescens at high and low Taq polymerase concentrations. Bacterial detection with 0.5 Units (A) or 0.05 Units (B) Qiagen Taq DNA polymerase with 16S350B assay on samples containing 10 3 , 10 2 , 10 1 and zero Pseudomonas fluorescens bacteria. A composite of A and B is shown in C.

    Article Snippet: Taq polymerases The following DNA polymerases from commercial vendors designed for qPCR were used in the experiments reported here: Amplitaq Gold (ABI, CA; Roche lot # J02913); Platinum Taq (Invitrogen, CA; cat # 10966–026; lot 1169610); Platinum HiFi Taq (Invitrogen, CA; cat# 11304–011; lot# 1267490); HotStar Taq (Qiagen, CA; Mat # 1007837; lot # 124125007); JumpStart Taq (Sigma, MO; cat # D-6558; lot # 71K9029).

    Techniques:

    Detection of bacterial DNA in six Taq polymerases. Four dilutions of six DNA polymerase were tested with primers for 16S rDNA in the presence of 100 pg (∼10̂5 16S rDNA) E. coli genomic DNA (circled) or H 2 O.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Detection of bacterial DNA in six Taq polymerases. Four dilutions of six DNA polymerase were tested with primers for 16S rDNA in the presence of 100 pg (∼10̂5 16S rDNA) E. coli genomic DNA (circled) or H 2 O.

    Article Snippet: Taq polymerases The following DNA polymerases from commercial vendors designed for qPCR were used in the experiments reported here: Amplitaq Gold (ABI, CA; Roche lot # J02913); Platinum Taq (Invitrogen, CA; cat # 10966–026; lot 1169610); Platinum HiFi Taq (Invitrogen, CA; cat# 11304–011; lot# 1267490); HotStar Taq (Qiagen, CA; Mat # 1007837; lot # 124125007); JumpStart Taq (Sigma, MO; cat # D-6558; lot # 71K9029).

    Techniques:

    Detection of the beta-lactamase gene in commercial Taq polymerase. Four dilutions of Amplitaq DNA polymerase were tested with primers for the beta-lactamase gene in the presence of 10 3 pUC19 plasmids (labeled 1000 pUC19 genomes) or H 2 O.

    Journal: PLoS ONE

    Article Title: Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

    doi: 10.1371/journal.pone.0007010

    Figure Lengend Snippet: Detection of the beta-lactamase gene in commercial Taq polymerase. Four dilutions of Amplitaq DNA polymerase were tested with primers for the beta-lactamase gene in the presence of 10 3 pUC19 plasmids (labeled 1000 pUC19 genomes) or H 2 O.

    Article Snippet: Taq polymerases The following DNA polymerases from commercial vendors designed for qPCR were used in the experiments reported here: Amplitaq Gold (ABI, CA; Roche lot # J02913); Platinum Taq (Invitrogen, CA; cat # 10966–026; lot 1169610); Platinum HiFi Taq (Invitrogen, CA; cat# 11304–011; lot# 1267490); HotStar Taq (Qiagen, CA; Mat # 1007837; lot # 124125007); JumpStart Taq (Sigma, MO; cat # D-6558; lot # 71K9029).

    Techniques: Labeling

    POLβ is specifically enriched at CAG expansions in the striatum but not in the cerebellum of HD mice. ChIP of CAG-expanded and Hdh loci from striatum and cerebellum of R6/1 (A) and R6/2 (B) mice using α-POLβ antibody (upper panels) and as control, α-AcH3K9/14 antibody (lower panels). (A) R6/1 mice at both 6 and 37 weeks of age and R6/2 mice at 12 weeks of age were analyzed. Each ChIP experiment was performed by pooling striata and cerebella from 2 to 4 mice. The values plotted on the graphs represent the mean values obtained from 3 to 4 independent experiments. The values correspond to percentage of enrichment, calculated as follows: % enrichment = (relative DNA concentration after ChIP with POLβ or AcH3—relative DNA concentration after ChIP with no antibody)/input. Relative DNA concentrations were measured using quantitative PCR. Error bars are sem.

    Journal: PLoS Genetics

    Article Title: Stoichiometry of Base Excision Repair Proteins Correlates with Increased Somatic CAG Instability in Striatum over Cerebellum in Huntington's Disease Transgenic Mice

    doi: 10.1371/journal.pgen.1000749

    Figure Lengend Snippet: POLβ is specifically enriched at CAG expansions in the striatum but not in the cerebellum of HD mice. ChIP of CAG-expanded and Hdh loci from striatum and cerebellum of R6/1 (A) and R6/2 (B) mice using α-POLβ antibody (upper panels) and as control, α-AcH3K9/14 antibody (lower panels). (A) R6/1 mice at both 6 and 37 weeks of age and R6/2 mice at 12 weeks of age were analyzed. Each ChIP experiment was performed by pooling striata and cerebella from 2 to 4 mice. The values plotted on the graphs represent the mean values obtained from 3 to 4 independent experiments. The values correspond to percentage of enrichment, calculated as follows: % enrichment = (relative DNA concentration after ChIP with POLβ or AcH3—relative DNA concentration after ChIP with no antibody)/input. Relative DNA concentrations were measured using quantitative PCR. Error bars are sem.

    Article Snippet: PCR reactions were performed using the expand high fidelity DNA polymerase (Roche), according to manufacturer's instructions.

    Techniques: Mouse Assay, Chromatin Immunoprecipitation, Concentration Assay, Real-time Polymerase Chain Reaction

    Characteristics and applications of the USER technique. ( a ) A comparison of the ability of DNA polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm Taq: HotMaster™ Taq DNA Polymerase, Taq: Platinum ® Taq DNA Polymerase, Pwo: Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo ® C x Hotstart DNA polymerase. ( b ) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus -specific USER compatible vector, pNB1u. H 2 O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. ( c ) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.

    Journal: Nucleic Acids Research

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    doi: 10.1093/nar/gkl635

    Figure Lengend Snippet: Characteristics and applications of the USER technique. ( a ) A comparison of the ability of DNA polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm Taq: HotMaster™ Taq DNA Polymerase, Taq: Platinum ® Taq DNA Polymerase, Pwo: Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo ® C x Hotstart DNA polymerase. ( b ) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus -specific USER compatible vector, pNB1u. H 2 O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. ( c ) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.

    Article Snippet: PCR conditions PCR with the following DNA polymerases: HotMaster™ Taq DNA Polymerase (Eppendorf), Platinum® Taq DNA Polymerase (Invitrogen), Pwo DNA Polymerase (Roche), Phusion™ DNA Polymerase (Finnzymes), and PfuTurbo® Cx Hotstart DNA polymerase (PfuCx) (Stratagene) was performed according to manufacturers' instructions on pBAD-TOPO® (Invitrogen) containing the gene, At5g43440 (Accession no. AY143873).

    Techniques: Functional Assay, Expressing, Plasmid Preparation, Injection, Fluorescence