Structured Review

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RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) <t>TaqMan</t> chemistries.
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1) Product Images from "Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology"

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology

Journal: PLoS ONE

doi: 10.1371/journal.pone.0159155

RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) TaqMan chemistries.
Figure Legend Snippet: RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) TaqMan chemistries.

Techniques Used: Quantitative RT-PCR, Polymerase Chain Reaction, In Vitro, SYBR Green Assay

2) Product Images from "The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis"

Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/erw181

Functional characterization of VvibZIPC22 in tobacco overexpressing lines. (A) Stamens and corollas of wild-type (WT) plants and transgenic lines (L), and (B) average VvibZIPC22 relative expression in stamens (S) and petal limbs (P) of three WT and six L at two developmental stages (S1=open flower without pollen, S2= mature flower). (C) Variation of cyanidin 3-rutinoside (Cyan 3-rut), quercetin 3-rutinoside (Que 3-rut), and kaempferol 3-rutinoside (Kaempf 3-rut) in stamens and of proanthocyanidins (PAs) in petal limbs of L and WT plants at the two stages. Anthocyanin and flavonol contents were determined by HPLC-DAD, while PA content was determined by DMACA. (D) Fold induction in gene expression of tobacco flavonoid structural genes between L and WT stamens and petal limbs. Transcript levels in both (B) and (D) were determined by qRT-PCR using gene-specific primers ( Supplementary Table S1 ), calibration against the average expression value in all the lines at each stage, and normalization against NteIF4a and NtUbiquitin relative expression. Average values with error bars indicating the SE are shown. Asterisks indicate significant changes ( P
Figure Legend Snippet: Functional characterization of VvibZIPC22 in tobacco overexpressing lines. (A) Stamens and corollas of wild-type (WT) plants and transgenic lines (L), and (B) average VvibZIPC22 relative expression in stamens (S) and petal limbs (P) of three WT and six L at two developmental stages (S1=open flower without pollen, S2= mature flower). (C) Variation of cyanidin 3-rutinoside (Cyan 3-rut), quercetin 3-rutinoside (Que 3-rut), and kaempferol 3-rutinoside (Kaempf 3-rut) in stamens and of proanthocyanidins (PAs) in petal limbs of L and WT plants at the two stages. Anthocyanin and flavonol contents were determined by HPLC-DAD, while PA content was determined by DMACA. (D) Fold induction in gene expression of tobacco flavonoid structural genes between L and WT stamens and petal limbs. Transcript levels in both (B) and (D) were determined by qRT-PCR using gene-specific primers ( Supplementary Table S1 ), calibration against the average expression value in all the lines at each stage, and normalization against NteIF4a and NtUbiquitin relative expression. Average values with error bars indicating the SE are shown. Asterisks indicate significant changes ( P

Techniques Used: Functional Assay, Transgenic Assay, Expressing, High Performance Liquid Chromatography, Quantitative RT-PCR

(A) Profiles of VvibZIPC22 relative expression and of six flavonol aglycons at different Pinot Noir berry developmental stages. (B) VvibZIPC22 relative expression in a panel of 15 Pinot Noir organs. Transcript levels (NRQs) were determined by qRT-PCR using gene-specific primers ( Supplementary Table S1 ), calibration against the mean expression value in all the stages, and normalization against VviGADPH and VviACTIN relative expression. Flavonol content was determined by HPLC-DAD analysis. Each value corresponds to the mean and SE of three different biological replicates. F, flowering (50% opened flowers, E-L 23); P, pepper corn (E-L 29); PV, pre-véraison (hard green berries, E-L 33); V, véraison (50% coloured berries, E-L 35); 16°, post-véraison (berries at 16° Brix, E-L 36); 18°, maturity (berries at 18° Brix, E-L 38); NRQ, normalized relative quantity; FW, fresh weight.
Figure Legend Snippet: (A) Profiles of VvibZIPC22 relative expression and of six flavonol aglycons at different Pinot Noir berry developmental stages. (B) VvibZIPC22 relative expression in a panel of 15 Pinot Noir organs. Transcript levels (NRQs) were determined by qRT-PCR using gene-specific primers ( Supplementary Table S1 ), calibration against the mean expression value in all the stages, and normalization against VviGADPH and VviACTIN relative expression. Flavonol content was determined by HPLC-DAD analysis. Each value corresponds to the mean and SE of three different biological replicates. F, flowering (50% opened flowers, E-L 23); P, pepper corn (E-L 29); PV, pre-véraison (hard green berries, E-L 33); V, véraison (50% coloured berries, E-L 35); 16°, post-véraison (berries at 16° Brix, E-L 36); 18°, maturity (berries at 18° Brix, E-L 38); NRQ, normalized relative quantity; FW, fresh weight.

Techniques Used: Expressing, Quantitative RT-PCR, High Performance Liquid Chromatography

UV light responsiveness of VvibZIPC22 and flavonol pathway genes. (A) Induction of VvibZIPC22 , VviFLS1 , and VviMYBF1 in Chardonnay leaves after UV light treatment. Each bar corresponds to the fold induction in gene expression between treated and control leaves as determined by qRT-PCR. Transcript levels were measured using gene-specific primers ( Supplementary Table S1 ), calibration against the expression value in the control sample at 0h post-treatment, and normalization against VviGADPH and VviUbiquitin relative expression. (B) Accumulation of flavonols in Chardonnay leaves after UV light treatment. Each bar corresponds to the fold induction in the total content of flavonol aglycons between treated and control leaves as detected by HPLC-DAD analysis. For both measurements, each data point corresponds to the mean and SE of three independent extractions of the same biological material. (C) Putative light regulatory elements identified in the promoter of VvibZIPC22 (PN, Ch) in this study (underlined) and in the promoters of VviMYBF1 and VviFLS1 ( Czemmel et al. , 2009 ). These cis -elements were aligned with the corresponding Arabidopsis elements taken from the PLACE database ( Higo et al. , 1999 ) using GeneDoc software (v. 1.6). Nucleotides are labelled from black to light grey according to their conservation. Numbers in front of the alignment indicate the relative distance of each element from the putative transcriptional start site (+1). CHS, chalcone synthase; FLS, flavonol synthase; PN, Pinot Noir; Ch, Chardonnay; ACS, ACGT-containing sequence similar to ACE in the Arabidopsis CHS promoter (accession no. S000355); MRS, MYB recognition sequence similar to MRE in the Arabidopsis CHS promoter (S000356); MYBCORE (S000176), IBOXCORE element (S000199); RRS, R response sequence similar to RRE in the Arabidopsis CHS promoter (S000407).
Figure Legend Snippet: UV light responsiveness of VvibZIPC22 and flavonol pathway genes. (A) Induction of VvibZIPC22 , VviFLS1 , and VviMYBF1 in Chardonnay leaves after UV light treatment. Each bar corresponds to the fold induction in gene expression between treated and control leaves as determined by qRT-PCR. Transcript levels were measured using gene-specific primers ( Supplementary Table S1 ), calibration against the expression value in the control sample at 0h post-treatment, and normalization against VviGADPH and VviUbiquitin relative expression. (B) Accumulation of flavonols in Chardonnay leaves after UV light treatment. Each bar corresponds to the fold induction in the total content of flavonol aglycons between treated and control leaves as detected by HPLC-DAD analysis. For both measurements, each data point corresponds to the mean and SE of three independent extractions of the same biological material. (C) Putative light regulatory elements identified in the promoter of VvibZIPC22 (PN, Ch) in this study (underlined) and in the promoters of VviMYBF1 and VviFLS1 ( Czemmel et al. , 2009 ). These cis -elements were aligned with the corresponding Arabidopsis elements taken from the PLACE database ( Higo et al. , 1999 ) using GeneDoc software (v. 1.6). Nucleotides are labelled from black to light grey according to their conservation. Numbers in front of the alignment indicate the relative distance of each element from the putative transcriptional start site (+1). CHS, chalcone synthase; FLS, flavonol synthase; PN, Pinot Noir; Ch, Chardonnay; ACS, ACGT-containing sequence similar to ACE in the Arabidopsis CHS promoter (accession no. S000355); MRS, MYB recognition sequence similar to MRE in the Arabidopsis CHS promoter (S000356); MYBCORE (S000176), IBOXCORE element (S000199); RRS, R response sequence similar to RRE in the Arabidopsis CHS promoter (S000407).

Techniques Used: Expressing, Quantitative RT-PCR, High Performance Liquid Chromatography, Software, Sequencing

3) Product Images from "Peruvian Maca (Lepidium peruvianum): (I) Phytochemical and Genetic Differences in Three Maca Phenotypes"

Article Title: Peruvian Maca (Lepidium peruvianum): (I) Phytochemical and Genetic Differences in Three Maca Phenotypes

Journal: International Journal of Biomedical Science : IJBS

doi:

DNA extracted from three fresh Maca hypocotyls ( L. peruvianum Chacon) Lb = Black; Lcz = Red; Lz = Yellow. Lines observed in RAPD analysis, particularly in wells 4 (Figure 5a: primer A-02 5'-TGCCGAGCTG-3') and well 57 (4th from the right - Figure 5b: primer A-19 5'-CAAACGTCGG-3') pointing with arrows at phenotypes Black (Lb) and Yellow (Lz) respectively may indicate an existence of genetic polymorphism. *Boxes above each of the three phenotypes sequence (Lb, Lcz and Lz) indicate well numbers (from – to) and corresponding primer code used for the sequence. The reaction used: 50 ng DNA, 20 pmol of each primer (OPL Kit OPERON or single RAPD primer OLIGO), and 7.5 μl of PCR Mix (Fermentas 2×). The final volume of the reactions was 15 μl.
Figure Legend Snippet: DNA extracted from three fresh Maca hypocotyls ( L. peruvianum Chacon) Lb = Black; Lcz = Red; Lz = Yellow. Lines observed in RAPD analysis, particularly in wells 4 (Figure 5a: primer A-02 5'-TGCCGAGCTG-3') and well 57 (4th from the right - Figure 5b: primer A-19 5'-CAAACGTCGG-3') pointing with arrows at phenotypes Black (Lb) and Yellow (Lz) respectively may indicate an existence of genetic polymorphism. *Boxes above each of the three phenotypes sequence (Lb, Lcz and Lz) indicate well numbers (from – to) and corresponding primer code used for the sequence. The reaction used: 50 ng DNA, 20 pmol of each primer (OPL Kit OPERON or single RAPD primer OLIGO), and 7.5 μl of PCR Mix (Fermentas 2×). The final volume of the reactions was 15 μl.

Techniques Used: Sequencing, Polymerase Chain Reaction

4) Product Images from "OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics"

Article Title: OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics

Journal: Journal of Immunology Research

doi: 10.1155/2018/2505818

Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.
Figure Legend Snippet: Identification of mice with B cell-specific deletion of ILK. Mice with B cell-specific deletion of ILK were identified through PCR (a) and flow cytometry (b) and Western blot (c). (a) DNA from the tail was amplified by PCR and then separated and visualized on 2% agarose gel to identify the mice with floxed ILK genes: L: TrackIt™ 100 bp DNA ladder; WT: ILK WT/WT ; H: ILK flox/WT ; and F: ILK flox/flox . (b) Identification of mice with CD19 WT/WT , CD19 Cre/WT , or CD19 Cre/Cre genotype was done by flow cytometric analysis on the blood that was double-stained with CD19 (FITC) and CD45R/B220 (PE/Cy5). Mice with CD19 Cre/Cre genotype did not display CD19 on the outer surface of the B cells. CD19 Cre/WT mice still expressed CD19 on the outer surface of the B cells, but at a lower density than CD19 WT/WT mice which consequently translated in a lower MFI compared to CD19 WT/WT mice. The similar percentages of gated CD45R/B220 + cells with equal MFI demonstrated that there was no loss in the B cell population in CD19 Cre/WT mice and CD19 Cre/Cre mice compared to CD19 WT/WT mice. (c) Western blot was performed on splenocytes and on splenic B cells from mice identified as being ILK wild type (WT, CD19 WT/WT /ILK flox/flox ) or ILK knockout (KO, CD19 Cre/WT /ILK flox/flox ). High expression of ILK was observed in the splenocytes of WT and KO mice, but ILK was not detected in the B lymphocytes of the latter.

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Western Blot, Amplification, Agarose Gel Electrophoresis, Staining, Knock-Out, Expressing

5) Product Images from "Bovine Papillomavirus Type 13 DNA in Equine Sarcoids"

Article Title: Bovine Papillomavirus Type 13 DNA in Equine Sarcoids

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.00371-13

PCR amplification of partial fragments of the L1 and E1 genes.
Figure Legend Snippet: PCR amplification of partial fragments of the L1 and E1 genes.

Techniques Used: Polymerase Chain Reaction, Amplification

6) Product Images from "MLGA--a rapid and cost-efficient assay for gene copy-number analysis"

Article Title: MLGA--a rapid and cost-efficient assay for gene copy-number analysis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm651

( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme. (I) Genomic DNA is digested by restriction enzyme to generate targets with defined ends. (II) Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization. (III) To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I). (IV) Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.
Figure Legend Snippet: ( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme. (I) Genomic DNA is digested by restriction enzyme to generate targets with defined ends. (II) Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization. (III) To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I). (IV) Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.

