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Galanin methylation analysis using the <t>MSP</t> assay and galanin expression by quantitative <t>RT-PCR</t> in HNSCC tumor samples. (A) Representative examples of MSP of galanin in primary tumors from Hamamatsu University Hospital, showing samples that are methylated
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1) Product Images from "Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1"

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1

Journal: Translational Oncology

doi:

Galanin methylation analysis using the MSP assay and galanin expression by quantitative RT-PCR in HNSCC tumor samples. (A) Representative examples of MSP of galanin in primary tumors from Hamamatsu University Hospital, showing samples that are methylated
Figure Legend Snippet: Galanin methylation analysis using the MSP assay and galanin expression by quantitative RT-PCR in HNSCC tumor samples. (A) Representative examples of MSP of galanin in primary tumors from Hamamatsu University Hospital, showing samples that are methylated

Techniques Used: Methylation, MSP Assay, Expressing, Quantitative RT-PCR

2) Product Images from "Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori"

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

Journal: Iranian Journal of Basic Medical Sciences

doi:

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

Techniques Used: Amplification, Polymerase Chain Reaction, Marker

3) Product Images from "Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor"

Article Title: Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.14475

HPV Nested PCR. The presence of HPV in the OSCC samples were tested using (a) HPV primer PGMY09/11 and (b) nested HPV primer GP5 + /6 + . A representative gel is shown from 84 samples tested with the two consensus primers displaying positive PCR amplification for PGMY09/11 (450bp) and nested GP5 + /6 + (150bp). Positive Control (PC) using DNA from HeLa cells and negative control (NC) were included in the experiments. Marker and sample ID are indicated.
Figure Legend Snippet: HPV Nested PCR. The presence of HPV in the OSCC samples were tested using (a) HPV primer PGMY09/11 and (b) nested HPV primer GP5 + /6 + . A representative gel is shown from 84 samples tested with the two consensus primers displaying positive PCR amplification for PGMY09/11 (450bp) and nested GP5 + /6 + (150bp). Positive Control (PC) using DNA from HeLa cells and negative control (NC) were included in the experiments. Marker and sample ID are indicated.

Techniques Used: Nested PCR, Polymerase Chain Reaction, Amplification, Positive Control, Negative Control, Marker

4) Product Images from "CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1"

Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi:

CD8+ cells express IL-17 within the allografts of T-bet-/- recipients. A , Intragraft expression of IL-17 was assessed by real-time RT-PCR. From top to bottom, RNA was harvested from allografts transplanted into the following mice: unmodified WT, unmodified
Figure Legend Snippet: CD8+ cells express IL-17 within the allografts of T-bet-/- recipients. A , Intragraft expression of IL-17 was assessed by real-time RT-PCR. From top to bottom, RNA was harvested from allografts transplanted into the following mice: unmodified WT, unmodified

Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

5) Product Images from "Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents"

Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents

Journal: Clinical Epigenetics

doi: 10.1186/s13148-018-0594-9

Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)
Figure Legend Snippet: Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

Techniques Used: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Derivative Assay, Positive Control, Molecular Weight

6) Product Images from "T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires"

Article Title: T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20021945

Single cell RT-PCR analysis reveals clusters of CW3-specific clones expressing β chains encoded by identical VDJ-β nucleotide sequences paired with different TCR-α chains. The cDNA from tubes containing single CW3-specific CD8 T cells sorted from PBL of M-2 and M-3 or M-33 was subjected to RT-PCR, and PCR products were sequenced and assigned a TCR sequence code (NS). The complete analysis is shown in Figs. S1, S2, and S3, available at http://www.jem.org/cgi/content/full/jem.20021945/DC1 . Represented here are those cells for which both a CW3-like Vβ10 sequence and a CW3-like Vα3, Vα4, or Vα8 sequence were amplified. The deduced aa sequences of the TCR junctions are shown. All TCR-α sequences incorporate the Jα35 sequence. Also shown are the number (#) of cells found for each αβ TCR clone and its corresponding percentage (%) within the αβ TCR repertoire defined here for each mouse. Each cluster of αβ TCR clones sharing an identical Vβ10 TCR nucleotide sequence is framed. Cells for which an in-frame (IF) or out of frame (OF) second TCR-α rearrangement was also amplified are indicated.
Figure Legend Snippet: Single cell RT-PCR analysis reveals clusters of CW3-specific clones expressing β chains encoded by identical VDJ-β nucleotide sequences paired with different TCR-α chains. The cDNA from tubes containing single CW3-specific CD8 T cells sorted from PBL of M-2 and M-3 or M-33 was subjected to RT-PCR, and PCR products were sequenced and assigned a TCR sequence code (NS). The complete analysis is shown in Figs. S1, S2, and S3, available at http://www.jem.org/cgi/content/full/jem.20021945/DC1 . Represented here are those cells for which both a CW3-like Vβ10 sequence and a CW3-like Vα3, Vα4, or Vα8 sequence were amplified. The deduced aa sequences of the TCR junctions are shown. All TCR-α sequences incorporate the Jα35 sequence. Also shown are the number (#) of cells found for each αβ TCR clone and its corresponding percentage (%) within the αβ TCR repertoire defined here for each mouse. Each cluster of αβ TCR clones sharing an identical Vβ10 TCR nucleotide sequence is framed. Cells for which an in-frame (IF) or out of frame (OF) second TCR-α rearrangement was also amplified are indicated.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Expressing, Polymerase Chain Reaction, Sequencing, Amplification

Repertoire analysis by single cell DNA-PCR for coamplification of CW3-specific TCR VDJ-β and VJ-α sequences together with DJ-β rearrangements as potential clonal markers. For each of three mice (M-41, M-42, and M-43), single pCW3Kd + Vβ10 + CD8 + splenocytes were sorted 2 wk after immunization with P815-CW3 tumor cells. The rearranged TCR-α and TCR-β nucleotide sequences were amplified by single cell DNA-PCR. PCR products from cells with successful amplifications for TCR-α and TCR-β rearrangements were sequenced to identify paired αβ TCRs. Other details are as described for Fig. 2 .
Figure Legend Snippet: Repertoire analysis by single cell DNA-PCR for coamplification of CW3-specific TCR VDJ-β and VJ-α sequences together with DJ-β rearrangements as potential clonal markers. For each of three mice (M-41, M-42, and M-43), single pCW3Kd + Vβ10 + CD8 + splenocytes were sorted 2 wk after immunization with P815-CW3 tumor cells. The rearranged TCR-α and TCR-β nucleotide sequences were amplified by single cell DNA-PCR. PCR products from cells with successful amplifications for TCR-α and TCR-β rearrangements were sequenced to identify paired αβ TCRs. Other details are as described for Fig. 2 .

Techniques Used: Polymerase Chain Reaction, Mouse Assay, Amplification

7) Product Images from "A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression"

Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063618

Colony PCR showing the cDNA derived maternal and paternal fragment of Nvtra . Panels A – D and E – H indicate the four replicate PCR runs. The maternal origin and age of the embryos is indicated above each set of PCR fragments. White arrow indicates cDNA fragment, black arrow indicates Russia Bait cDNA fragment and dotted arrow indicates gDNA fragment. M is 100 bp molecular standard, ranging from 300 bp (lowest band) to 500 bp (higest band).
Figure Legend Snippet: Colony PCR showing the cDNA derived maternal and paternal fragment of Nvtra . Panels A – D and E – H indicate the four replicate PCR runs. The maternal origin and age of the embryos is indicated above each set of PCR fragments. White arrow indicates cDNA fragment, black arrow indicates Russia Bait cDNA fragment and dotted arrow indicates gDNA fragment. M is 100 bp molecular standard, ranging from 300 bp (lowest band) to 500 bp (higest band).