Techniques Used: Multiplex Assay, Ligation, Amplification, Plasmid Preparation, Hybridization, Polymerase Chain Reaction, Sequencing, Electrophoresis

7) Product Images from "Expression of Arabidopsis MYB transcription factor, AtMYB111, in tobacco requires light to modulate flavonol content"

Article Title: Expression of Arabidopsis MYB transcription factor, AtMYB111, in tobacco requires light to modulate flavonol content

Journal: Scientific Reports

doi: 10.1038/srep05018

Development of AtMYB111 -expressing transgenic tobacco lines and their phenotypic characterization. (a) Schematic representation of T-DNA region of plant expression construct carrying AtMYB111 cDNA in pBI121 vector used for tobacco transformation. (b) Flower color alterations in different transgenic lines. The pigmentation in petals of transgenic lines expressing AtMYB111 (1, 2 and 3) was compared with WT and empty vector (EV) transformed tobacco lines. (c) Confirmation of the presence of the transgene by PCR amplification of transgene using forward and reverse primers of AtMYB111 in different transgenic lines. (d) Expression analysis of transgene through semiquantitative RT-PCR using total RNA from leaves of WT, EV and different transgenic lines. (e) Total anthocyanin content in petals of WT, EV and different transgenic lines. Data are expressed as means ± SE of at least 3 independent experiments, each experiment consisting of 3 technical replicates. In (c) and (d), gels have been cropped for clarity and conciseness of the presentation. *** indicate values that differ significantly from WT at P
Figure Legend Snippet: Development of AtMYB111 -expressing transgenic tobacco lines and their phenotypic characterization. (a) Schematic representation of T-DNA region of plant expression construct carrying AtMYB111 cDNA in pBI121 vector used for tobacco transformation. (b) Flower color alterations in different transgenic lines. The pigmentation in petals of transgenic lines expressing AtMYB111 (1, 2 and 3) was compared with WT and empty vector (EV) transformed tobacco lines. (c) Confirmation of the presence of the transgene by PCR amplification of transgene using forward and reverse primers of AtMYB111 in different transgenic lines. (d) Expression analysis of transgene through semiquantitative RT-PCR using total RNA from leaves of WT, EV and different transgenic lines. (e) Total anthocyanin content in petals of WT, EV and different transgenic lines. Data are expressed as means ± SE of at least 3 independent experiments, each experiment consisting of 3 technical replicates. In (c) and (d), gels have been cropped for clarity and conciseness of the presentation. *** indicate values that differ significantly from WT at P

Techniques Used: Expressing, Transgenic Assay, Construct, Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction

8) Product Images from "Correlation between Point Mutations in NS2 and the Viability and Cytopathogenicity of Bovine Viral Diarrhea Virus Strain Oregon Analyzed with an Infectious cDNA Clone"

Article Title: Correlation between Point Mutations in NS2 and the Viability and Cytopathogenicity of Bovine Viral Diarrhea Virus Strain Oregon Analyzed with an Infectious cDNA Clone

Journal: Journal of Virology

doi:

Analyses following the transfection of RNA derived from pOR with the S codon at position 1555 of the ORF exchanged for an F codon. (A) Detection of cells containing replicating virus resulting from RNA transfection. Cells were transfected with equal doses of the in vitro-transcribed RNAs by the DEAE-dextran method and seeded at a sufficient density that 3 days posttransfection a closed layer of cells was obtained. At this time point, cells were fixed and virus-containing cells were visualized by MAb-mediated peroxidase staining. Below the dishes, the NS2-3 cleavage efficiency determined for the respective polyprotein after transient expression is indicated. (B) Analysis of part of the NS2 sequence from RNA obtained at different time points after transfection of RNA derived from pOR/S-F. Cells were split every 3 to 4 days after transfection, and total RNA was prepared from each passage and used as starting material for RT-PCR. RNA from passage 0 was isolated 3 days posttransfection. The amplified fragments were directly sequenced to look for the mutation introduced into pOR/S-F (arrowhead). In lanes C, the sequence of the RT-PCR product derived from the in vitro-transcribed RNA is shown as a control.
Figure Legend Snippet: Analyses following the transfection of RNA derived from pOR with the S codon at position 1555 of the ORF exchanged for an F codon. (A) Detection of cells containing replicating virus resulting from RNA transfection. Cells were transfected with equal doses of the in vitro-transcribed RNAs by the DEAE-dextran method and seeded at a sufficient density that 3 days posttransfection a closed layer of cells was obtained. At this time point, cells were fixed and virus-containing cells were visualized by MAb-mediated peroxidase staining. Below the dishes, the NS2-3 cleavage efficiency determined for the respective polyprotein after transient expression is indicated. (B) Analysis of part of the NS2 sequence from RNA obtained at different time points after transfection of RNA derived from pOR/S-F. Cells were split every 3 to 4 days after transfection, and total RNA was prepared from each passage and used as starting material for RT-PCR. RNA from passage 0 was isolated 3 days posttransfection. The amplified fragments were directly sequenced to look for the mutation introduced into pOR/S-F (arrowhead). In lanes C, the sequence of the RT-PCR product derived from the in vitro-transcribed RNA is shown as a control.

Techniques Used: Transfection, Derivative Assay, In Vitro, Staining, Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Mutagenesis

9) Product Images from "Analysis of Endocannabinoid System in Rat Testis During the First Spermatogenetic Wave"

Article Title: Analysis of Endocannabinoid System in Rat Testis During the First Spermatogenetic Wave

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2018.00269

Expression of cannabinoid receptors in fetal and post-natal rat testis. Representative fetal testis section from 19.5 days post coitum (dpc) old rats stained with hematoxylin eosin and immunostained for CB1. Arrowheads indicate immunopositive gonocytes. Scale bar: 20 µm (A) . Representative images of RT-PCR analyses showing Cb1 and Actin mRNA expression in rat testes from 1-to-14 days post partum ( dpp ). Cb1 expression was quantified by densitometry analysis, normalized against Actin signals and expressed in OD values (B) . Representative images of RT-PCR analyses showing Cb2 and Actin mRNA expression in rat testes, from 7 to 60 dpp . Cb2 expression was quantified by densitometry analysis, normalized against Actin signals and expressed in OD values (C) . Representative agarose gel image of RT + cDNA (testes from 90 dpp rats), RT− cDNA (testes from 1 to 60 dpp rats) and water (H 2 O) analyzed by PCR using actin primers (D) . All data are reported as the mean ± SEM. Different letters indicate statistically significant differences ( p
Figure Legend Snippet: Expression of cannabinoid receptors in fetal and post-natal rat testis. Representative fetal testis section from 19.5 days post coitum (dpc) old rats stained with hematoxylin eosin and immunostained for CB1. Arrowheads indicate immunopositive gonocytes. Scale bar: 20 µm (A) . Representative images of RT-PCR analyses showing Cb1 and Actin mRNA expression in rat testes from 1-to-14 days post partum ( dpp ). Cb1 expression was quantified by densitometry analysis, normalized against Actin signals and expressed in OD values (B) . Representative images of RT-PCR analyses showing Cb2 and Actin mRNA expression in rat testes, from 7 to 60 dpp . Cb2 expression was quantified by densitometry analysis, normalized against Actin signals and expressed in OD values (C) . Representative agarose gel image of RT + cDNA (testes from 90 dpp rats), RT− cDNA (testes from 1 to 60 dpp rats) and water (H 2 O) analyzed by PCR using actin primers (D) . All data are reported as the mean ± SEM. Different letters indicate statistically significant differences ( p

Techniques Used: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Polymerase Chain Reaction

RT-PCR analysis of the main AEA and 2-arachidonoylglycerol metabolizing enzymes in rat testis during the first wave of spermatogenesis. Representative images of RT-PCR analyses showing Nape-pld, Faah, Dagl, Magl , and Actin mRNA expression in rat testes from 7 to 60 days post partum ( dpp ) (A) . Representative agarose gel image of RT + cDNA (testes from 90 dpp rats), RT− cDNA (testes from 7 to 60 dpp rats) and water (H 2 O) analyzed by PCR using actin primers (B) . Gene expression was quantified by densitometry analysis and graphed relatively to germ cells appearance or activities (C) . Values for Nape-pld, Faah, Dagl , and Magl signals were normalized against Actin and are expressed as OD values (D) . All data are expressed as the mean ± SEM. Different letters indicate statistically significant differences ( p
Figure Legend Snippet: RT-PCR analysis of the main AEA and 2-arachidonoylglycerol metabolizing enzymes in rat testis during the first wave of spermatogenesis. Representative images of RT-PCR analyses showing Nape-pld, Faah, Dagl, Magl , and Actin mRNA expression in rat testes from 7 to 60 days post partum ( dpp ) (A) . Representative agarose gel image of RT + cDNA (testes from 90 dpp rats), RT− cDNA (testes from 7 to 60 dpp rats) and water (H 2 O) analyzed by PCR using actin primers (B) . Gene expression was quantified by densitometry analysis and graphed relatively to germ cells appearance or activities (C) . Values for Nape-pld, Faah, Dagl , and Magl signals were normalized against Actin and are expressed as OD values (D) . All data are expressed as the mean ± SEM. Different letters indicate statistically significant differences ( p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction

10) Product Images from "Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ▿ ▿ †"

Article Title: Sequencing and Transcriptional Analysis of the Biosynthesis Gene Cluster of Putrescine-Producing Lactococcus lactis ▿ ▿ †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.05507-11

DNA manipulation and PCR amplification.
Figure Legend Snippet: DNA manipulation and PCR amplification.

Techniques Used: Polymerase Chain Reaction, Amplification

11) Product Images from "Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer"

Article Title: Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkz811

Effect of LT-RPA on stutter peak formation using mono- to tetra-nucleotide repeat microsatellites and CEPH 0144711 cell line. ( A ) Stutter ratio obtained with PCR (20 ng of DNA) and RPA (5 ng DNA, 10.5 mM Mg(OAc) 2 and 40 min incubation at 32°C). ( B ) Microsatellite profiles of the tested mono- to tetra-nucleotide repeat microsatellites using CEPH 0144711 cell line. ( C ) Multiplexing of PCR and low temperature RPA using NR24, HT17 and BAT26 assays and CEPH 0144711 cell line. n (S) and n (L) indicate the small and large allele of a same microsatellite respectively. NRMS, nucleotide repeat microsatellites. All experiments were performed in quadruplicates.
Figure Legend Snippet: Effect of LT-RPA on stutter peak formation using mono- to tetra-nucleotide repeat microsatellites and CEPH 0144711 cell line. ( A ) Stutter ratio obtained with PCR (20 ng of DNA) and RPA (5 ng DNA, 10.5 mM Mg(OAc) 2 and 40 min incubation at 32°C). ( B ) Microsatellite profiles of the tested mono- to tetra-nucleotide repeat microsatellites using CEPH 0144711 cell line. ( C ) Multiplexing of PCR and low temperature RPA using NR24, HT17 and BAT26 assays and CEPH 0144711 cell line. n (S) and n (L) indicate the small and large allele of a same microsatellite respectively. NRMS, nucleotide repeat microsatellites. All experiments were performed in quadruplicates.

Techniques Used: Recombinase Polymerase Amplification, Polymerase Chain Reaction, Incubation, Multiplexing

Effect of the variation of RPA conditions on stutter ratio (SR) compared to PCR using HT17 microsatellite. ( A ) RPA of HT17 using 5 ng of DNA of CEPH 0144711 T17/T17 cell line and 40 min incubation and variable concentrations of Mg(OAc) 2 (5 mM to 14 mM) and of isothermal amplification temperatures (25–42°C). ( B ) PCR amplification of HT17 using 50 pg to 20 ng of DNA of CEPH 0144711 T17/T17 cell line. ( C ) RPA of HT17 using 50 pg to 10 ng of DNA of CEPH 0144711 T17/T17 cell line, 10.5 mM of Mg(OAc) 2 at 32°C during 40 min. ( D ) RPA of HT17 using 5 ng of DNA of CEPH 0144711 T17/T17 cell line, 10.5 mM of Mg(OAc) 2 at 32°C during 10 to 40 min. ( E ) HT17 microsatellite profiles obtained after PCR and RPA (32°C incubation during 40 min with 10.5 mM of Mg(OAc) 2 ) using 20 ng and 5 ng DNA respectively of CEPH cell line harboring the three WT genotypes. Blue dotted lines indicate the HT17 alleles present in the WT cell lines used. Blue and orange peaks correspond to HT17 microsatellite and 140 nucleotide size standard respectively. All experiments were performed in triplicate (B–D), quadruplicate (A) or sextuplicate (B-C, for 50 pg).
Figure Legend Snippet: Effect of the variation of RPA conditions on stutter ratio (SR) compared to PCR using HT17 microsatellite. ( A ) RPA of HT17 using 5 ng of DNA of CEPH 0144711 T17/T17 cell line and 40 min incubation and variable concentrations of Mg(OAc) 2 (5 mM to 14 mM) and of isothermal amplification temperatures (25–42°C). ( B ) PCR amplification of HT17 using 50 pg to 20 ng of DNA of CEPH 0144711 T17/T17 cell line. ( C ) RPA of HT17 using 50 pg to 10 ng of DNA of CEPH 0144711 T17/T17 cell line, 10.5 mM of Mg(OAc) 2 at 32°C during 40 min. ( D ) RPA of HT17 using 5 ng of DNA of CEPH 0144711 T17/T17 cell line, 10.5 mM of Mg(OAc) 2 at 32°C during 10 to 40 min. ( E ) HT17 microsatellite profiles obtained after PCR and RPA (32°C incubation during 40 min with 10.5 mM of Mg(OAc) 2 ) using 20 ng and 5 ng DNA respectively of CEPH cell line harboring the three WT genotypes. Blue dotted lines indicate the HT17 alleles present in the WT cell lines used. Blue and orange peaks correspond to HT17 microsatellite and 140 nucleotide size standard respectively. All experiments were performed in triplicate (B–D), quadruplicate (A) or sextuplicate (B-C, for 50 pg).