Techniques Used: Polymerase Chain Reaction, Derivative Assay

8) Product Images from "Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿"

Article Title: Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿

Journal:

doi: 10.1128/AEM.00370-09

PCR-RFLP analysis of the rRNA IGS region for differentiating the lineages of F. oxysporum f. sp. cubense . For this procedure, DNA obtained from a putative F. oxysporum f. sp. cubense isolate is used as a template for amplication of the IGS region (A),
Figure Legend Snippet: PCR-RFLP analysis of the rRNA IGS region for differentiating the lineages of F. oxysporum f. sp. cubense . For this procedure, DNA obtained from a putative F. oxysporum f. sp. cubense isolate is used as a template for amplication of the IGS region (A),

Techniques Used: Polymerase Chain Reaction

9) Product Images from "Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site"

Article Title: Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site

Journal: Nucleic Acids Research

doi:

MDR1 quantification using SRS-cPCR. Competitive PCR and restriction digest were carried out as described in Materials and Methods. Lanes 1 and 12 in each panel contain size marker (100 bp ladder). Lanes 10 and 11 represent amplification products of only competitor and target mRNA, respectively. ( A ) Twenty microliters PCR product was loaded on a 2% agarose gel. The target and the competitor products were identical in size (299 bp). Lanes 2–9 contain 10 6 molecules of an in vitro transcribed cloned human wild-type MDR1 fragment and a semi-logarithmic dilution of the competitor RNA ranging from 10 4 to 3 × 10 7 molecules . ( B ) After digestion with the restriction endonuclease Pvu II, the wild-type PCR products were cleaved into 127 and 172 bp fragments, and the competitor PCR products into 100 and 199 bp fragments. The EQP is indicated by an arrow (lane 5). It was determined by comparison of signal intensities of the 100 and 127 bp fragments, or of the 172 and 199 bp fragments, respectively. The competitive amplification of an external control gene, β 2-MG , permitting correction of target quantification (29) is not displayed. In competitive PCR reactions, the subdominant template yields visible products if it represents at least 1% of the total. Three competitor dilutions covering a range of 3 logs (lanes 3, 6 and 9, indicated by arrows) usually permit the visualization of target- and competitor-derived products in at least one of the reactions (lanes 3 and 6), thus permitting calculation of the target molecules. ( C ) The band intensities of competitor- and target-derived fragments were digitalized and measured by using the 1D Image Analysis Software. In the graph, the mean intensities from the larger competitor (line B) and the target (line A) fragments were used for the performance of a regression analysis. The EQP is indicated by the intersection of the two lines. The numbers on the abscissa correspond to the lane numbers on the agarose gel. Elimination of any data points between the extreme values has no effect on the EQP. This demonstrates that it is possible to reduce the number of competitor dilutions needed for quantitative analysis of target transcripts, thus rendering the assay less laborious.
Figure Legend Snippet: MDR1 quantification using SRS-cPCR. Competitive PCR and restriction digest were carried out as described in Materials and Methods. Lanes 1 and 12 in each panel contain size marker (100 bp ladder). Lanes 10 and 11 represent amplification products of only competitor and target mRNA, respectively. ( A ) Twenty microliters PCR product was loaded on a 2% agarose gel. The target and the competitor products were identical in size (299 bp). Lanes 2–9 contain 10 6 molecules of an in vitro transcribed cloned human wild-type MDR1 fragment and a semi-logarithmic dilution of the competitor RNA ranging from 10 4 to 3 × 10 7 molecules . ( B ) After digestion with the restriction endonuclease Pvu II, the wild-type PCR products were cleaved into 127 and 172 bp fragments, and the competitor PCR products into 100 and 199 bp fragments. The EQP is indicated by an arrow (lane 5). It was determined by comparison of signal intensities of the 100 and 127 bp fragments, or of the 172 and 199 bp fragments, respectively. The competitive amplification of an external control gene, β 2-MG , permitting correction of target quantification (29) is not displayed. In competitive PCR reactions, the subdominant template yields visible products if it represents at least 1% of the total. Three competitor dilutions covering a range of 3 logs (lanes 3, 6 and 9, indicated by arrows) usually permit the visualization of target- and competitor-derived products in at least one of the reactions (lanes 3 and 6), thus permitting calculation of the target molecules. ( C ) The band intensities of competitor- and target-derived fragments were digitalized and measured by using the 1D Image Analysis Software. In the graph, the mean intensities from the larger competitor (line B) and the target (line A) fragments were used for the performance of a regression analysis. The EQP is indicated by the intersection of the two lines. The numbers on the abscissa correspond to the lane numbers on the agarose gel. Elimination of any data points between the extreme values has no effect on the EQP. This demonstrates that it is possible to reduce the number of competitor dilutions needed for quantitative analysis of target transcripts, thus rendering the assay less laborious.

Techniques Used: Polymerase Chain Reaction, Marker, Amplification, Agarose Gel Electrophoresis, In Vitro, Clone Assay, Derivative Assay, Software

10) Product Images from "Small Variant STEVOR Antigen Is Uniquely Located within Maurer's Clefts in Plasmodium falciparum-Infected Red Blood Cells"

Article Title: Small Variant STEVOR Antigen Is Uniquely Located within Maurer's Clefts in Plasmodium falciparum-Infected Red Blood Cells

Journal: Eukaryotic Cell

doi: 10.1128/EC.1.6.926-935.2002

(A) Schematic representation of stevor (1,000 bp). The external (smkf1 and smkr1) and internal (RepF1, RepF2, and RepR) primers used in this study are indicated. Dark grey shading, moderately polymorphic. Light grey shading, highly polymorphic. Broken line, weakly predicted transmembrane domain. Solid line (above sequence and at right), strongly predicted transmembrane domain. AS, position of alternative splice site. Positions of peptides (Pep) 1 and 2 are also shown. (B) PCR and RT-PCR analyses of stevor . (I) PCR products of 3D7 genomic DNA amplified with smkf1 and smkr1 and with RepF1, RepF2, and RepR. (II) RT-PCR products obtained from RNA of asynchronous parasites with smkf1 and smkr1 and with RepF1, RepF2, and RepR. The products were separated on Metaphor 3% agarose gels. Lanes +, RT added. Lanes −, no RT added. Lanes M, 100-bp ladder (Bio-Rad). (C) Southern blot analysis of P. falciparum 3D7 chromosomes probed with PCR or RT-PCR products. The left panel shows PFGE of 3D7 chromosomes stained with ethidium bromide (EtBr) and photographed before the DNA from the gels was transferred to nylon membranes and probed. Numbers at left refer to P. falciparum chromosomes. Probing was performed with the PCR product obtained from DNA (middle panel) and with the RT-PCR product obtained from RNA (cDNA) (right panel) of 3D7 parasites.
Figure Legend Snippet: (A) Schematic representation of stevor (1,000 bp). The external (smkf1 and smkr1) and internal (RepF1, RepF2, and RepR) primers used in this study are indicated. Dark grey shading, moderately polymorphic. Light grey shading, highly polymorphic. Broken line, weakly predicted transmembrane domain. Solid line (above sequence and at right), strongly predicted transmembrane domain. AS, position of alternative splice site. Positions of peptides (Pep) 1 and 2 are also shown. (B) PCR and RT-PCR analyses of stevor . (I) PCR products of 3D7 genomic DNA amplified with smkf1 and smkr1 and with RepF1, RepF2, and RepR. (II) RT-PCR products obtained from RNA of asynchronous parasites with smkf1 and smkr1 and with RepF1, RepF2, and RepR. The products were separated on Metaphor 3% agarose gels. Lanes +, RT added. Lanes −, no RT added. Lanes M, 100-bp ladder (Bio-Rad). (C) Southern blot analysis of P. falciparum 3D7 chromosomes probed with PCR or RT-PCR products. The left panel shows PFGE of 3D7 chromosomes stained with ethidium bromide (EtBr) and photographed before the DNA from the gels was transferred to nylon membranes and probed. Numbers at left refer to P. falciparum chromosomes. Probing was performed with the PCR product obtained from DNA (middle panel) and with the RT-PCR product obtained from RNA (cDNA) (right panel) of 3D7 parasites.

Techniques Used: Sequencing, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Southern Blot, Staining

11) Product Images from "Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †"

Article Title: Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00040-10

Dendrograms showing the similarity of PCR-DGGE profiles generated with the bacterial 16S rRNA DGGE system for soil B at the flowering (A) and senescence (B) stages and for soil V at the flowering stage (C). B, Buinen soil; A, cultivar Aveka; Av, cultivar
Figure Legend Snippet: Dendrograms showing the similarity of PCR-DGGE profiles generated with the bacterial 16S rRNA DGGE system for soil B at the flowering (A) and senescence (B) stages and for soil V at the flowering stage (C). B, Buinen soil; A, cultivar Aveka; Av, cultivar

Techniques Used: Polymerase Chain Reaction, Denaturing Gradient Gel Electrophoresis, Generated

12) Product Images from "Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription"

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm1149

Production of HIV-1 DIS RNA mutants from PBMCs partially rescues defects in viral cDNA synthesis. WT and DIS mutant HIV-1 viruses were produced either by transfection of 293T cells or infection of PHA-stimulated freshly isolated PBMCs. Equivalent amounts of DNase treated virus were used to infect either SupT1 cells ( A ) or PBMCs ( B ). Cells were lysed 24 h postinfection and HIV-1 cDNA was measured by real-time PCR. HIV-1 DNA copies were normalized by CCR5. Results are reported as a percentage of the WT control and represent means (±SE) of separate experiments.
Figure Legend Snippet: Production of HIV-1 DIS RNA mutants from PBMCs partially rescues defects in viral cDNA synthesis. WT and DIS mutant HIV-1 viruses were produced either by transfection of 293T cells or infection of PHA-stimulated freshly isolated PBMCs. Equivalent amounts of DNase treated virus were used to infect either SupT1 cells ( A ) or PBMCs ( B ). Cells were lysed 24 h postinfection and HIV-1 cDNA was measured by real-time PCR. HIV-1 DNA copies were normalized by CCR5. Results are reported as a percentage of the WT control and represent means (±SE) of separate experiments.

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

13) Product Images from "Sarcocystis cruzi: First Molecular Identification from Cattle in Iran"

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran

Journal: International Journal of Molecular and Cellular Medicine

doi:

Electrophoresis of PCR product of Sarcocystis isolated from goat and calf in northern Iran. Lanes: 1 2, Sarcocystis isolated from goat; 3, Sarcocystis isolated from calf muscle sample; M, 100 bp ladder DNA size marker; and N, negative control
Figure Legend Snippet: Electrophoresis of PCR product of Sarcocystis isolated from goat and calf in northern Iran. Lanes: 1 2, Sarcocystis isolated from goat; 3, Sarcocystis isolated from calf muscle sample; M, 100 bp ladder DNA size marker; and N, negative control

Techniques Used: Electrophoresis, Polymerase Chain Reaction, Isolation, Northern Blot, Marker, Negative Control

14) Product Images from "Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway"

Article Title: Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601184

Effect of treatment of C 2 C 12 myotubes with 15( S )-HETE for 4 h on expression of mRNA for ( A ) C2 proteasome subunit, ( B ) C5 proteasome subunit, ( C ) E2 14k , as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m. from three separate determinations performed on different occasions with different samples. Differences are indicated as (a) P
Figure Legend Snippet: Effect of treatment of C 2 C 12 myotubes with 15( S )-HETE for 4 h on expression of mRNA for ( A ) C2 proteasome subunit, ( B ) C5 proteasome subunit, ( C ) E2 14k , as determined by quantitative competitive RT–PCR. Results represent mean±s.e.m. from three separate determinations performed on different occasions with different samples. Differences are indicated as (a) P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

15) Product Images from "Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways"

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.12.3882-3888.2004

Disruption of the dspA gene. (A) PCR analysis of the dspA gene in potential mutants in which the dspA gene was insertionally inactivated with the cat gene. WT, wild type; M, DNA size marker. (B and C) Depiction of the constructs used to generate the dspA-cat strain (B) and the nblS + dspA mutant strain (C). To generate the dspA-cat disruption, an internal 29-bp dspA fragment from the dspA gene (from Sp to Bm; shaded area) was deleted and replaced by a 0.85-kbp chloramphenicol resistance cartridge (Cat); in panel C, the cat gene shown in panel B was replaced by a 2.2-kb nblS gene linked with a 1.2-kb kanamycin resistance cartridge (Kan); the nblS and kan r genes are transcribed in opposite directions. In panel A, both the intact dspA (∼2.0 kbp) and the dspA-cat (∼2.8 kbp) loci were efficiently amplified from genomic DNA preparations by PCR. A total of 5 chloramphenicol-resistant transformants were isolated (lanes 1 to 5) and analyzed for a disruption of the dspA gene; transformants 3 and 4 showed no detectable wild-type dspA PCR product, and transformant 4 was used in all subsequent analyses. Sp, SphI; Bg, BglII; Bm, BamHI; Xm, XmnI; Sm, SmaI; Pt, PstI.
Figure Legend Snippet: Disruption of the dspA gene. (A) PCR analysis of the dspA gene in potential mutants in which the dspA gene was insertionally inactivated with the cat gene. WT, wild type; M, DNA size marker. (B and C) Depiction of the constructs used to generate the dspA-cat strain (B) and the nblS + dspA mutant strain (C). To generate the dspA-cat disruption, an internal 29-bp dspA fragment from the dspA gene (from Sp to Bm; shaded area) was deleted and replaced by a 0.85-kbp chloramphenicol resistance cartridge (Cat); in panel C, the cat gene shown in panel B was replaced by a 2.2-kb nblS gene linked with a 1.2-kb kanamycin resistance cartridge (Kan); the nblS and kan r genes are transcribed in opposite directions. In panel A, both the intact dspA (∼2.0 kbp) and the dspA-cat (∼2.8 kbp) loci were efficiently amplified from genomic DNA preparations by PCR. A total of 5 chloramphenicol-resistant transformants were isolated (lanes 1 to 5) and analyzed for a disruption of the dspA gene; transformants 3 and 4 showed no detectable wild-type dspA PCR product, and transformant 4 was used in all subsequent analyses. Sp, SphI; Bg, BglII; Bm, BamHI; Xm, XmnI; Sm, SmaI; Pt, PstI.