Techniques Used: Recombinase Polymerase Amplification, Polymerase Chain Reaction, Incubation, Amplification

Microsatellite profiles of BAT26, D18S61 and D12ATA63 obtained with four blood samples (named B1, B2, B3 and B4) from healthy individuals using PCR (20 ng of DNA in 20 μl) and LT-RPA (5 ng of DNA and 10.5 mM of Mg(OAc) 2 in 10 μl at 32°C). ( A ) Profiles obtained by capillary electrophoresis fragment analysis. ( B ) Profiles obtained by amplicon sequencing. Capillary electrophoresis experiments were performed in duplicates and a representative profile is shown for each duplicated experiment. For amplicon sequencing experiments, the y-axis indicates the read counts for each alleles on a scale of 0–100. NRMS, nucleotide repeat microsatellite.
Figure Legend Snippet: Microsatellite profiles of BAT26, D18S61 and D12ATA63 obtained with four blood samples (named B1, B2, B3 and B4) from healthy individuals using PCR (20 ng of DNA in 20 μl) and LT-RPA (5 ng of DNA and 10.5 mM of Mg(OAc) 2 in 10 μl at 32°C). ( A ) Profiles obtained by capillary electrophoresis fragment analysis. ( B ) Profiles obtained by amplicon sequencing. Capillary electrophoresis experiments were performed in duplicates and a representative profile is shown for each duplicated experiment. For amplicon sequencing experiments, the y-axis indicates the read counts for each alleles on a scale of 0–100. NRMS, nucleotide repeat microsatellite.

Techniques Used: Polymerase Chain Reaction, Recombinase Polymerase Amplification, Electrophoresis, Amplification, Sequencing

HT17 LT-RPA improves the limit of detection of MSI compared to PCR. PCR (20 ng of DNA) and LT-RPA (5 ng of DNA and 10.5 mM of Mg(OAc) 2 at 32°C during 40 min) experiments were conducted on dilution series of cancer cell lines (50%, 25%, 10%, 5% and 1%) harboring T10 to T15 HT17 mutant alleles mixed with T16/T16 WT CEPH cell lines. Only HT17 profiles of the fractions corresponding to the limit of detection of the mutant alleles are shown. Arrows indicate for each cancer cell line used the lowest amount of mutant alleles visible in mutant profiles compared to WT, defining the limit of detection of MSI for each type of mutation. Red and blue dotted lines indicate HT17 genotype of cancer and WT cell lines used respectively. Orange peaks correspond to 140 nucleotide size standard. All experiments were performed in triplicates.
Figure Legend Snippet: HT17 LT-RPA improves the limit of detection of MSI compared to PCR. PCR (20 ng of DNA) and LT-RPA (5 ng of DNA and 10.5 mM of Mg(OAc) 2 at 32°C during 40 min) experiments were conducted on dilution series of cancer cell lines (50%, 25%, 10%, 5% and 1%) harboring T10 to T15 HT17 mutant alleles mixed with T16/T16 WT CEPH cell lines. Only HT17 profiles of the fractions corresponding to the limit of detection of the mutant alleles are shown. Arrows indicate for each cancer cell line used the lowest amount of mutant alleles visible in mutant profiles compared to WT, defining the limit of detection of MSI for each type of mutation. Red and blue dotted lines indicate HT17 genotype of cancer and WT cell lines used respectively. Orange peaks correspond to 140 nucleotide size standard. All experiments were performed in triplicates.

Techniques Used: Recombinase Polymerase Amplification, Polymerase Chain Reaction, Mutagenesis

12) Product Images from "Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis"

Article Title: Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis

Journal: Parasites & Vectors

doi: 10.1186/1756-3305-3-113

Transcription of the bovipain-2 gene . Bovipain-2 transcripts were detected by total RNA extraction of in vitro cultured B. bovis merozoites (BboS2P strain), followed by incubation with reverse transcriptase and PCR amplification of the complete ORF (RT+). To rule out DNA contamination, an equal amount of RNA was not incubated with reverse transcriptase and then treated as above (RT-). 1 kb: DNA size standard. The size of the obtained amplicon is marked at the left.
Figure Legend Snippet: Transcription of the bovipain-2 gene . Bovipain-2 transcripts were detected by total RNA extraction of in vitro cultured B. bovis merozoites (BboS2P strain), followed by incubation with reverse transcriptase and PCR amplification of the complete ORF (RT+). To rule out DNA contamination, an equal amount of RNA was not incubated with reverse transcriptase and then treated as above (RT-). 1 kb: DNA size standard. The size of the obtained amplicon is marked at the left.

Techniques Used: RNA Extraction, In Vitro, Cell Culture, Incubation, Polymerase Chain Reaction, Amplification

13) Product Images from "Potential complications when developing gene deletion clones in Xylella fastidiosa"

Article Title: Potential complications when developing gene deletion clones in Xylella fastidiosa

Journal: BMC Research Notes

doi: 10.1186/s13104-015-1117-9

Mixture of wild-type and mutant Xylella fastidiosa strains after second isolation. The XfΔ pilJ mutant confirmed in Figure 3 (isolate 12) was streaked onto periwinkle agar plates amended with kanamycin and the genotype assessed for 32 single colonies. Each number denotes a single colony. A . The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent bands for the transformed XfΔ pilJ mutant strains or the deletion plasmid (P). B . The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔ pilJ strain and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H 2 O, was used as negative controls for each PCR reaction.
Figure Legend Snippet: Mixture of wild-type and mutant Xylella fastidiosa strains after second isolation. The XfΔ pilJ mutant confirmed in Figure 3 (isolate 12) was streaked onto periwinkle agar plates amended with kanamycin and the genotype assessed for 32 single colonies. Each number denotes a single colony. A . The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent bands for the transformed XfΔ pilJ mutant strains or the deletion plasmid (P). B . The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔ pilJ strain and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H 2 O, was used as negative controls for each PCR reaction.

Techniques Used: Mutagenesis, Isolation, Amplification, Transformation Assay, Plasmid Preparation, Positive Control, Polymerase Chain Reaction

Protection of wild-type Xylella fastidiosa from antibiotic selection pressure. Wild-type X. fastidiosa (wt), XfΔ pilJ mutant (J), or an equal mixture of both (M) were grown in PD2 liquid media before being plated onto agar plates with 0, 4, 10, 25, or 50 μg/mL kanamycin, and tested by PCR. The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for wild-type cells and a 2200 bp fragment from the XfΔ pilJ strain. The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for wild-type bacteria and no equivalent bands for the XfΔ pilJ mutant strains. For mixed samples, the AD primers amplified a Xf pilJ strain fragment and the EF primers amplified a wild-type band. The RST31/33 (RST) primers confirmed that the bacteria were X. fastidiosa . Wells of each cell type and kanamycin concentration condition are numbered as follows: (1) AD amplification, (2) EF amplification, and (3) RST amplification.
Figure Legend Snippet: Protection of wild-type Xylella fastidiosa from antibiotic selection pressure. Wild-type X. fastidiosa (wt), XfΔ pilJ mutant (J), or an equal mixture of both (M) were grown in PD2 liquid media before being plated onto agar plates with 0, 4, 10, 25, or 50 μg/mL kanamycin, and tested by PCR. The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for wild-type cells and a 2200 bp fragment from the XfΔ pilJ strain. The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for wild-type bacteria and no equivalent bands for the XfΔ pilJ mutant strains. For mixed samples, the AD primers amplified a Xf pilJ strain fragment and the EF primers amplified a wild-type band. The RST31/33 (RST) primers confirmed that the bacteria were X. fastidiosa . Wells of each cell type and kanamycin concentration condition are numbered as follows: (1) AD amplification, (2) EF amplification, and (3) RST amplification.

Techniques Used: Selection, Mutagenesis, Polymerase Chain Reaction, Amplification, Concentration Assay

Mixture of wild-type and mutant Xylella fastidiosa strains after first isolation. The XfΔ pilJ mutant confirmed in Figure 2 was stored at -80°C, streaked onto periwinkle agar plates amended with kanamycin, and the genotype assessed for 12 single colonies. Each number denotes a single colony. The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed X. fastidiosa and no band for the XfΔ pilJ mutant. Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reaction, while primer reaction without template DNA represented by H 2 O, was used as a negative control.
Figure Legend Snippet: Mixture of wild-type and mutant Xylella fastidiosa strains after first isolation. The XfΔ pilJ mutant confirmed in Figure 2 was stored at -80°C, streaked onto periwinkle agar plates amended with kanamycin, and the genotype assessed for 12 single colonies. Each number denotes a single colony. The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed X. fastidiosa and no band for the XfΔ pilJ mutant. Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reaction, while primer reaction without template DNA represented by H 2 O, was used as a negative control.

Techniques Used: Mutagenesis, Isolation, Amplification, Transformation Assay, Positive Control, Polymerase Chain Reaction, Negative Control

Mixture of wild-type and mutant Xylella fastidiosa strains after third isolation. The XfΔ pilJ mutants confirmed in Figure 4 (isolates 4 and 17) were streaked onto PW agar plates amended with kanamycin and the genotype assessed for 16 single colonies. Each number denotes a single colony. A . The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent band for the XfΔ pilJ strains or the deletion plasmid (P). B . The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔ pilJ strains and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H 2 O, was used as negative controls for both PCR reactions.
Figure Legend Snippet: Mixture of wild-type and mutant Xylella fastidiosa strains after third isolation. The XfΔ pilJ mutants confirmed in Figure 4 (isolates 4 and 17) were streaked onto PW agar plates amended with kanamycin and the genotype assessed for 16 single colonies. Each number denotes a single colony. A . The pilJ E/ pilJ F (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent band for the XfΔ pilJ strains or the deletion plasmid (P). B . The pilJ A/ pilJ D (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔ pilJ strains and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H 2 O, was used as negative controls for both PCR reactions.

Techniques Used: Mutagenesis, Isolation, Amplification, Transformation Assay, Plasmid Preparation, Positive Control, Polymerase Chain Reaction

Orientation of primers for Xylella fastidiosa pilJ gene deletion. Location of binding sites for PCR primers and length of resulting PCR products for transformed XfΔ pilJ strains and for wild-type control or non-transformed cells. RST31/33 are primers specific to X. fastidiosa and used for bacteria confirmation.
Figure Legend Snippet: Orientation of primers for Xylella fastidiosa pilJ gene deletion. Location of binding sites for PCR primers and length of resulting PCR products for transformed XfΔ pilJ strains and for wild-type control or non-transformed cells. RST31/33 are primers specific to X. fastidiosa and used for bacteria confirmation.

Techniques Used: Binding Assay, Polymerase Chain Reaction, Transformation Assay

14) Product Images from "Multiple antibiotic resistances among Shiga toxin producing Escherichia coli O157 in feces of dairy cattle farms in Eastern Cape of South Africa"

Article Title: Multiple antibiotic resistances among Shiga toxin producing Escherichia coli O157 in feces of dairy cattle farms in Eastern Cape of South Africa

Journal: BMC Microbiology

doi: 10.1186/s12866-015-0553-y

Agarose gel images of amplicons obtained from PCR with primers designed for str A resistance gene of E. coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are str A (548 bp) gene from O157 strains isolated in this study
Figure Legend Snippet: Agarose gel images of amplicons obtained from PCR with primers designed for str A resistance gene of E. coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are str A (548 bp) gene from O157 strains isolated in this study

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Isolation

Gel image of amplicons obtained from multiplex PCR with primers designed for bla - CMY and bla - CXT-M resistance genes of E coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla- CMY (507bp) and bla- CXT-M (158 bp) genes from O157 strains isolated in this study
Figure Legend Snippet: Gel image of amplicons obtained from multiplex PCR with primers designed for bla - CMY and bla - CXT-M resistance genes of E coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla- CMY (507bp) and bla- CXT-M (158 bp) genes from O157 strains isolated in this study

Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Negative Control, Isolation

Agarose gel image of amplicons obtained from PCR with primers designed for bla - ampC resistance gene of E . coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla- ampC (198 bp) gene from O157 strains isolated in this study
Figure Legend Snippet: Agarose gel image of amplicons obtained from PCR with primers designed for bla - ampC resistance gene of E . coli isolates recovered from this study. Lane 1 is molecular size markers (100 bp), lane 2 is negative control (PCR mix without DNA) while lanes 3 to 18 are bla- ampC (198 bp) gene from O157 strains isolated in this study

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Negative Control, Isolation

Gel image of amplified PCR products from study isolates with primers designed for stx1 virulent gene. Lane 1 is the MWM (100 bp), lane 2 is the negative control (PCR mix without DNA) with lane 3 as positive control ( E . coli ATCC 35150) while lanes 4 to 13 are stx1 (555 bp) gene amplified from O157 isolates
Figure Legend Snippet: Gel image of amplified PCR products from study isolates with primers designed for stx1 virulent gene. Lane 1 is the MWM (100 bp), lane 2 is the negative control (PCR mix without DNA) with lane 3 as positive control ( E . coli ATCC 35150) while lanes 4 to 13 are stx1 (555 bp) gene amplified from O157 isolates

Techniques Used: Amplification, Polymerase Chain Reaction, Negative Control, Positive Control

15) Product Images from "Identification of New Repetitive Element in Leptospira interrogans Serovar Copenhageni and Its Application to PCR-Based Differentiation of Leptospira Serogroups"

Article Title: Identification of New Repetitive Element in Leptospira interrogans Serovar Copenhageni and Its Application to PCR-Based Differentiation of Leptospira Serogroups

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.39.1.191-195.2001

iRep1 PCR-based molecular typing of reference strains from the genus Leptospira . DNA from boiled culture pellets was used in PCRs with the iRep1 primer for L. interrogans serovar autumnalis strain Akiyami A (lane 2), serovar icterohaemorrhagiae strain RGA (lane 3), serovar copenhageni strain Winjberg (lane 4), serovar wolfii strain 3705 (lane 5), and serovar hardjo strain Hardjoprajitno (lane 6); L. borgpetersenii serovar hardjo strain Lely 607 (lane 7), serovar sejroe strain M84 (lane 8), and serovar castellonis strain Castellon 3 (lane 9); L. weilii serovar celledoni strain Celledoni (lane 10); L. noguchi serovar panama strain CZ 214 K (lane 11); L. santarosai serovar shermani strain 1342 (lane 12); L. kirshneri serovar grippotyphosa strain Moskva V (lane 13); L. wolbachi serovar codice strain CDC (lane 14); L. inadai serovar lyme strain 10 (lane 15); L. fainei serovar hurstbridge strain BUT6 (lane 16); L. meyeri serovar ranarum strain ranae (strain 17); and L. biflexa serovar patoc strain Patoc1 (lane 18). A negative control sample without DNA is shown in lane 19. The positions of the 100-bp size markers are represented in lanes 1 and 20.
Figure Legend Snippet: iRep1 PCR-based molecular typing of reference strains from the genus Leptospira . DNA from boiled culture pellets was used in PCRs with the iRep1 primer for L. interrogans serovar autumnalis strain Akiyami A (lane 2), serovar icterohaemorrhagiae strain RGA (lane 3), serovar copenhageni strain Winjberg (lane 4), serovar wolfii strain 3705 (lane 5), and serovar hardjo strain Hardjoprajitno (lane 6); L. borgpetersenii serovar hardjo strain Lely 607 (lane 7), serovar sejroe strain M84 (lane 8), and serovar castellonis strain Castellon 3 (lane 9); L. weilii serovar celledoni strain Celledoni (lane 10); L. noguchi serovar panama strain CZ 214 K (lane 11); L. santarosai serovar shermani strain 1342 (lane 12); L. kirshneri serovar grippotyphosa strain Moskva V (lane 13); L. wolbachi serovar codice strain CDC (lane 14); L. inadai serovar lyme strain 10 (lane 15); L. fainei serovar hurstbridge strain BUT6 (lane 16); L. meyeri serovar ranarum strain ranae (strain 17); and L. biflexa serovar patoc strain Patoc1 (lane 18). A negative control sample without DNA is shown in lane 19. The positions of the 100-bp size markers are represented in lanes 1 and 20.

Techniques Used: Polymerase Chain Reaction, Negative Control

iRep1 PCR-based molecular typing of human and rat Leptospira strains isolated from an urban epidemic in Salvador, Brazil. DNA from boiled Leptospira culture pellets was amplified with the iRep1 primer. The following samples were identified as L. interrogans serovar copenhageni except for L1-133, shown in lane 3 ( L. interrogans serovar canicola). The following clinical isolates of L. interrogans serovar copenhageni were evaluated: L1-130 (lane 5), L1-212 (lane 6), L8-38 (lane 7), L8-118 (lane 8), and L8-163 (lane 9). Rat isolates included R1-15 (lane 10), R1-98 (lane 11), and R1-147 (lane 12), and R1-152 (lane 13). Lanes 2 and 4 represent reference strains L. interrogans serovar canicola strain Hond Utrecht IV and L. interrogans serovar copenhageni strain Winjberg, respectively. The positions of the 100-bp size markers are represented in lanes 1 and 14.
Figure Legend Snippet: iRep1 PCR-based molecular typing of human and rat Leptospira strains isolated from an urban epidemic in Salvador, Brazil. DNA from boiled Leptospira culture pellets was amplified with the iRep1 primer. The following samples were identified as L. interrogans serovar copenhageni except for L1-133, shown in lane 3 ( L. interrogans serovar canicola). The following clinical isolates of L. interrogans serovar copenhageni were evaluated: L1-130 (lane 5), L1-212 (lane 6), L8-38 (lane 7), L8-118 (lane 8), and L8-163 (lane 9). Rat isolates included R1-15 (lane 10), R1-98 (lane 11), and R1-147 (lane 12), and R1-152 (lane 13). Lanes 2 and 4 represent reference strains L. interrogans serovar canicola strain Hond Utrecht IV and L. interrogans serovar copenhageni strain Winjberg, respectively. The positions of the 100-bp size markers are represented in lanes 1 and 14.

Techniques Used: Polymerase Chain Reaction, Isolation, Amplification

16) Product Images from "Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens"

Article Title: Direct Amplification and Genotyping of Dientamoeba fragilis from Human Stool Specimens

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.42.2.631-635.2004

Restriction endonuclease digestion of DF1/DF4 PCR products from nine patient samples (lanes 1 to 9) and of PCR products of D. fragilis genotype 1 (lane 11) and genotype 2 (lane 10) with Rsa I (top panel) and Dde I (bottom panel). The size marker (lane M) is a 100-bp ladder.
Figure Legend Snippet: Restriction endonuclease digestion of DF1/DF4 PCR products from nine patient samples (lanes 1 to 9) and of PCR products of D. fragilis genotype 1 (lane 11) and genotype 2 (lane 10) with Rsa I (top panel) and Dde I (bottom panel). The size marker (lane M) is a 100-bp ladder.

Techniques Used: Polymerase Chain Reaction, Marker

17) Product Images from "Analysis of 2-LTR circle junctions of Viral DNA in infected cells"

Article Title: Analysis of 2-LTR circle junctions of Viral DNA in infected cells

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-59745-170-3_6

Quantitation of 2-LTR circle Junction DNA by Real-time PCR. Jurkat cells were infected with WT R3B virus and infected cells DNA was isolated at 2h, 24h and 48h post-infection. Virus infected cell DNA was analyzed for amount of 2-LTR circle junction DNA
Figure Legend Snippet: Quantitation of 2-LTR circle Junction DNA by Real-time PCR. Jurkat cells were infected with WT R3B virus and infected cells DNA was isolated at 2h, 24h and 48h post-infection. Virus infected cell DNA was analyzed for amount of 2-LTR circle junction DNA

Techniques Used: Quantitation Assay, Real-time Polymerase Chain Reaction, Infection, Isolation

18) Product Images from "RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and ?-Actin Genes"

Article Title: RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and ?-Actin Genes

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.6.1961-1967.2003

Distribution of RNA Pol II on γ-actin gene. Schematic diagram of the γ-actin gene is shown at the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene. Amplification of an intergenic region served as an internal background control. (A) Specific antibodies used for chromatin IP are shown on top of each gel. Chromatin IP without any antibody or with a nonspecific antibody, Oct2, served as negative controls. PCR analyses of chromatin IP for different regions of the gene are shown. (B) Real-time PCR results are shown for quantification of DNA associated with specific proteins.
Figure Legend Snippet: Distribution of RNA Pol II on γ-actin gene. Schematic diagram of the γ-actin gene is shown at the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene. Amplification of an intergenic region served as an internal background control. (A) Specific antibodies used for chromatin IP are shown on top of each gel. Chromatin IP without any antibody or with a nonspecific antibody, Oct2, served as negative controls. PCR analyses of chromatin IP for different regions of the gene are shown. (B) Real-time PCR results are shown for quantification of DNA associated with specific proteins.

Techniques Used: Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Capping enzyme is concentrated at the promoter of the DHFR gene. HeLa cells containing high numbers of copies of the DHFR gene were cross-linked with formaldehyde. Following sonication, a specific antibody recognizing capping enzyme was added to the chromatin solution for immunoprecipitation of a DNA-capping enzyme complex. PCR was performed to analyze amounts of DNA along the gene that were associated with the capping enzyme. (A) Sonication efficiency of chromatin DNA. The sonicated chromatin solution was analyzed on an ethidium bromide-stained agarose gel after proteinase K treatment and reversal of the cross-links. Two samples are shown, with a 1-kb DNA ladder on the left of the gel. (B) Schematic diagram of the DHFR gene. Black boxes represent exons and thin lines represent introns. DNA fragments for PCR amplification (about 150 nucleotides [nt] long) are depicted as bars under the gene. A pair of primers amplifying an intergenic region downstream of the DHFR gene served as an internal background control. Sequences of primer pairs are presented in Materials and Methods. Numbers below each gel correspond to individual primer sets along the DHFR gene. (C) Chromatin IP with a capping enzyme antibody. PCRs with input DNA (before immunoprecipitation) were performed as controls for amplification efficiency of individual PCR primer sets. Chromatin IP without any antibody and with a nonspecific antibody (Oct2) served as negative controls. Agarose gel analyses of PCR products are shown, with lane numbers corresponding to regions of the gene.
Figure Legend Snippet: Capping enzyme is concentrated at the promoter of the DHFR gene. HeLa cells containing high numbers of copies of the DHFR gene were cross-linked with formaldehyde. Following sonication, a specific antibody recognizing capping enzyme was added to the chromatin solution for immunoprecipitation of a DNA-capping enzyme complex. PCR was performed to analyze amounts of DNA along the gene that were associated with the capping enzyme. (A) Sonication efficiency of chromatin DNA. The sonicated chromatin solution was analyzed on an ethidium bromide-stained agarose gel after proteinase K treatment and reversal of the cross-links. Two samples are shown, with a 1-kb DNA ladder on the left of the gel. (B) Schematic diagram of the DHFR gene. Black boxes represent exons and thin lines represent introns. DNA fragments for PCR amplification (about 150 nucleotides [nt] long) are depicted as bars under the gene. A pair of primers amplifying an intergenic region downstream of the DHFR gene served as an internal background control. Sequences of primer pairs are presented in Materials and Methods. Numbers below each gel correspond to individual primer sets along the DHFR gene. (C) Chromatin IP with a capping enzyme antibody. PCRs with input DNA (before immunoprecipitation) were performed as controls for amplification efficiency of individual PCR primer sets. Chromatin IP without any antibody and with a nonspecific antibody (Oct2) served as negative controls. Agarose gel analyses of PCR products are shown, with lane numbers corresponding to regions of the gene.

Techniques Used: Sonication, Immunoprecipitation, Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Amplification, Chromatin Immunoprecipitation

RNA Pol II density determined by nuclear run-on analysis of the DHFR gene. Nuclear run-on analysis was performed as described in Material and Methods. Newly synthesized, 32 P-radiolabeled transcripts were hybridized to PCR products containing specific regions of the DHFR gene. The locations of PCR fragments are diagramed at the top and indicated on the left of the filters. PCR fragments 1 and 3 are genomic DNA, whereas 2 and 4 are cDNA encompassing parts of two adjacent exons. Radiolabeled DHFR RNA was transcribed with SP6 polymerase from each of the four fragments and then pooled and hybridized to the filter as a positive control for hybridization efficiencies among different DHFR probes. Analysis of labeled RNA from the nuclear run-on assay is shown on the right.
Figure Legend Snippet: RNA Pol II density determined by nuclear run-on analysis of the DHFR gene. Nuclear run-on analysis was performed as described in Material and Methods. Newly synthesized, 32 P-radiolabeled transcripts were hybridized to PCR products containing specific regions of the DHFR gene. The locations of PCR fragments are diagramed at the top and indicated on the left of the filters. PCR fragments 1 and 3 are genomic DNA, whereas 2 and 4 are cDNA encompassing parts of two adjacent exons. Radiolabeled DHFR RNA was transcribed with SP6 polymerase from each of the four fragments and then pooled and hybridized to the filter as a positive control for hybridization efficiencies among different DHFR probes. Analysis of labeled RNA from the nuclear run-on assay is shown on the right.

Techniques Used: Synthesized, Polymerase Chain Reaction, Positive Control, Hybridization, Labeling, Nuclear Run-on Assay

19) Product Images from "Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization"

Article Title: Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035438

Differences in the hybridization kinetics of a target specific reporter probe and a non-target specific reporter probe. A. The diagram shows the kinetics of reporter probe hr-H1-683 in presence of different amounts of hr-H1-683 specific target amplicon. As indicated in the diagram, hybridization speed strongly depends upon the amount of target amplicon present in the reaction. With no target amplicon present in the reaction, reporter probes are available to bind to their complementary capture probes on the array. With increasing amounts of target amplicon, the speed of reporter probe binding to the respective capture probe decreases. Finally, in the presence of approximately the target amplicon concentration expected for a completed PCR- Reaction (150 nM), the reaction speed is at its lowest point. B. The data illustrate that the effect is sequence specific. The speed of hybridization of non-target specific reporter probes to their respective capture probes does not depend on the concentration of target amplicons in solution.
Figure Legend Snippet: Differences in the hybridization kinetics of a target specific reporter probe and a non-target specific reporter probe. A. The diagram shows the kinetics of reporter probe hr-H1-683 in presence of different amounts of hr-H1-683 specific target amplicon. As indicated in the diagram, hybridization speed strongly depends upon the amount of target amplicon present in the reaction. With no target amplicon present in the reaction, reporter probes are available to bind to their complementary capture probes on the array. With increasing amounts of target amplicon, the speed of reporter probe binding to the respective capture probe decreases. Finally, in the presence of approximately the target amplicon concentration expected for a completed PCR- Reaction (150 nM), the reaction speed is at its lowest point. B. The data illustrate that the effect is sequence specific. The speed of hybridization of non-target specific reporter probes to their respective capture probes does not depend on the concentration of target amplicons in solution.