Techniques Used: Polymerase Chain Reaction, Marker, Construct, Mutagenesis, Amplification, Isolation

16) Product Images from "Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells"

Article Title: Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells

Journal: Human Reproduction (Oxford, England)

doi: 10.1093/humrep/dex348

eGFP is expressed in transplanted testis, but not in benign and malignant lesions outside of the testis. Gel electrophoresis of a standard PCR for eGFP performed on genomic DNA from adult testis and lesions found in transplanted animals. Genomic DNA of adult B6D2F1 actin-eGFP testis serves as a positive control. Mouse 541570 serves as an example for all transplanted mice, and was transplanted unilaterally with eGFP positive GS cells, which can be confirmed by PCR 14 months after transplantation with a positive eGFP signal in the transplanted testis and no signal in the contralateral untransplanted testis. Mouse 541555 developed a hematoma in the transplanted testis, explaining a positive read-out in this case. eGFP could not be detected in all other benign and malignant lesions. An amplicon for β-actin is present in all samples. LN, lymph node; LCL, diffuse large cell lymphoma.
Figure Legend Snippet: eGFP is expressed in transplanted testis, but not in benign and malignant lesions outside of the testis. Gel electrophoresis of a standard PCR for eGFP performed on genomic DNA from adult testis and lesions found in transplanted animals. Genomic DNA of adult B6D2F1 actin-eGFP testis serves as a positive control. Mouse 541570 serves as an example for all transplanted mice, and was transplanted unilaterally with eGFP positive GS cells, which can be confirmed by PCR 14 months after transplantation with a positive eGFP signal in the transplanted testis and no signal in the contralateral untransplanted testis. Mouse 541555 developed a hematoma in the transplanted testis, explaining a positive read-out in this case. eGFP could not be detected in all other benign and malignant lesions. An amplicon for β-actin is present in all samples. LN, lymph node; LCL, diffuse large cell lymphoma.

Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Positive Control, Mouse Assay, Transplantation Assay, Amplification

17) Product Images from "Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway"

Article Title: Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601184

( A ) Representative gel illustrating quantitative competitive RT–PCR for C5 mRNA in control cultures at 4 h incubation. Lanes: M ; 100-bp DNA ladder: RT-ve; without reverse transcriptase, PCR-ve: without template. Lanes 1–5 each contain 0.25 μ g target RNA and a specific amount of the C5 competitor RNA ranging from 0.01875 to 0.3 ng in two-fold dilutions. ( B ) Dose–response curve from the gel shown in ( A ). ▪, target volume; ▴, competitor volume. The amount of unknown determined from where the two lines intersect=0.0445 ng mRNA.
Figure Legend Snippet: ( A ) Representative gel illustrating quantitative competitive RT–PCR for C5 mRNA in control cultures at 4 h incubation. Lanes: M ; 100-bp DNA ladder: RT-ve; without reverse transcriptase, PCR-ve: without template. Lanes 1–5 each contain 0.25 μ g target RNA and a specific amount of the C5 competitor RNA ranging from 0.01875 to 0.3 ng in two-fold dilutions. ( B ) Dose–response curve from the gel shown in ( A ). ▪, target volume; ▴, competitor volume. The amount of unknown determined from where the two lines intersect=0.0445 ng mRNA.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Incubation, Polymerase Chain Reaction

18) Product Images from "Dexamethasone inhibits IL-9 production by human T cells"

Article Title: Dexamethasone inhibits IL-9 production by human T cells

Journal: Journal of Inflammation (London, England)

doi: 10.1186/1476-9255-2-3

IL-9 RT-PCR. A. Time course. PBMC were incubated for various times with OKT3, RNA was extracted, IL-9 real time RT-PCR was performed, and the mean threshold cycle (Ct) was determined. The data shown are from an experiment on one representative individual. The values are means of duplicate determinations. B. Gel electrophoresis. PBMC were incubated for 24 h with or without OKT3 and with or without Dex (10-6 M). RNA was extracted and IL-9 RT-PCR performed for 40 cycles. For each condition, duplicate PCRs were performed on cDNA from one representative individual. Products were analysed in a 2% agarose gel. The left lane contains HaeIII cut ΦX174 molecular size markers (Roche); the arrow indicates the position of the 281/271 bp markers.
Figure Legend Snippet: IL-9 RT-PCR. A. Time course. PBMC were incubated for various times with OKT3, RNA was extracted, IL-9 real time RT-PCR was performed, and the mean threshold cycle (Ct) was determined. The data shown are from an experiment on one representative individual. The values are means of duplicate determinations. B. Gel electrophoresis. PBMC were incubated for 24 h with or without OKT3 and with or without Dex (10-6 M). RNA was extracted and IL-9 RT-PCR performed for 40 cycles. For each condition, duplicate PCRs were performed on cDNA from one representative individual. Products were analysed in a 2% agarose gel. The left lane contains HaeIII cut ΦX174 molecular size markers (Roche); the arrow indicates the position of the 281/271 bp markers.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Incubation, Quantitative RT-PCR, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis

19) Product Images from "Induction of 4-1BB (CD137) expression by DNA damaging agents in human T lymphocytes"

Article Title: Induction of 4-1BB (CD137) expression by DNA damaging agents in human T lymphocytes

Journal: Immunology

doi: 10.1046/j.1365-2567.2002.01538.x

4-1BB mRNA expression is increased by anticancer drugs. Purified peripheral blood T cells from two healthy donors were cultured with bleomycin (60 µg/ml) or mitomycin C (1000 ng/ml) (a), doxorubicin (200 ng/ml) or Con A (10 µg/ml) (b). The cells were harvested at 0, 12, 24, 36, and 48 hr. The total RNA was extracted and analysed for relative levels of 4-1BB mRNA by RT–PCR as described in Materials and Methods. GAPDH was used to normalize for template quantity. Three-fold serial dilutions of cDNA at each time were subjected to PCR and equal amount of product for 4-1BB (368 bp) and GAPDH (500 bp) were resolved on 1·5% agarose gels stained with ethidium bromide. Data shown were the representative results from 15 independent experiments with the samples from five donors. (c) The first band intensity of each time point was divided by the corresponding GAPDH band intensity and the results were represented as intensity fold when 0 hr value in each reagent group was designated as 1.
Figure Legend Snippet: 4-1BB mRNA expression is increased by anticancer drugs. Purified peripheral blood T cells from two healthy donors were cultured with bleomycin (60 µg/ml) or mitomycin C (1000 ng/ml) (a), doxorubicin (200 ng/ml) or Con A (10 µg/ml) (b). The cells were harvested at 0, 12, 24, 36, and 48 hr. The total RNA was extracted and analysed for relative levels of 4-1BB mRNA by RT–PCR as described in Materials and Methods. GAPDH was used to normalize for template quantity. Three-fold serial dilutions of cDNA at each time were subjected to PCR and equal amount of product for 4-1BB (368 bp) and GAPDH (500 bp) were resolved on 1·5% agarose gels stained with ethidium bromide. Data shown were the representative results from 15 independent experiments with the samples from five donors. (c) The first band intensity of each time point was divided by the corresponding GAPDH band intensity and the results were represented as intensity fold when 0 hr value in each reagent group was designated as 1.