Techniques Used: Hybridization, Amplification, Binding Assay, Concentration Assay, Polymerase Chain Reaction, Sequencing

20) Product Images from "Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing"

Article Title: Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing

Journal: BMC Genomics

doi: 10.1186/s12864-016-2437-4

The scheme illustrating the developed workflow of lampbrush chromosomes (LBC) microdissection. The main steps are as follows: 1. Mechanical microdissection of lampbrush chromosome regions; 2. Primary amplification of isolated DNA material by DOP-PCR. Alternatively, for investigation of RNA-component of chromosomal loci, before the amplification the dissected material was pretreated with DNAse I followed by reverse transcription; 3. FISH-probe generation via reamplification and labeling; 4. Verification of specificity and brightness of the probes by FISH on metaphase chromosomes; 5. High-resolution FISH-mapping of dissected regions on lampbrush chromosomes; 6. Preparation of DNA-libraries for high-throughput sequencing. Processing, visualization and analysis of the sequencing data using web-based bioinformatic platform Galaxy [ 39 ] and genome browser IGV [ 43 , 44 ]. The sequence of actions is depicted by arrows
Figure Legend Snippet: The scheme illustrating the developed workflow of lampbrush chromosomes (LBC) microdissection. The main steps are as follows: 1. Mechanical microdissection of lampbrush chromosome regions; 2. Primary amplification of isolated DNA material by DOP-PCR. Alternatively, for investigation of RNA-component of chromosomal loci, before the amplification the dissected material was pretreated with DNAse I followed by reverse transcription; 3. FISH-probe generation via reamplification and labeling; 4. Verification of specificity and brightness of the probes by FISH on metaphase chromosomes; 5. High-resolution FISH-mapping of dissected regions on lampbrush chromosomes; 6. Preparation of DNA-libraries for high-throughput sequencing. Processing, visualization and analysis of the sequencing data using web-based bioinformatic platform Galaxy [ 39 ] and genome browser IGV [ 43 , 44 ]. The sequence of actions is depicted by arrows

Techniques Used: Laser Capture Microdissection, Amplification, Isolation, Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Labeling, Next-Generation Sequencing, Sequencing

21) Product Images from "Association of Protein Expression and Methylation of DAPK1 with Clinicopathological Features in Invasive Ductal Carcinoma Patients from Kashmir"

Article Title: Association of Protein Expression and Methylation of DAPK1 with Clinicopathological Features in Invasive Ductal Carcinoma Patients from Kashmir

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

doi: 10.31557/APJCP.2019.20.3.839

MSP Analysis of DAPK1 Gene in Breast Cancer Tissues. Existence of PCR products in lane M indicates the presence of methylation. MSP product in lane U indicates the presence of nonmethylation alleles. In vitro SssI methyltransferase–treated and –untreated DNA from normal lymphocytes were used as the positive controls (PC) for methylation and nonmethylation (NC), respectively. L, 100-bp DNA marker ladder; The methylated allele was detected in Cases 1-3, 5, 7 and 8. Unmethylated allele was detected in Cases 4, 6 and 9.
Figure Legend Snippet: MSP Analysis of DAPK1 Gene in Breast Cancer Tissues. Existence of PCR products in lane M indicates the presence of methylation. MSP product in lane U indicates the presence of nonmethylation alleles. In vitro SssI methyltransferase–treated and –untreated DNA from normal lymphocytes were used as the positive controls (PC) for methylation and nonmethylation (NC), respectively. L, 100-bp DNA marker ladder; The methylated allele was detected in Cases 1-3, 5, 7 and 8. Unmethylated allele was detected in Cases 4, 6 and 9.

Techniques Used: Polymerase Chain Reaction, Methylation, In Vitro, Marker

22) Product Images from "Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma"

Article Title: Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2014.00655

Graphical overview of the methods used to detect latent viral forms. (A) Method used to detect episomal genomic DNA. On the top the genome organization of the Sl.L-PV-1, on the bottom a representation of the predicted circular form. Red arrows represent primer binding sites. The use of reverse primer annealing at the beginning of NS1 and forward primers annealing at the end of VP1 allows the amplification the connecting genomic sequence. (B) Restriction enzyme based methods to detect integrated virus. The purple lines represent the host genomic sequence, the orange flashes indicate restriction enzyme sites and the orange boxed indicate artificially ligated adaptors. (B1) It is shown how, after overnight ligation of the digested DNA, the obtained fragment would self-ligate in a circular form and how a specific amplification would allow to amplify the integration site. (B2) It is shown how, after the ligation of specific adaptors to the obtained digested fragments, a semi specific PCR would allow the amplification of the integration site. (C) Method to detect the integrated virus based on repeated Alu sequences in the host genome. Purple boxes represent Alu sequences and red arrows represent primer binding sites. A comparison of the PCR results obtained amplifying the template with either a mixture of Alu binding primers and specific primers or Alu binding primers alone (negative control) would allow to amplify the integration site and determine which amplified fragment on gel corresponds to it.
Figure Legend Snippet: Graphical overview of the methods used to detect latent viral forms. (A) Method used to detect episomal genomic DNA. On the top the genome organization of the Sl.L-PV-1, on the bottom a representation of the predicted circular form. Red arrows represent primer binding sites. The use of reverse primer annealing at the beginning of NS1 and forward primers annealing at the end of VP1 allows the amplification the connecting genomic sequence. (B) Restriction enzyme based methods to detect integrated virus. The purple lines represent the host genomic sequence, the orange flashes indicate restriction enzyme sites and the orange boxed indicate artificially ligated adaptors. (B1) It is shown how, after overnight ligation of the digested DNA, the obtained fragment would self-ligate in a circular form and how a specific amplification would allow to amplify the integration site. (B2) It is shown how, after the ligation of specific adaptors to the obtained digested fragments, a semi specific PCR would allow the amplification of the integration site. (C) Method to detect the integrated virus based on repeated Alu sequences in the host genome. Purple boxes represent Alu sequences and red arrows represent primer binding sites. A comparison of the PCR results obtained amplifying the template with either a mixture of Alu binding primers and specific primers or Alu binding primers alone (negative control) would allow to amplify the integration site and determine which amplified fragment on gel corresponds to it.

Techniques Used: Binding Assay, Amplification, Sequencing, Ligation, Polymerase Chain Reaction, Negative Control

23) Product Images from "Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons"

Article Title: Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons

Journal: Neuron

doi: 10.1016/j.neuron.2015.07.030

Lineage Analysis of Nkx2.1+ Progenitors Using Barcode Retroviral Library (A) Schematic of the QmGFP-OL murine retroviral library. Each retrovirus expresses membrane GFP and contains a 24 bp barcode sequence. (B) EnvA pseudotyped retrovirus libraries were intraventricularly delivered into Nkx2.1-Cre;LSL-Tva embryos at E12.5; brains were harvested and analyzed at P28. (C) Stained neuron outlined for laser capture microdissection and catapulting. (D) Example gel of nested PCR products of dissected cells and GFP negative tissue sections used as controls. (E) Example barcode sequence alignment showing two four-cell clones with matching barcodes. (F) Three dimensional map of two four-cell clones shown in red and blue. Green spheres show cells which did not return a barcode sequence.
Figure Legend Snippet: Lineage Analysis of Nkx2.1+ Progenitors Using Barcode Retroviral Library (A) Schematic of the QmGFP-OL murine retroviral library. Each retrovirus expresses membrane GFP and contains a 24 bp barcode sequence. (B) EnvA pseudotyped retrovirus libraries were intraventricularly delivered into Nkx2.1-Cre;LSL-Tva embryos at E12.5; brains were harvested and analyzed at P28. (C) Stained neuron outlined for laser capture microdissection and catapulting. (D) Example gel of nested PCR products of dissected cells and GFP negative tissue sections used as controls. (E) Example barcode sequence alignment showing two four-cell clones with matching barcodes. (F) Three dimensional map of two four-cell clones shown in red and blue. Green spheres show cells which did not return a barcode sequence.

Techniques Used: Sequencing, Staining, Laser Capture Microdissection, Nested PCR, Clone Assay

24) Product Images from "hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1"

Article Title: hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1

Journal: Nature

doi: 10.1038/nature14280

Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1 a , Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a , presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex 12 , 14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold 24 (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log 2 ) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61 .
Figure Legend Snippet: Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1 a , Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a , presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex 12 , 14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold 24 (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log 2 ) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61 .

Techniques Used: Autoradiography, Isolation, Ligation, Labeling, Nucleic Acid Electrophoresis, Sequencing, Construct, Immunoprecipitation, Luciferase, Reverse Transcription Polymerase Chain Reaction

25) Product Images from "Cyclin-dependent Kinase 2-associating Protein 1 Commits Murine Embryonic Stem Cell Differentiation through Retinoblastoma Protein Regulation *"

Article Title: Cyclin-dependent Kinase 2-associating Protein 1 Commits Murine Embryonic Stem Cell Differentiation through Retinoblastoma Protein Regulation *

Journal:

doi: 10.1074/jbc.M109.026088

Gene expression profiles of Cdk2ap1 −/− mESC embryoid bodies. The molecular effects of the deletion of Cdk2ap1 on the regulation of stem cell genes or differentiation-related genes were examined by quantitative reverse transcription-PCR
Figure Legend Snippet: Gene expression profiles of Cdk2ap1 −/− mESC embryoid bodies. The molecular effects of the deletion of Cdk2ap1 on the regulation of stem cell genes or differentiation-related genes were examined by quantitative reverse transcription-PCR

Techniques Used: Expressing, Polymerase Chain Reaction

26) Product Images from "A new and simple approach for genotyping Alzheimer's disease presenilin-1 mutant knockin mice"

Article Title: A new and simple approach for genotyping Alzheimer's disease presenilin-1 mutant knockin mice

Journal: Journal of neuroscience methods

doi: 10.1016/j.jneumeth.2009.05.009

Repeatability approaches Two different PCR amplifications were realized from one DNA extraction for the samples 1 to 33. Each PCR product was denaturated and loaded on gels four separate times. (A) Samples 1 to 19 were loaded on two gels migrating in a same electrophoresis (1) as for samples 20 to 33 (electrophoresis 2). (B) Samples 1 to 33 were loaded on two gels (1 to 19 and 20 to 33). Two electrophoresis were realized (3 and 4).
Figure Legend Snippet: Repeatability approaches Two different PCR amplifications were realized from one DNA extraction for the samples 1 to 33. Each PCR product was denaturated and loaded on gels four separate times. (A) Samples 1 to 19 were loaded on two gels migrating in a same electrophoresis (1) as for samples 20 to 33 (electrophoresis 2). (B) Samples 1 to 33 were loaded on two gels (1 to 19 and 20 to 33). Two electrophoresis were realized (3 and 4).

Techniques Used: Polymerase Chain Reaction, DNA Extraction, Electrophoresis

Agarose electrophoresis of PS1 DNA amplified by PCR using the designed primers The specific set of primers designed for PS1 gene generated a 231 bp PCR product in three different DNA samples tested whose genotypes were determined using the classical method (+/+, +/KI, KI/KI). PCR was performed in triplicate for each sample. No PCR product was detected in the negative control lane (T−).
Figure Legend Snippet: Agarose electrophoresis of PS1 DNA amplified by PCR using the designed primers The specific set of primers designed for PS1 gene generated a 231 bp PCR product in three different DNA samples tested whose genotypes were determined using the classical method (+/+, +/KI, KI/KI). PCR was performed in triplicate for each sample. No PCR product was detected in the negative control lane (T−).

Techniques Used: Electrophoresis, Amplification, Polymerase Chain Reaction, Generated, Negative Control

27) Product Images from "High expression of DDR1 is associated with the poor prognosis in Chinese patients with pancreatic ductal adenocarcinoma"

Article Title: High expression of DDR1 is associated with the poor prognosis in Chinese patients with pancreatic ductal adenocarcinoma

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-015-0202-1

DDR1 expression is increased in PDAC at mRNA level. a increased DDR1 mRNA expression in 30 matched tumor (T) and non-tumor tissue (N) was detected by real-time quantitative PCR. b DDR1 expression in Buchholz pancreas grouped by normal pancreatic duct (1) and PDAC (2)
Figure Legend Snippet: DDR1 expression is increased in PDAC at mRNA level. a increased DDR1 mRNA expression in 30 matched tumor (T) and non-tumor tissue (N) was detected by real-time quantitative PCR. b DDR1 expression in Buchholz pancreas grouped by normal pancreatic duct (1) and PDAC (2)

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

28) Product Images from "rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia, Treponema, and Leptospira"

Article Title: rpoB Gene Analysis as a Novel Strategy for Identification of Spirochetes from the Genera Borrelia, Treponema, and Leptospira

Journal: Journal of Clinical Microbiology

doi:

Specific PCR amplification of the spirochetal rpoB gene. The PCR assay was performed by using Taq polymerase with an annealing temperature of 52°C. Lanes: 1, molecular mass markers (DNA molecular weight marker VI; Boehringer); 2, Borrelia burgdorferi ; 3, Borrelia recurrentis ; 4, Treponema pallidum ; 5, Leptospira biflexa serovar patoc; 6, Leptospira interrogans , serovar australis; 7, Leptospira interrogans , serovar icterohaemmorragiae; 8, Escherichia coli ; 9, Staphylococcus aureus ; 10, Streptococcus salivarius ; 11, Pseudomonas aeruginosa ; 12, negative control without DNA. (A) rpoB gene primers LTB 1730F and LTB 2900R. (B) rpoB gene primers LTB 1730F and LTB 3700R. (C) 16S rRNA gene primers FD1 and RP2.
Figure Legend Snippet: Specific PCR amplification of the spirochetal rpoB gene. The PCR assay was performed by using Taq polymerase with an annealing temperature of 52°C. Lanes: 1, molecular mass markers (DNA molecular weight marker VI; Boehringer); 2, Borrelia burgdorferi ; 3, Borrelia recurrentis ; 4, Treponema pallidum ; 5, Leptospira biflexa serovar patoc; 6, Leptospira interrogans , serovar australis; 7, Leptospira interrogans , serovar icterohaemmorragiae; 8, Escherichia coli ; 9, Staphylococcus aureus ; 10, Streptococcus salivarius ; 11, Pseudomonas aeruginosa ; 12, negative control without DNA. (A) rpoB gene primers LTB 1730F and LTB 2900R. (B) rpoB gene primers LTB 1730F and LTB 3700R. (C) 16S rRNA gene primers FD1 and RP2.

Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Negative Control

Related Articles

Clone Assay:

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Centrifugation:

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Article Snippet: A 1 ml aliquot was harvested by centrifugation at 13,700xg and the cell pellet was suspended in 0.5 ml PBS buffer. .. 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM.

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
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Amplification:

Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
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Article Title: CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis
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Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments
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Article Title: Dual transcripts of BCR-ABL different polymorphisms in chronic myeloid leukaemia patients
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Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
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Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
Article Snippet: Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. .. One-tenth of the amplified reaction mixture was mixed with a gel-loading buffer and electrophoresed on a 0.8% (wt/vol) agarose gel; the reaction products were visualized by ethidium bromide staining.

Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
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Positive Control:

Article Title: CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis
Article Snippet: The PCR mixture contained 50 ng of DNA, 0.08 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 1 mM MgCl2 , 5 pmol of each oligonucleotide and 1 U of Platinium Taq DNA Polymerase (Invitrogen, Carlsbad, USA), in a final volume of 15 μL. .. DNA from strain ATCC 43504 (cagA + , cag PAI+ ) was used as a second negative control, and strain UEGE-644 (cagA − , cag PAI− ) as positive control.

Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments
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Polymerase Chain Reaction:

Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
Article Snippet: .. The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold). ..

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
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Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene
Article Snippet: .. For set III, the PCR mixture contained 1X PCR buffer, dNTPs (each at 1.28 mmol/L; Invitrogen), primers (4 ng/μl; Sigma Genosys), HotStarTaq DNA polymerase (1.5U; Qiagen), and bisulfite-modified DNA (∼100 ng) or unmodified DNA (50 to 100 ng) in a final volume of 25 μl. ..

Article Title: CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis
Article Snippet: .. The PCR mixture contained 50 ng of DNA, 0.08 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 1 mM MgCl2 , 5 pmol of each oligonucleotide and 1 U of Platinium Taq DNA Polymerase (Invitrogen, Carlsbad, USA), in a final volume of 15 μL. .. The amplification conditions were: 1 cycle at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 61 °C for 30 s and 72 °C for 45 s; and one final extension cycle at 72 °C for 7 min. PCR products were subjected to 2% agarose gel electrophoresis, followed by ethidium bromide staining and UV light observation.

Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
Article Snippet: .. 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM. .. Primers, listed in , were chosen using Primer Express 3.0 software (Applied Biosystems) and were designed to amplify a region of about 150–200 bp within each transcript.

Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments
Article Snippet: .. Approximately 40 ng of template DNA was used in a 100-μl PCR mixture; the concentration of template DNA was measured by using the PicoGreen double-stranded DNA quantification kit (Molecular Probes) and a spectrofluorophotometer (RF-5300PC; Shimadzu). .. An approximately 1,900-bp fragment of dsrAB was amplified by using the primers DSR1Fdeg and DSR4Rdeg ( ).

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
Article Snippet: .. The PCR mixture contained 4 μL DNA, 34 μL H2 O, 10 μL 5X Phusion HF Buffer, 1 μL 10 mM dNTPs, 0.5 μL (1 mg/mL) VNTR oligonucleotide, and 0.5 μL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA). .. The PCR reaction was prepared in a Bio-Rad hard-shell 96-well PCR plate (Bio-Rad Laboratories, Hercules, CA) on ice and was carried out in a PTC-225 Tetrad Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) using the following conditions: hold at 96 °C for 5 min, cycle at 96 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min, repeated for 30 times, followed by 72 °C for 7 min and a 4 °C hold.

Article Title: Dual transcripts of BCR-ABL different polymorphisms in chronic myeloid leukaemia patients
Article Snippet: .. For amplification of the breakpoint region (BCR -ABL ), BCR gene, ABL gene and ABL kinase domain from the cDNA, the PCR mixture (15 μl) contained 100 mM Tris HCl (p H 8.8), 50 mM KCl, 1.5 mM MgCl2 , 0.1 per cent triton X-100, 10 mM dNTPs (deoxynucleotides), 10 pmol of the forward and reverse primers, 2 units of DyNAzyme II DNA polymerase (Thermo Scientific, USA). .. The products were resolved in agarose gel and the desired amplicons were purified using GenElute gel extraction kit (Sigma-Aldrich, USA) and sequenced on Applied Biosystems Genetic analyzer 3500 (Life Technologies, USA).

Article Title: Peruvian Maca (Lepidium peruvianum): (I) Phytochemical and Genetic Differences in Three Maca Phenotypes
Article Snippet: .. RAPD/ISSR-PCR: The reaction consisted of 50 ng DNA, 20 pmol of each primer (OPL Kit, OPERON or single RAPD primer, OLIGO), and 7.5 μl of PCR Mix (Fermentas, 2×). ..

Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
Article Snippet: .. The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The PCR reactions were performed using the iCycler thermocycler (Bio-Rad, Hercules, CA, USA) with the following cycling parameters: 1 cycle at 95°C for 3 minutes, followed by 45 cycles with a denaturation step at 95°C for 30 seconds, an annealing step at 55°C for 30 seconds, and an extension step at 68°C for 2 minutes.

Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
Article Snippet: .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA). ..

Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
Article Snippet: .. Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. ..

Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
Article Snippet: .. The PCR mixture for Ampli Taq Gold was composed of the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× TaqMan Universal PCR Master Mix (including deoxynucleoside triphosphates [dNTPs; 200 μM dATP, 200 μM dCTP, 200 μM dGTP, and 400 μM dUTP]; Applied Biosystems), 0.01 U of uracil- N -glycosylase per μl, 2.5 mM MgCl2 , and 0.025 U of Ampli Taq Gold per μl. .. The PCR mixture for r Tth DNA polymerase had the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× chelating buffer (N808-0098; Applied Biosytems), 0.05 U of r Tth DNA polymerase per μl, dNTPs (dATP, dCTP, dGTP, and dTTP, each at a concentration of 200 μM; Applied Biosystems), 2.5 mM MgCl2 , and 8% (vol/vol) glycerol (Merck).

Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
Article Snippet: .. One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD). ..

Quantitative RT-PCR:

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
Article Snippet: Paragraph title: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry ... The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

SYBR Green Assay:

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
Article Snippet: Paragraph title: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry ... The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
Article Snippet: .. 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM. .. Primers, listed in , were chosen using Primer Express 3.0 software (Applied Biosystems) and were designed to amplify a region of about 150–200 bp within each transcript.

Incubation:

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
Article Snippet: Each strain was cultured in 100 mL LNG liquid medium, incubated at 30 °C for 2 days, and the cells were pelleted by centrifugation at 4,000×g for 10 min. .. The PCR mixture contained 4 μL DNA, 34 μL H2 O, 10 μL 5X Phusion HF Buffer, 1 μL 10 mM dNTPs, 0.5 μL (1 mg/mL) VNTR oligonucleotide, and 0.5 μL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA).

In Silico:

Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
Article Snippet: In silico restriction digestions were carried out, and predicted restriction patterns were determined for each species (genotype). .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA).

Modification:

Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene
Article Snippet: Genomic DNA (18 μl) obtained from tissues was treated with 5 mol/L sodium metabisulfite (Fisher Scientific Ltd., Ottawa, Canada) for 16 hours (protocol kindly provided by Peter Laird, University of Southern California), while cell line DNA (1 μg) was modified using the CpGenome DNA Modification Kit (Intergen Company, Purchase, NY). .. For set III, the PCR mixture contained 1X PCR buffer, dNTPs (each at 1.28 mmol/L; Invitrogen), primers (4 ng/μl; Sigma Genosys), HotStarTaq DNA polymerase (1.5U; Qiagen), and bisulfite-modified DNA (∼100 ng) or unmodified DNA (50 to 100 ng) in a final volume of 25 μl.

Transformation Assay:

Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
Article Snippet: Paragraph title: Cloning of VvibZIPC22 and tobacco transformation ... The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold).

Derivative Assay:

Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
Article Snippet: The VvibZIPC22 sequence considered in this study was derived from the 12Xv1 assembly of the Pinot Noir genome (VIT_07s0005g01450, from http://genomes.cribi.unipd.it/ DATA/V1, last accessed 11 December 2015) and confirmed by EST sequences (EE065899.1, EC946004.1, and EC945791.1). .. The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold).

Concentration Assay:

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
Article Snippet: The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water. .. Serial 10-fold dilutions of the cDNA transcripts from IVT RNA were prepared in nuclease free water starting with highest concentration of 1.7 x 108 copies/μl.

Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
Article Snippet: .. 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM. .. Primers, listed in , were chosen using Primer Express 3.0 software (Applied Biosystems) and were designed to amplify a region of about 150–200 bp within each transcript.

Article Title: Non-Sulfate-Reducing, Syntrophic Bacteria Affiliated with Desulfotomaculum Cluster I Are Widely Distributed in Methanogenic Environments
Article Snippet: .. Approximately 40 ng of template DNA was used in a 100-μl PCR mixture; the concentration of template DNA was measured by using the PicoGreen double-stranded DNA quantification kit (Molecular Probes) and a spectrofluorophotometer (RF-5300PC; Shimadzu). .. An approximately 1,900-bp fragment of dsrAB was amplified by using the primers DSR1Fdeg and DSR4Rdeg ( ).

Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
Article Snippet: .. Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. ..

Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
Article Snippet: The PCR mixture for Ampli Taq Gold was composed of the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× TaqMan Universal PCR Master Mix (including deoxynucleoside triphosphates [dNTPs; 200 μM dATP, 200 μM dCTP, 200 μM dGTP, and 400 μM dUTP]; Applied Biosystems), 0.01 U of uracil- N -glycosylase per μl, 2.5 mM MgCl2 , and 0.025 U of Ampli Taq Gold per μl. .. The PCR mixture for r Tth DNA polymerase had the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× chelating buffer (N808-0098; Applied Biosytems), 0.05 U of r Tth DNA polymerase per μl, dNTPs (dATP, dCTP, dGTP, and dTTP, each at a concentration of 200 μM; Applied Biosystems), 2.5 mM MgCl2 , and 8% (vol/vol) glycerol (Merck).

Cell Culture:

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
Article Snippet: Each strain was cultured in 100 mL LNG liquid medium, incubated at 30 °C for 2 days, and the cells were pelleted by centrifugation at 4,000×g for 10 min. .. The PCR mixture contained 4 μL DNA, 34 μL H2 O, 10 μL 5X Phusion HF Buffer, 1 μL 10 mM dNTPs, 0.5 μL (1 mg/mL) VNTR oligonucleotide, and 0.5 μL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA).

Generated:

Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
Article Snippet: To develop a PCR-RFLP assay for species- or genotype-specific identification of Prototheca algae, single nucleotide polymorphisms (SNPs) were detected upon alignment of the cytb gene partial sequences generated from each Prototheca species (genotype), as described above. .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA).

Negative Control:

Article Title: CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis
Article Snippet: The PCR mixture contained 50 ng of DNA, 0.08 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 1 mM MgCl2 , 5 pmol of each oligonucleotide and 1 U of Platinium Taq DNA Polymerase (Invitrogen, Carlsbad, USA), in a final volume of 15 μL. .. As negative control, DNA was replaced with sterile deionized water.

Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
Article Snippet: One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD). .. The purified PCR products of plasmids C-wt and C-mut1, a mixture of the two at a 1:1 ratio (wt/wt), and a no-target PCR negative control (NC) were used to validate each of the WT and Mut ASPE primers.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
Article Snippet: The melting curve analysis was performed from 60°C to 95°C in 0.1°C/s increments to assess the specificity of the RT-PCR products. .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

Article Title: Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma
Article Snippet: Paragraph title: RT-PCR ... The PCR mixture contained: 100 ng of cDNA, 1× PCR Rxn buffer (Invitrogen Life technologies), 0.2 mmol/L dNTP, 2 mmol/L MgCl2 , 0.2 µmol/L of each primer, and 1 U platinum Taq polymerase (Invitrogen Life technologies).

TaqMan Assay:

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
Article Snippet: .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water. .. The PCR amplification consisted of 1 cycle of enzyme activation for 2 min at 50°C followed by denaturation at 95°C for 20 s, and 40 cycles of denaturation at 95°C for 3 s, 30 s annealing at 60°C.

Nucleic Acid Electrophoresis:

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
Article Snippet: The PCR mixture contained 4 μL DNA, 34 μL H2 O, 10 μL 5X Phusion HF Buffer, 1 μL 10 mM dNTPs, 0.5 μL (1 mg/mL) VNTR oligonucleotide, and 0.5 μL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA). .. The amplified DNA was analyzed by gel electrophoresis on 1 % (w /v ) agarose gels stained with ethidium bromide using lambda DNA-Hin d III/phiX-174 RF DNA-Hae III marker (72 bp to 23 kb; Thermo Fisher Scientific, Waltham, MA).

Methylation:

Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene
Article Snippet: Paragraph title: Methylation-Specific Polymerase Chain Reaction (MSP) ... For set III, the PCR mixture contained 1X PCR buffer, dNTPs (each at 1.28 mmol/L; Invitrogen), primers (4 ng/μl; Sigma Genosys), HotStarTaq DNA polymerase (1.5U; Qiagen), and bisulfite-modified DNA (∼100 ng) or unmodified DNA (50 to 100 ng) in a final volume of 25 μl.

Mutagenesis:

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
Article Snippet: The PCR mixture contained 4 μL DNA, 34 μL H2 O, 10 μL 5X Phusion HF Buffer, 1 μL 10 mM dNTPs, 0.5 μL (1 mg/mL) VNTR oligonucleotide, and 0.5 μL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA). .. The procedure amplified the genomic sequence between two VNTR sequences to determine alterations in the minisatellite regions in the genome caused by UV-C mutagenesis.