Techniques Used: Expressing, Purification, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining

20) Product Images from "Receptors for NPY and PACAP differ in expression and activity during adipogenesis in the murine 3T3-L1 fibroblast cell line"

Article Title: Receptors for NPY and PACAP differ in expression and activity during adipogenesis in the murine 3T3-L1 fibroblast cell line

Journal: British Journal of Pharmacology

doi: 10.1111/j.1476-5381.2009.00164.x

RT-PCR products of mouse brain mRNA for the PAC 1 variants. Upper row: Amplicons of 329 and 413 bp corresponding to the HIP-isoform of PAC 1 (left lane) and 259 bp band generated when using HOP-specific primers. Second row: Primers for HOP-cDNA produced
Figure Legend Snippet: RT-PCR products of mouse brain mRNA for the PAC 1 variants. Upper row: Amplicons of 329 and 413 bp corresponding to the HIP-isoform of PAC 1 (left lane) and 259 bp band generated when using HOP-specific primers. Second row: Primers for HOP-cDNA produced

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Generated, Produced

21) Product Images from "Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India"

Article Title: Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India

Journal: Journal of Mid-Life Health

doi: 10.4103/0976-7800.127786

L1 (My09/11) polymerase chain reaction products. Lane 1: 50 bp deoxyribonucleic acid ladder; lanes 2-7: Samples showing specific band for L1 at 450 bp; lane 8: L1 (–)ve sample; lane 9: Positive control; lane 10: Negative control
Figure Legend Snippet: L1 (My09/11) polymerase chain reaction products. Lane 1: 50 bp deoxyribonucleic acid ladder; lanes 2-7: Samples showing specific band for L1 at 450 bp; lane 8: L1 (–)ve sample; lane 9: Positive control; lane 10: Negative control

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

Representative electropherograms showing aligned sequences of L1 obtained by polymerase chain reaction with MY09/11 primers
Figure Legend Snippet: Representative electropherograms showing aligned sequences of L1 obtained by polymerase chain reaction with MY09/11 primers

Techniques Used: Polymerase Chain Reaction

22) Product Images from "Detection of Borrelia burgdorferi Nucleic Acids after Antibiotic Treatment Does Not Confirm Viability"

Article Title: Detection of Borrelia burgdorferi Nucleic Acids after Antibiotic Treatment Does Not Confirm Viability

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.02785-12

DNA extraction and PCR.
Figure Legend Snippet: DNA extraction and PCR.

Techniques Used: DNA Extraction, Polymerase Chain Reaction

23) Product Images from "Selective inhibition of cell growth by activin in SNU-16 cells"

Article Title: Selective inhibition of cell growth by activin in SNU-16 cells

Journal: World Journal of Gastroenterology : WJG

doi: 10.3748/wjg.v12.i19.3000

Effects of activin A on activin receptor mRNA expression (A), smad mRNA expression (B), and p21 CIP1/WAF1 mRNA expression (C) in SNU-16 cells. Cells (5 × 10 4 cells/mL) were cultured with activin A (100 ng/mL) in a time-dependent manner and mRNA levels were measured by RT-PCR. Values are mean ± SD of three individual experiments and reported as the ratio of activin receptors to β-actin signals, the ratio of Smad to β-actin signals, and the ratio of p21 CIP1/WAF1 to β-actin signals, respectively. a P
Figure Legend Snippet: Effects of activin A on activin receptor mRNA expression (A), smad mRNA expression (B), and p21 CIP1/WAF1 mRNA expression (C) in SNU-16 cells. Cells (5 × 10 4 cells/mL) were cultured with activin A (100 ng/mL) in a time-dependent manner and mRNA levels were measured by RT-PCR. Values are mean ± SD of three individual experiments and reported as the ratio of activin receptors to β-actin signals, the ratio of Smad to β-actin signals, and the ratio of p21 CIP1/WAF1 to β-actin signals, respectively. a P

Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

mRNA expressions of inhibin/activin subunits, activin receptors, Smads, and p21 CIP1/WAF1 in human gastric cancer cell lines. The mRNA levels were analyzed by RT-PCR. M: DNA size marker; P: positive control; lane 1: AGS; lane 2: KATO III; lane 3: SNU-1; lane 4: SNU-5; lane 5: SNU-16; lane 6: SNU-484; lane 7: SNU-601; lane 8: SNU-638; lane 9: SNU-668; lane 10: SNU-719. Positive control was used for inhibin/activin subunits (mouse ovary), activin receptor (K562), Smad and p21 CIP1/WAF1 (human keratinocytes) respectively.
Figure Legend Snippet: mRNA expressions of inhibin/activin subunits, activin receptors, Smads, and p21 CIP1/WAF1 in human gastric cancer cell lines. The mRNA levels were analyzed by RT-PCR. M: DNA size marker; P: positive control; lane 1: AGS; lane 2: KATO III; lane 3: SNU-1; lane 4: SNU-5; lane 5: SNU-16; lane 6: SNU-484; lane 7: SNU-601; lane 8: SNU-638; lane 9: SNU-668; lane 10: SNU-719. Positive control was used for inhibin/activin subunits (mouse ovary), activin receptor (K562), Smad and p21 CIP1/WAF1 (human keratinocytes) respectively.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control

24) Product Images from "Role of T cell Transforming Growth Factor ? Signaling and IL-17 in Allograft Acceptance and Fibrosis Associated with Chronic Rejection 1"

Article Title: Role of T cell Transforming Growth Factor ? Signaling and IL-17 in Allograft Acceptance and Fibrosis Associated with Chronic Rejection 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0902446

In recipients that fail to produce IL-17, allografts are protected from fibrosis (A) WT (squares) and IL-17-/- (circles) mice were transplanted with BALB/c cardiac allografts and were either left untreated (closed symbols) or transiently depleted of CD4+ cells (open symbols). Graft function was monitored by palpation. Unmodified recipients were harvested at time of rejection, while inductive anti-CD4 mAb treated recipients were harvested at day 50 post-transplantation. Numbers in parentheses represent the number of recipients in each group. (B) Sections of grafts from recipients transiently depleted of CD4+ T cells (day 50 post-transplant) were stained with Masson's trichrome stain. Frames are of grafts from WT and IL-17-/- recipients and are representative of at least 6 WT and 9 IL-17-/- recipient allografts. 200× magnification. (C) Quantification of fibrosis by morphometric analysis. Bars represent the average percentage (+/- S.E.M.) of graft area positive for fibrosis in at least 5 WT and IL-17-/- recipients treated with anti-CD4 mAb. WT (open bars) and IL-17-/- (closed bars). (D) On day 50 post-transplant, RNA was harvested from allografts of WT and IL-17-/- recipients transiently depleted of CD4+ T cells. Intragraft transcript levels of FoxP3 and IL-17 were assessed by real-time RT-PCR. Bars depict the means of RNA from 5 WT and 9 IL-17-/- grafts.
Figure Legend Snippet: In recipients that fail to produce IL-17, allografts are protected from fibrosis (A) WT (squares) and IL-17-/- (circles) mice were transplanted with BALB/c cardiac allografts and were either left untreated (closed symbols) or transiently depleted of CD4+ cells (open symbols). Graft function was monitored by palpation. Unmodified recipients were harvested at time of rejection, while inductive anti-CD4 mAb treated recipients were harvested at day 50 post-transplantation. Numbers in parentheses represent the number of recipients in each group. (B) Sections of grafts from recipients transiently depleted of CD4+ T cells (day 50 post-transplant) were stained with Masson's trichrome stain. Frames are of grafts from WT and IL-17-/- recipients and are representative of at least 6 WT and 9 IL-17-/- recipient allografts. 200× magnification. (C) Quantification of fibrosis by morphometric analysis. Bars represent the average percentage (+/- S.E.M.) of graft area positive for fibrosis in at least 5 WT and IL-17-/- recipients treated with anti-CD4 mAb. WT (open bars) and IL-17-/- (closed bars). (D) On day 50 post-transplant, RNA was harvested from allografts of WT and IL-17-/- recipients transiently depleted of CD4+ T cells. Intragraft transcript levels of FoxP3 and IL-17 were assessed by real-time RT-PCR. Bars depict the means of RNA from 5 WT and 9 IL-17-/- grafts.

Techniques Used: Mouse Assay, Transplantation Assay, Staining, Quantitative RT-PCR

Reduction of TGFβ-dependent intragraft gene expression from CD4-DNR recipients transiently depleted of CD4+ T cells RNA was harvested from allografts of WT and CD4-DNR recipients transiently depleted of CD4+ T cells. Intragraft transcript levels of FoxP3 and IL-17 were assessed by real-time RT-PCR. Allografts were recovered between days 35-40 post-transplant. Bars depict the means of RNA expression from 6 WT and 9 CD4-DNR grafts.
Figure Legend Snippet: Reduction of TGFβ-dependent intragraft gene expression from CD4-DNR recipients transiently depleted of CD4+ T cells RNA was harvested from allografts of WT and CD4-DNR recipients transiently depleted of CD4+ T cells. Intragraft transcript levels of FoxP3 and IL-17 were assessed by real-time RT-PCR. Allografts were recovered between days 35-40 post-transplant. Bars depict the means of RNA expression from 6 WT and 9 CD4-DNR grafts.

Techniques Used: Expressing, Quantitative RT-PCR, RNA Expression

Related Articles

Clone Assay:

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template. .. The gene-specific PCR products were originally generated from plasmid clones or from genomic DNA by two cycles of PCR.

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. Cloning of VacA gene in bacterial expression vector and transformation The PCR product was purified from the agarose gel by high pure PCR product purification kit (Roche, Germany) according to the manufacturer guideline.

Amplification:

Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression
Article Snippet: Paragraph title: RT-PCR Amplification of Nvtra Transcript ... One l 1∶100 diluted cDNA was mixed with 10x PCR buffer (Roche) (10x concentrate; 100 mM Tris-HCl, 500 mM KCl; pH 8.3), 200 M dNTPs, 2 units Taq polymerase (Roche), 400 nM of primers NvTra_poly_F1 and NvTra_poly_R1 and supplemented with milliQ water to 25 l. PCR profile was as follows: initial denaturing step of 95 C for 3 min, followed by 20 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s, ending with a final extension step of 72 C for 7 min. As negative control one l of milliQ water was used.

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template. .. In the first cycle, a larger fragment was amplified, which was purified from agarose gels and used as the template for a second PCR to generate gene-specific probes.

Article Title: Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿
Article Snippet: .. Each amplification reaction mixture contained ∼5 ng/μl DNA, 0.3 μM of each primer, 250 μM deoxynucleoside triphosphates (dNTPs; Fermantas, Nunningen, Switzerland), 0.04 U/μl Taq DNA polymerase (Roche Molecular Biochemicals, Manheim, Germany), and PCR buffer with MgCl2 (Roche). .. Cycling conditions consisted of 35 cycles at 94°C for 45 s, 60°C (TEF), 53°C (MtSSU), 50°C (IGS), or 59°C (MtR) for 45 s and 72°C for 90 s. Each PCR was preceded by an initial denaturation step at 94°C for 2 min and concluded with a final extension step at 72°C for 5 min. PCR products were purified using the High Pure PCR product purification kit (Roche Applied Biochemicals) and sequenced using the Big Dye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 377 automated sequencer (Applied Biosystems).

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

Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents
Article Snippet: Each exon was amplified with polymerase chain reaction (PCR) amplification using specific primer pairs, as previously reported [ ]. .. PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche).

Article Title: Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †
Article Snippet: Paragraph title: PCR amplification for DGGE community fingerprinting. ... Briefly, each 25-μl PCR mixture used for bacterial PCR-denaturing gradient gel electrophoresis (DGGE) analysis contained 5 μl PCR buffer [60 mM Tris-HCl, 15 mM (NH4 )2 SO4 , 5.5 mM MgCl2 ; pH 9.0], 0.5 μl formamide, 0.5 μg T4 gene 32 protein (Roche, Almere, Netherlands), 10 nmol of each deoxyribonucleoside triphosphate, 10 pmol of each primer (GC-341F and 518R) , and 5 U of AmpliTaq DNA polymerase (Stoffel fragment; Applied Biosystems, Foster City, CA) in pure water.

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: The part of ssurRNA gene was amplified by PCR by a pair of Primers, Primer 1L, Forward, CCA TGC ATG TCT AAG TAT AAG C and Primer 3H, Reverse, GGC AAA TGC TTT CGC AGT AG (BIONEER, Korea) ( ). .. One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl.

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: For unmethylated DNA amplification, the following unmethylated MSP (UMSP) primers were used: UMSP- galanin -F (5′-TGA TGT GAT TTT GGG TGG TT-3′) and UMSP- galanin -R (5′-TAT CCA CCA CCC AAT ATA AC-3′). .. MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl.

Positive Control:

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. DNA was extracted from macro-cysts of S. moulei isolated from infected goat sample used as positive control and water were used as negative control.

Construct:

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. Forward primer was used to construct a continuous sequence of the inserted DNA.

Enzyme-linked Immunosorbent Assay:

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: PBMCs or SupT1 cells were infected with equivalent amounts of virus as determined by a HIV-1 antigen (p24 CA) Micro ELISA assay. .. Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche).

Modification:

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: Paragraph title: Bisulfite Modification and Methylation-Specific Polymerase Chain Reaction Analysis ... MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl.

Incubation:

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche). .. Samples were incubated at 56°C for 1 h before proteinase K was inactivated at 95°C for 10 min, samples were then stored at –20°C.

Formalin-fixed Paraffin-Embedded:

Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents
Article Snippet: Briefly, FFPE slices (three 10-μm-thick slices for each sample) were digested overnight at 56 °C in ATL buffer with the addition of proteinase K (Qiagen). .. PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche).

Activity Assay:

Article Title: Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site
Article Snippet: The competitor- and target-derived MDR1 amplicons were digested in the PCR buffer using the restriction enzyme Pvu II (Roche). .. Most of the restriction endonucleases revealed excellent enzymatic activity in PCR buffer.

Expressing:

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. Cloning of VacA gene in bacterial expression vector and transformation The PCR product was purified from the agarose gel by high pure PCR product purification kit (Roche, Germany) according to the manufacturer guideline.