Isolation:

Article Title: Dual transcripts of BCR-ABL different polymorphisms in chronic myeloid leukaemia patients
Article Snippet: RNA was isolated from the peripheral blood of the control and patients using Ambion's mirVana miRNA isolation kit (Life technologies, USA) following the manufacture's instruction. .. For amplification of the breakpoint region (BCR -ABL ), BCR gene, ABL gene and ABL kinase domain from the cDNA, the PCR mixture (15 μl) contained 100 mM Tris HCl (p H 8.8), 50 mM KCl, 1.5 mM MgCl2 , 0.1 per cent triton X-100, 10 mM dNTPs (deoxynucleotides), 10 pmol of the forward and reverse primers, 2 units of DyNAzyme II DNA polymerase (Thermo Scientific, USA).

Article Title: Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma
Article Snippet: RT-PCR Total RNA was isolated using Trizol according to the manufacturer's protocol (Invitrogen Life technologies). .. The PCR mixture contained: 100 ng of cDNA, 1× PCR Rxn buffer (Invitrogen Life technologies), 0.2 mmol/L dNTP, 2 mmol/L MgCl2 , 0.2 µmol/L of each primer, and 1 U platinum Taq polymerase (Invitrogen Life technologies).

Size-exclusion Chromatography:

Article Title: Peruvian Maca (Lepidium peruvianum): (I) Phytochemical and Genetic Differences in Three Maca Phenotypes
Article Snippet: RAPD/ISSR-PCR: The reaction consisted of 50 ng DNA, 20 pmol of each primer (OPL Kit, OPERON or single RAPD primer, OLIGO), and 7.5 μl of PCR Mix (Fermentas, 2×). .. The cycler was programmed as follows: 20 cycles of 95°C for 300 sec, 92°C for 90 sec, 35°C for 90 sec and 72°C for 120 sec followed by 20 cycles of 90 sec at 92°C, 90 sec. at 38°C and 120 sec at 72°C for denaturing, primer annealing and primer extension, respectively.

Labeling:

Article Title: Modeling of 5? Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
Article Snippet: The TaqMan probe was labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye). .. The PCR mixture for Ampli Taq Gold was composed of the following reagents and concentrations: 100 nM TaqMan probe, forward and reverse primers at concentrations of 900 nM each, 1× TaqMan Universal PCR Master Mix (including deoxynucleoside triphosphates [dNTPs; 200 μM dATP, 200 μM dCTP, 200 μM dGTP, and 400 μM dUTP]; Applied Biosystems), 0.01 U of uracil- N -glycosylase per μl, 2.5 mM MgCl2 , and 0.025 U of Ampli Taq Gold per μl.

Purification:

Article Title: Dual transcripts of BCR-ABL different polymorphisms in chronic myeloid leukaemia patients
Article Snippet: For amplification of the breakpoint region (BCR -ABL ), BCR gene, ABL gene and ABL kinase domain from the cDNA, the PCR mixture (15 μl) contained 100 mM Tris HCl (p H 8.8), 50 mM KCl, 1.5 mM MgCl2 , 0.1 per cent triton X-100, 10 mM dNTPs (deoxynucleotides), 10 pmol of the forward and reverse primers, 2 units of DyNAzyme II DNA polymerase (Thermo Scientific, USA). .. The products were resolved in agarose gel and the desired amplicons were purified using GenElute gel extraction kit (Sigma-Aldrich, USA) and sequenced on Applied Biosystems Genetic analyzer 3500 (Life Technologies, USA).

Article Title: Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma
Article Snippet: The RNA was further purified using iso-propanol precipitation and was concentrated using the RNeasy MinElute Cleanup kit (Qiagen). .. The PCR mixture contained: 100 ng of cDNA, 1× PCR Rxn buffer (Invitrogen Life technologies), 0.2 mmol/L dNTP, 2 mmol/L MgCl2 , 0.2 µmol/L of each primer, and 1 U platinum Taq polymerase (Invitrogen Life technologies).

Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
Article Snippet: One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD). .. The PCR products were purified using the ExoSAP-IT PCR cleanup kit (USB Co., Cleveland, OH).

Sequencing:

Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
Article Snippet: .. The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold). ..

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
Article Snippet: The PCR mixture contained 4 μL DNA, 34 μL H2 O, 10 μL 5X Phusion HF Buffer, 1 μL 10 mM dNTPs, 0.5 μL (1 mg/mL) VNTR oligonucleotide, and 0.5 μL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA). .. The procedure amplified the genomic sequence between two VNTR sequences to determine alterations in the minisatellite regions in the genome caused by UV-C mutagenesis.

Staining:

Article Title: CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis
Article Snippet: The PCR mixture contained 50 ng of DNA, 0.08 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 1 mM MgCl2 , 5 pmol of each oligonucleotide and 1 U of Platinium Taq DNA Polymerase (Invitrogen, Carlsbad, USA), in a final volume of 15 μL. .. The amplification conditions were: 1 cycle at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 61 °C for 30 s and 72 °C for 45 s; and one final extension cycle at 72 °C for 7 min. PCR products were subjected to 2% agarose gel electrophoresis, followed by ethidium bromide staining and UV light observation.

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
Article Snippet: The PCR mixture contained 4 μL DNA, 34 μL H2 O, 10 μL 5X Phusion HF Buffer, 1 μL 10 mM dNTPs, 0.5 μL (1 mg/mL) VNTR oligonucleotide, and 0.5 μL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA). .. The amplified DNA was analyzed by gel electrophoresis on 1 % (w /v ) agarose gels stained with ethidium bromide using lambda DNA-Hin d III/phiX-174 RF DNA-Hae III marker (72 bp to 23 kb; Thermo Fisher Scientific, Waltham, MA).

Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
Article Snippet: Paragraph title: Gomori Methenamine Silver (GMS) Staining and PCR Detection for Fusarium Species ... The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water.

Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
Article Snippet: Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. .. One-tenth of the amplified reaction mixture was mixed with a gel-loading buffer and electrophoresed on a 0.8% (wt/vol) agarose gel; the reaction products were visualized by ethidium bromide staining.

Plasmid Preparation:

Article Title: The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis
Article Snippet: The complete coding DNA sequence (CDS) of VvibZIPC22 was amplified in a 12.5 µl PCR mix containing primers (200 pM each), 1U of Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), dNTPs (200 µM), PCR buffer (1×), and cDNA (diluted 10-fold). .. A PCR product of 438bp was obtained and subcloned into a pGEM-T Easy Vector (Promega, Mannheim, Germany) for sequencing.

Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
Article Snippet: .. One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD). ..

Software:

Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
Article Snippet: 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM. .. Primers, listed in , were chosen using Primer Express 3.0 software (Applied Biosystems) and were designed to amplify a region of about 150–200 bp within each transcript.

Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
Article Snippet: To check if the SNPs located within the restriction enzyme recognition sites that would produce an RFLP, the sequences were screened with Clone Manager software, version 9.0 (Sci-Ed Software, Denver, CO, USA). .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA).

Real-time Polymerase Chain Reaction:

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix. .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

Article Title: Phase Variation in Myxococcus xanthus Yields Cells Specialized for Iron Sequestration
Article Snippet: Paragraph title: Real-time PCR ... 5 µl of diluted cDNA sample was added to 20 µl of a PCR mixture prepared from Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA), which contained each primer at a concentration of 160 nM.

Multiplex Assay:

Article Title: Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay
Article Snippet: Paragraph title: Development of the multiplex allele-specific HIVDR assay for HIV-1 subtype C. ... One microliter of 10 ng/μl plasmid DNAs (equivalent to 1,618 viral DNA copies) was added to 100 μl of the PCR mixture, containing 0.2 μM primers M13F (5′-CCCAG TCACG ACGTT GTAAA ACG-3′) and M13R (5′-AGCGG ATAAC AATTT CACAC AGG-3′), 1× High Fidelity PCR buffer, 2.0 mM MgSO4 , and 2.0 U of Platinum Taq High Fidelity (Life Technologies, Bethesda, MD).

Agarose Gel Electrophoresis:

Article Title: CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis
Article Snippet: The PCR mixture contained 50 ng of DNA, 0.08 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 1 mM MgCl2 , 5 pmol of each oligonucleotide and 1 U of Platinium Taq DNA Polymerase (Invitrogen, Carlsbad, USA), in a final volume of 15 μL. .. The amplification conditions were: 1 cycle at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 61 °C for 30 s and 72 °C for 45 s; and one final extension cycle at 72 °C for 7 min. PCR products were subjected to 2% agarose gel electrophoresis, followed by ethidium bromide staining and UV light observation.

Article Title: Dual transcripts of BCR-ABL different polymorphisms in chronic myeloid leukaemia patients
Article Snippet: For amplification of the breakpoint region (BCR -ABL ), BCR gene, ABL gene and ABL kinase domain from the cDNA, the PCR mixture (15 μl) contained 100 mM Tris HCl (p H 8.8), 50 mM KCl, 1.5 mM MgCl2 , 0.1 per cent triton X-100, 10 mM dNTPs (deoxynucleotides), 10 pmol of the forward and reverse primers, 2 units of DyNAzyme II DNA polymerase (Thermo Scientific, USA). .. The products were resolved in agarose gel and the desired amplicons were purified using GenElute gel extraction kit (Sigma-Aldrich, USA) and sequenced on Applied Biosystems Genetic analyzer 3500 (Life Technologies, USA).

Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
Article Snippet: The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The detection of amplified products (550 base pairs [bp]) was performed by electrophoresis of an aliquot of 5 μL of each amplicon in a 1% agarose gel with ethidium bromide 0.02% in 1 × Tris-acetate-EDTA (TAE) buffer.

Article Title: Role of Biofilm-Associated Protein Bap in the Pathogenesis of Bovine Staphylococcus aureus
Article Snippet: Briefly, 25 μl of the PCR mixture consisted of 250 ng of DNA; a 0.2 μM concentration of each of the forward and reverse primers; a 200 μM concentration each of dATP, dCTP, dGTP, and dTTP; 2.5 mM MgCl2 ; and 2.5 U of DyNAzyme EXT (Finnzymes) in a 1× reaction buffer. .. One-tenth of the amplified reaction mixture was mixed with a gel-loading buffer and electrophoresed on a 0.8% (wt/vol) agarose gel; the reaction products were visualized by ethidium bromide staining.

In Vitro:

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix. .. The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water.

Electrophoresis:

Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
Article Snippet: The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The detection of amplified products (550 base pairs [bp]) was performed by electrophoresis of an aliquot of 5 μL of each amplicon in a 1% agarose gel with ethidium bromide 0.02% in 1 × Tris-acetate-EDTA (TAE) buffer.

Electron Paramagnetic Resonance:

Article Title: CagL polymorphisms D58/K59 are predominant in Helicobacter pylori strains isolated from Mexican patients with chronic gastritis
Article Snippet: Confirmation of cag PAI empty site The absence of cagA and the pathogenicity island cag PAI in the cagA − strains was confirmed by the empty-site assay by conventional PCR, using the ESf and ESr oligonucleotides (Table ), which bind upstream and downstream, respectively, of the region where the cag PAI is inserted in the genome of the reference strain NCTC 12455 (NCTC: National Collection of Type Culture) [ ]. .. The PCR mixture contained 50 ng of DNA, 0.08 mM dNTPs (Invitrogen, Carlsbad, CA, USA), 1 mM MgCl2 , 5 pmol of each oligonucleotide and 1 U of Platinium Taq DNA Polymerase (Invitrogen, Carlsbad, USA), in a final volume of 15 μL.

Spectrophotometry:

Article Title: Transcriptional Silencing of the Wnt-Antagonist DKK1 by Promoter Methylation Is Associated with Enhanced Wnt Signaling in Advanced Multiple Myeloma
Article Snippet: The quantity of total RNA was measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). .. The PCR mixture contained: 100 ng of cDNA, 1× PCR Rxn buffer (Invitrogen Life technologies), 0.2 mmol/L dNTP, 2 mmol/L MgCl2 , 0.2 µmol/L of each primer, and 1 U platinum Taq polymerase (Invitrogen Life technologies).

Produced:

Article Title: cytb as a New Genetic Marker for Differentiation of Prototheca Species
Article Snippet: .. Experimentally, 644-bp amplimers of the partial cytb gene, produced with the PCR mixture and cycling conditions identical to those described above, were doubly digested with FastDigest RsaI and TaiI (Thermo Fisher Scientific, Waltham, MA, USA). ..

Activation Assay:

Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology
Article Snippet: The PCR mix for the Taqman assay consisted of 5 μl of cDNA dilution and 15 μl of master mix prepared using 1 μl of a 20X TaqMan probe/primer mix (5 μM probe: FAM-labelled ISAV segment 8 and 18 μM each primers) [ ], 10 μl TaqMan master mix (Applied Biosystems) and 4 μl of nuclease free water. .. The PCR amplification consisted of 1 cycle of enzyme activation for 2 min at 50°C followed by denaturation at 95°C for 20 s, and 40 cycles of denaturation at 95°C for 3 s, 30 s annealing at 60°C.

Marker:

Article Title: Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates
Article Snippet: The PCR mixture contained 4 μL DNA, 34 μL H2 O, 10 μL 5X Phusion HF Buffer, 1 μL 10 mM dNTPs, 0.5 μL (1 mg/mL) VNTR oligonucleotide, and 0.5 μL Phusion Enzyme (Finnzymes Phusion High-Fidelity PCR kit; New England Biolabs, Ipswich, MA). .. The amplified DNA was analyzed by gel electrophoresis on 1 % (w /v ) agarose gels stained with ethidium bromide using lambda DNA-Hin d III/phiX-174 RF DNA-Hae III marker (72 bp to 23 kb; Thermo Fisher Scientific, Waltham, MA).

Gel Extraction:

Article Title: Dual transcripts of BCR-ABL different polymorphisms in chronic myeloid leukaemia patients
Article Snippet: For amplification of the breakpoint region (BCR -ABL ), BCR gene, ABL gene and ABL kinase domain from the cDNA, the PCR mixture (15 μl) contained 100 mM Tris HCl (p H 8.8), 50 mM KCl, 1.5 mM MgCl2 , 0.1 per cent triton X-100, 10 mM dNTPs (deoxynucleotides), 10 pmol of the forward and reverse primers, 2 units of DyNAzyme II DNA polymerase (Thermo Scientific, USA). .. The products were resolved in agarose gel and the desired amplicons were purified using GenElute gel extraction kit (Sigma-Aldrich, USA) and sequenced on Applied Biosystems Genetic analyzer 3500 (Life Technologies, USA).

Hood:

Article Title: Human Trabecular Meshwork Sphingolipid and Ceramide Profiles and Potential Latent Fungal Commensalism
Article Snippet: The PCR reactions were performed with approximately 50 ng of genomic DNA in a 25-μl PCR mixture, which included 1 μL (200 ng) of DNA template, 2.5 μL of buffer solution (containing the dNTPs and MgCl2 ), 1 μL of each 10 μM primer (F18S and F28S), 1 μL of Taq DNA polymerase (Invitrogen), and 18.5 μL of distilled water. .. The DNA bands were visualized under UV illumination (Universal Hood II; Bio-Rad).

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  • 90
    Thermo Fisher sybr green i
    Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) <t>SYBR</t> <t>GREEN</t> I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).
    Sybr Green I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 803 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher qrt pcr reaction mixture
    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by <t>qRT-PCR</t> with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P
    Qrt Pcr Reaction Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 13 article reviews
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    99
    Thermo Fisher universal pcr master mix
    The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time <t>PCR;</t> n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.
    Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 152 article reviews
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    Thermo Fisher f13a1 gene pcr mix
    Gradient <t>PCR</t> for <t>F13A1</t> (F13) gene. Gradient PCR for F13 gene was carried out using specific primers as outlined in the M M section. Lane 1: 50 °C, lane 2: 51 °C, lane 3:52.9 °C, lane 4: 55.7 °C, lane 5: 59.1 °C, lane 6: 62 °C, lane 7 63.8 °C, lane 8: 65 °C. The F13 PCR showed maximum signal between 50–52.9 °C.M: DNA molecular weight maker (75 bp–10 Kb).
    F13a1 Gene Pcr Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) SYBR GREEN I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).

    Journal: PLoS ONE

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology

    doi: 10.1371/journal.pone.0159155

    Figure Lengend Snippet: Correlation of detection of virus by Bio-Plex and RT-qPCR. Correlation between qPCR (Ct value) and Bio-Plex (MFI) detection of in-vitro grown ISAV standard curve by (A) SYBR GREEN I and (B)TaqMan; Correlation of detection of copy number of ISAV in plasma samples by (C) SYBR Green I (n = 13) and (D) TaqMan chemistries (n = 8).

    Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix.

    Techniques: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, In Vitro, SYBR Green Assay

    RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) TaqMan chemistries.

    Journal: PLoS ONE

    Article Title: Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology

    doi: 10.1371/journal.pone.0159155

    Figure Lengend Snippet: RT-qPCR standard curves. Standard curve relating viral copy number to Ct value from RT-Polymerase chain reaction using 10-fold dilutions of in-vitro transcribed RNA using (a) SYBR ® Green I and (b) TaqMan chemistries.

    Article Snippet: 2.3.1 qRT-PCR with SYBR Green I chemistry or TaqMan® chemistry Real time PCR was performed on first strand cDNA (reverse transcribed from in-vitro transcribed RNA) using the Eppendorf® RealPlex2 Mastercycler gradient S instrument, using ISAV Segment 8 primer pair ISAV8F/ISAV8R (amplifying a 104 bp product), [ ], either with SYBR® Green I (Thermo Scientific) master mix or with TaqMan® master mix.

    Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, In Vitro, SYBR Green Assay

    Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: Effect of transient overexpression of G(G)PPS and GES in C. roseus leaves. mRNA expression (A,B) and metabolite (C,D) analyses in C. roseus leaves infiltrated with A. tumefaciens carrying pBI121 empty vector (black bar), pBI121:: G(G)PPS and co-infiltrated with pBI121:: G(G)PPS +pBI121:: GES (gray bar) constructs. Transcripts were analyzed by qRT-PCR with CrN227 as an endogenous reference gene. Expression levels were normalized to CrN227 and are represented as expression relative to the pBI121 controls that was set to 1. Relative amounts of secologanin, vindoline, and catharanthine in C. roseus . In all cases, first pair of infiltrated leaves were used for alkaloid extraction and quantified by HPLC. The levels of quantified metabolites are expressed in % relative to pBI121 vector infiltrated leaves. The bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Over Expression, Expressing, Plasmid Preparation, Construct, Quantitative RT-PCR, High Performance Liquid Chromatography

    Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: Analysis of gene expression in transgenic G(G)PPS and GES C. roseus plants. qRT-PCR analysis of G(G)PPS (gray bar) (A) and G(G)PPS (gray bar)/ GES (black bar) (B) in transgenic C. roseus plants. Expression levels of genes were normalized to the endogenous reference gene CrN227 and are represented relative to the wild type (WT) controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

    PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Journal: Frontiers in Plant Science

    Article Title: Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

    doi: 10.3389/fpls.2018.00942

    Figure Lengend Snippet: PRX1 expression in transgenic C. roseus plants. Analysis of PRX1 expression by qRT-PCR in G(G)PPS (A) and G(G)PPS + GES (B) overexpressing transgenic C. roseus plants. Expression levels of PRX1 were normalized to the endogenous reference gene CrN227 and are represented relative to the WT controls, which was set to 1. Error bars represent mean ± standard error (SE) of three independent experiments. Significant differences at P

    Article Snippet: Total RNA (2 μg) was used for first strand cDNA synthesis with random hexamer primers using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, United States) as per manufacturer’s instructions. qRT-PCR was performed with a linear range of cDNA using StepOne Real-Time PCR System (Applied Biosystems, United States) and expression of transcripts were normalized using N227 that was previously reported as an appropriate endogenous gene in C. roseus ( ). qRT-PCR reaction mixture of 5 μl contained 2.5 μl of 2X Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific, United States), 1:3 diluted cDNA and 2 μM gene-specific primers (Supplementary Table ).

    Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR

    The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time PCR; n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes

    doi: 10.1152/ajpgi.00238.2018

    Figure Lengend Snippet: The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time PCR; n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.

    Article Snippet: Real-time PCR was completed with Universal PCR Master Mix and TaqMan mRNA and miRNA assays (ThermoFisher).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Sequencing, MANN-WHITNEY

    Mediator complex subunit 1 (Med1) is regulated by microRNA (miR)-96/182/183 in mice. A : expression of Med1 by real-time PCR in mouse primary hepatocytes transfected with 50 nM of Med1 or scramble siRNA; n = 3 B : Western blot of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with Med1 or scramble siRNA. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. A.U., arbitrary units. C : hepatic Med1 mRNA levels in db/db mice treated with vehicle or 2% colesevelam (Col)-supplemented diet and PBS or locked-nucleic acid (LNA)-182-5p; real-time PCR; n = 8–9. D : hepatic Med1 and β-actin (loading control) protein levels from 3 representative liver protein samples for Chow + PBS, Col + PBS, and Col + LNA-182. Western blotting. Ratio of Med1/β-actin densitometry is indicated between the blots. E : densitometry quantification of Med1 protein levels normalized to β-actin; n = 8–9. F : expression of miR-96-5p, miR-182-5p, and miR-183-5p in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, miR-183-5p mimics individually or in combination; real-time PCR; n = 3. G : Med1 mRNA levels in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 3. H : protein levels of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with mock or 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. For comparisons between 2 groups, Students’ t- tests were used, and for more than 2 groups, Kruskal-Wallis one-way ANOVA with Dunn’s posttest was used (α = 0.05) or one-way ANOVA with Bonferonni’s posttest for comparison to mock group only was used (α = 0.05).

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes

    doi: 10.1152/ajpgi.00238.2018

    Figure Lengend Snippet: Mediator complex subunit 1 (Med1) is regulated by microRNA (miR)-96/182/183 in mice. A : expression of Med1 by real-time PCR in mouse primary hepatocytes transfected with 50 nM of Med1 or scramble siRNA; n = 3 B : Western blot of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with Med1 or scramble siRNA. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. A.U., arbitrary units. C : hepatic Med1 mRNA levels in db/db mice treated with vehicle or 2% colesevelam (Col)-supplemented diet and PBS or locked-nucleic acid (LNA)-182-5p; real-time PCR; n = 8–9. D : hepatic Med1 and β-actin (loading control) protein levels from 3 representative liver protein samples for Chow + PBS, Col + PBS, and Col + LNA-182. Western blotting. Ratio of Med1/β-actin densitometry is indicated between the blots. E : densitometry quantification of Med1 protein levels normalized to β-actin; n = 8–9. F : expression of miR-96-5p, miR-182-5p, and miR-183-5p in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, miR-183-5p mimics individually or in combination; real-time PCR; n = 3. G : Med1 mRNA levels in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 3. H : protein levels of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with mock or 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. For comparisons between 2 groups, Students’ t- tests were used, and for more than 2 groups, Kruskal-Wallis one-way ANOVA with Dunn’s posttest was used (α = 0.05) or one-way ANOVA with Bonferonni’s posttest for comparison to mock group only was used (α = 0.05).

    Article Snippet: Real-time PCR was completed with Universal PCR Master Mix and TaqMan mRNA and miRNA assays (ThermoFisher).

    Techniques: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot

    Mediator complex subunit 1 (MED1) is a direct target of microRNA (miR)-182-5p, miR-183-5p, and miR-96-5p in humans. A : schematic of human MED1 mRNA and the 3 putative miR-182/183/96 target sites in the 3′-untranslated region (3′-UTR). B : predicted MED1 3′-UTR target sites. Bases highlighted in dark gray represent mutations for gene reporter (luciferase) assays. C : normalized luciferase activity in HEK293 cells after dual transfection of miR-96/182/183 mimics and gene (luciferase) reporters harboring putative target sites for miR-96-5p, miR-182-5p, and/or miR-183-5p; n = 4. D : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. E : MED1 mRNA levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. F : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with mock or 50 nM locked-nucleic acids (LNAs) against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. G : MED1 mRNA levels in Huh7 cells transfected mock or 50 nM LNAs against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. H : protein levels of Med1 and β-actin (loading control) from protein lysates of Huh7 cells transfected with mock or 50 nM miR-96, miR-182, and miR-183 LNAs. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 6. A.U. arbitrary units. For comparisons between more than 2 groups to mock only, one-way ANOVA with Bonferonni’s posttest was used, α = 0.05. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes

    doi: 10.1152/ajpgi.00238.2018

    Figure Lengend Snippet: Mediator complex subunit 1 (MED1) is a direct target of microRNA (miR)-182-5p, miR-183-5p, and miR-96-5p in humans. A : schematic of human MED1 mRNA and the 3 putative miR-182/183/96 target sites in the 3′-untranslated region (3′-UTR). B : predicted MED1 3′-UTR target sites. Bases highlighted in dark gray represent mutations for gene reporter (luciferase) assays. C : normalized luciferase activity in HEK293 cells after dual transfection of miR-96/182/183 mimics and gene (luciferase) reporters harboring putative target sites for miR-96-5p, miR-182-5p, and/or miR-183-5p; n = 4. D : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. E : MED1 mRNA levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. F : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with mock or 50 nM locked-nucleic acids (LNAs) against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. G : MED1 mRNA levels in Huh7 cells transfected mock or 50 nM LNAs against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. H : protein levels of Med1 and β-actin (loading control) from protein lysates of Huh7 cells transfected with mock or 50 nM miR-96, miR-182, and miR-183 LNAs. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 6. A.U. arbitrary units. For comparisons between more than 2 groups to mock only, one-way ANOVA with Bonferonni’s posttest was used, α = 0.05. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used.

    Article Snippet: Real-time PCR was completed with Universal PCR Master Mix and TaqMan mRNA and miRNA assays (ThermoFisher).

    Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    Gradient PCR for F13A1 (F13) gene. Gradient PCR for F13 gene was carried out using specific primers as outlined in the M M section. Lane 1: 50 °C, lane 2: 51 °C, lane 3:52.9 °C, lane 4: 55.7 °C, lane 5: 59.1 °C, lane 6: 62 °C, lane 7 63.8 °C, lane 8: 65 °C. The F13 PCR showed maximum signal between 50–52.9 °C.M: DNA molecular weight maker (75 bp–10 Kb).

    Journal: MethodsX

    Article Title: Use of coagulation factor XIII (F13) gene as an internal control for normalization of genomic DNA’s for HLA typing

    doi: 10.1016/j.mex.2018.07.020

    Figure Lengend Snippet: Gradient PCR for F13A1 (F13) gene. Gradient PCR for F13 gene was carried out using specific primers as outlined in the M M section. Lane 1: 50 °C, lane 2: 51 °C, lane 3:52.9 °C, lane 4: 55.7 °C, lane 5: 59.1 °C, lane 6: 62 °C, lane 7 63.8 °C, lane 8: 65 °C. The F13 PCR showed maximum signal between 50–52.9 °C.M: DNA molecular weight maker (75 bp–10 Kb).

    Article Snippet: The F13A1 gene PCR mix comprised of 40 ng of human gDNA with 1 U of Taq DNA polymerase, 2.5 μl of 10 X ammonium sulfate buffer, pH 8.3 (Thermo Fisher Scientific, USA), 0.2 mM dNTP’s, 2% Tween-20, 0.32% BSA, 5% DMSO and 2.5 mM MgCl2 in a final volume of 25 μl master mix.

    Techniques: Polymerase Chain Reaction, Molecular Weight