Acrylamide Gel Assay:

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl. .. The 82-bp PCR products were separated by electrophoresis through a 9% acrylamide gel, stained with ethidium bromide.

Real-time Polymerase Chain Reaction:

Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression
Article Snippet: From the eight sample classes each replicate was used four times in a PCR reaction with similar parameters as described for the qPCR. .. One l 1∶100 diluted cDNA was mixed with 10x PCR buffer (Roche) (10x concentrate; 100 mM Tris-HCl, 500 mM KCl; pH 8.3), 200 M dNTPs, 2 units Taq polymerase (Roche), 400 nM of primers NvTra_poly_F1 and NvTra_poly_R1 and supplemented with milliQ water to 25 l. PCR profile was as follows: initial denaturing step of 95 C for 3 min, followed by 20 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s, ending with a final extension step of 72 C for 7 min. As negative control one l of milliQ water was used.

Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1
Article Snippet: Five μg of total RNA were reverse transcribed using 10× PCR buffer (Roche), 10 mM dNTPs, Oligo (dT), M-MLV-RT (all from Invitrogen), and RNAsin (Promega). .. Real-time PCR was performed on cDNA using a Rotor-Gene 3000 ™ (Corbett Life Science, San Francisco, CA).

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: Paragraph title: Real-time PCR ... Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche).

Transformation Assay:

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. Cloning of VacA gene in bacterial expression vector and transformation The PCR product was purified from the agarose gel by high pure PCR product purification kit (Roche, Germany) according to the manufacturer guideline.

Hybridization:

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: .. The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template. .. The gene-specific PCR products were originally generated from plasmid clones or from genomic DNA by two cycles of PCR.

Countercurrent Chromatography:

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: For unmethylated DNA amplification, the following unmethylated MSP (UMSP) primers were used: UMSP- galanin -F (5′-TGA TGT GAT TTT GGG TGG TT-3′) and UMSP- galanin -R (5′-TAT CCA CCA CCC AAT ATA AC-3′). .. MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl.

Transfection:

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: Concentrated virus stocks were treated with 50 U/ml of DNase (Roche) for 15 min at 37°C before infection to remove any contaminating plasmid DNA from the transfection procedure. .. Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche).

Northern Blot:

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: Paragraph title: RNA isolation and Northern hybridizations. ... The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template.

Infection:

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: Concentrated virus stocks were treated with 50 U/ml of DNase (Roche) for 15 min at 37°C before infection to remove any contaminating plasmid DNA from the transfection procedure. .. Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche).

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. DNA was extracted from macro-cysts of S. moulei isolated from infected goat sample used as positive control and water were used as negative control.

Reverse Transcription Polymerase Chain Reaction:

Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression
Article Snippet: Paragraph title: RT-PCR Amplification of Nvtra Transcript ... One l 1∶100 diluted cDNA was mixed with 10x PCR buffer (Roche) (10x concentrate; 100 mM Tris-HCl, 500 mM KCl; pH 8.3), 200 M dNTPs, 2 units Taq polymerase (Roche), 400 nM of primers NvTra_poly_F1 and NvTra_poly_R1 and supplemented with milliQ water to 25 l. PCR profile was as follows: initial denaturing step of 95 C for 3 min, followed by 20 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s, ending with a final extension step of 72 C for 7 min. As negative control one l of milliQ water was used.

Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1
Article Snippet: Paragraph title: RNA isolation and RT-PCR ... Five μg of total RNA were reverse transcribed using 10× PCR buffer (Roche), 10 mM dNTPs, Oligo (dT), M-MLV-RT (all from Invitrogen), and RNAsin (Promega).

Light Microscopy:

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: The sections were examined with a light microscope at 10 and 40ҳ magnifications. .. One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl.

Generated:

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template. .. The gene-specific PCR products were originally generated from plasmid clones or from genomic DNA by two cycles of PCR.

Droplet Countercurrent Chromatography:

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl. .. Previously described primers and conditions were used to analyze the methylation status of the GALR1 gene [ ], p16 gene [ ], RASSF1A gene [ ], MGMT gene [ ], DAPK gene [ ], and DCC gene [ ].

Polymerase Chain Reaction:

Article Title: T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires
Article Snippet: .. Cells gated as pCW3Kd+ Vβ10+ CD8+ were sorted as single cells into tubes containing 20 μl 1× PCR buffer (Roche) and 4 μg/ml 16S rRNA (Roche). .. After proteinase K digestion , a first PCR reaction that amplifies rearrangements of Vβ10, D1, or D2 gene segments to any J1 or J2 segment, or those of Vα3, Vα4, or Vα8 genes to Jα35 was performed.

Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression
Article Snippet: .. One l 1∶100 diluted cDNA was mixed with 10x PCR buffer (Roche) (10x concentrate; 100 mM Tris-HCl, 500 mM KCl; pH 8.3), 200 M dNTPs, 2 units Taq polymerase (Roche), 400 nM of primers NvTra_poly_F1 and NvTra_poly_R1 and supplemented with milliQ water to 25 l. PCR profile was as follows: initial denaturing step of 95 C for 3 min, followed by 20 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s, ending with a final extension step of 72 C for 7 min. As negative control one l of milliQ water was used. .. Since the amount of product was too low to visualize on gel, all six replicates for each sample class were pooled and one l of this was used in a similar PCR regime as above but with 40 cycles.

Article Title: Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor
Article Snippet: .. The DNA sample and primer mixture was complemented with an optimal concentration of PCR Buffer containing 2 mM MgCl2 , PCR grade deoxyribonucleoside triphosphates / dNTP mix (10 mM of each nucleotide), 2U of FastStart Taq DNA Polymerase (Roche, Germany), and nuclease free water. .. Amplification was performed with an Applied Biosystems® Veriti® 96-Well Thermal Cycler (USA).

Article Title: Small Variant STEVOR Antigen Is Uniquely Located within Maurer's Clefts in Plasmodium falciparum-Infected Red Blood Cells
Article Snippet: .. The PCR mixture used with primers smkf1 and smkr1 contained 1 μM each primer, 5 μl of PCR buffer (100 mM Tris-HCl, 500 mM KCl), 2 mM MgCl2 (Roche), 1 mM each deoxynucleoside triphosphate (Amersham Pharmacia Biotech), and 5 U of Ampli Taq polymerase (Roche) in a volume of 50 μl. .. The PCR program was 1 cycle of 94°C for 3 min; 35 cycles of 94°C for 1 min, 62.5°C for 1 min, and 72°C for 1 min 30 s; and finally one 10-min cycle at 72°C.

Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1
Article Snippet: .. Five μg of total RNA were reverse transcribed using 10× PCR buffer (Roche), 10 mM dNTPs, Oligo (dT), M-MLV-RT (all from Invitrogen), and RNAsin (Promega). ..

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: .. The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template. .. The gene-specific PCR products were originally generated from plasmid clones or from genomic DNA by two cycles of PCR.

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: .. Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche). .. Samples were incubated at 56°C for 1 h before proteinase K was inactivated at 95°C for 10 min, samples were then stored at –20°C.

Article Title: Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿
Article Snippet: .. Each amplification reaction mixture contained ∼5 ng/μl DNA, 0.3 μM of each primer, 250 μM deoxynucleoside triphosphates (dNTPs; Fermantas, Nunningen, Switzerland), 0.04 U/μl Taq DNA polymerase (Roche Molecular Biochemicals, Manheim, Germany), and PCR buffer with MgCl2 (Roche). .. Cycling conditions consisted of 35 cycles at 94°C for 45 s, 60°C (TEF), 53°C (MtSSU), 50°C (IGS), or 59°C (MtR) for 45 s and 72°C for 90 s. Each PCR was preceded by an initial denaturation step at 94°C for 2 min and concluded with a final extension step at 72°C for 5 min. PCR products were purified using the High Pure PCR product purification kit (Roche Applied Biochemicals) and sequenced using the Big Dye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 377 automated sequencer (Applied Biosystems).

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

Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents
Article Snippet: .. PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche). .. PCR conditions were an initial denaturation of 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 52–64 °C for 30 s, and 72 °C for 30 s. PCR products were purified with the Qiaquick PCR purification kit (Qiagen) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems).

Article Title: Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †
Article Snippet: .. Briefly, each 25-μl PCR mixture used for bacterial PCR-denaturing gradient gel electrophoresis (DGGE) analysis contained 5 μl PCR buffer [60 mM Tris-HCl, 15 mM (NH4 )2 SO4 , 5.5 mM MgCl2 ; pH 9.0], 0.5 μl formamide, 0.5 μg T4 gene 32 protein (Roche, Almere, Netherlands), 10 nmol of each deoxyribonucleoside triphosphate, 10 pmol of each primer (GC-341F and 518R) , and 5 U of AmpliTaq DNA polymerase (Stoffel fragment; Applied Biosystems, Foster City, CA) in pure water. .. After addition of about 5 ng of template DNA, the mixtures were placed in a GeneAmp PCR system 9700 cycler (Applied Biosystems, Foster City, CA), and thermal cycling was performed as follows: initial denaturation for 5 min at 94°C, followed by 35 cycles consisting of 1 min at 94°C, 1 min at 57°C, and 3 min at 72°C and then extension for 30 min at 72°C.

Article Title: Quantification of mRNA expression by competitive PCR using non-homologous competitors containing a shifted restriction site
Article Snippet: .. The competitor- and target-derived MDR1 amplicons were digested in the PCR buffer using the restriction enzyme Pvu II (Roche). .. To test the applicability of this approach to a broad spectrum of target sequences, we have investigated a number of restriction endonucleases for their ability to cut efficiently in PCR buffer.

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: .. One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. The PCR reactions were performed using Thermal Cycler (Bio-Rad, USA) using the following PCR protocol: initial hot start at 93 °C for 2 min, followed by 93 °C for 45 s, 55 °C for 45 s, 72 °C for 2 min; and the final extension at 72 °C for 10 min. PCR products were visualized by UV after electrophoresis on 1% agarose gel stained with Ethidium bromide.

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: .. MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl. ..

Binding Assay:

Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1
Article Snippet: Five μg of total RNA were reverse transcribed using 10× PCR buffer (Roche), 10 mM dNTPs, Oligo (dT), M-MLV-RT (all from Invitrogen), and RNAsin (Promega). .. Primer binding to DNA was detected by SYBR Green I™ dye (Roche, Indianapolis, IN).

DNA Extraction:

Article Title: Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿
Article Snippet: Paragraph title: DNA isolation, PCR amplification, and sequencing. ... Each amplification reaction mixture contained ∼5 ng/μl DNA, 0.3 μM of each primer, 250 μM deoxynucleoside triphosphates (dNTPs; Fermantas, Nunningen, Switzerland), 0.04 U/μl Taq DNA polymerase (Roche Molecular Biochemicals, Manheim, Germany), and PCR buffer with MgCl2 (Roche).

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: Chromosomal DNA isolation Genomic DNA of H. pylori was extracted from the colonies on the Brucella agar plates according to the standard CTAB/NaCl protocol ( ). .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany).

Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents
Article Snippet: DNA extraction was then continued with QIAamp DNA micro kit (Qiagen). .. PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche).

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: DNA extraction and PCR analysis 10 ml of the filtrated sample was centrifuged at 8000 rpm for 5 min. .. One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl.

Nucleic Acid Electrophoresis:

Article Title: Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †
Article Snippet: .. Briefly, each 25-μl PCR mixture used for bacterial PCR-denaturing gradient gel electrophoresis (DGGE) analysis contained 5 μl PCR buffer [60 mM Tris-HCl, 15 mM (NH4 )2 SO4 , 5.5 mM MgCl2 ; pH 9.0], 0.5 μl formamide, 0.5 μg T4 gene 32 protein (Roche, Almere, Netherlands), 10 nmol of each deoxyribonucleoside triphosphate, 10 pmol of each primer (GC-341F and 518R) , and 5 U of AmpliTaq DNA polymerase (Stoffel fragment; Applied Biosystems, Foster City, CA) in pure water. .. After addition of about 5 ng of template DNA, the mixtures were placed in a GeneAmp PCR system 9700 cycler (Applied Biosystems, Foster City, CA), and thermal cycling was performed as follows: initial denaturation for 5 min at 94°C, followed by 35 cycles consisting of 1 min at 94°C, 1 min at 57°C, and 3 min at 72°C and then extension for 30 min at 72°C.

Methylation:

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: Paragraph title: Bisulfite Modification and Methylation-Specific Polymerase Chain Reaction Analysis ... MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl.

Isolation:

Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1
Article Snippet: Paragraph title: RNA isolation and RT-PCR ... Five μg of total RNA were reverse transcribed using 10× PCR buffer (Roche), 10 mM dNTPs, Oligo (dT), M-MLV-RT (all from Invitrogen), and RNAsin (Promega).

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: Paragraph title: RNA isolation and Northern hybridizations. ... The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template.

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. DNA was extracted from macro-cysts of S. moulei isolated from infected goat sample used as positive control and water were used as negative control.

Purification:

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template. .. In the first cycle, a larger fragment was amplified, which was purified from agarose gels and used as the template for a second PCR to generate gene-specific probes.

Article Title: Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿
Article Snippet: Each amplification reaction mixture contained ∼5 ng/μl DNA, 0.3 μM of each primer, 250 μM deoxynucleoside triphosphates (dNTPs; Fermantas, Nunningen, Switzerland), 0.04 U/μl Taq DNA polymerase (Roche Molecular Biochemicals, Manheim, Germany), and PCR buffer with MgCl2 (Roche). .. Cycling conditions consisted of 35 cycles at 94°C for 45 s, 60°C (TEF), 53°C (MtSSU), 50°C (IGS), or 59°C (MtR) for 45 s and 72°C for 90 s. Each PCR was preceded by an initial denaturation step at 94°C for 2 min and concluded with a final extension step at 72°C for 5 min. PCR products were purified using the High Pure PCR product purification kit (Roche Applied Biochemicals) and sequenced using the Big Dye Terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 377 automated sequencer (Applied Biosystems).

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: Quality and quantity of the purified genomic DNA was assessed by 0.8% horizontal agarose gel electrophoresis in 1X TBE buffer (10X TBE: 890 mM Tris-base, 890 mM Boric acid, 25 mM EDTA) and visualized by ethidium bromide (1 µg/ml) staining on UV transilluminator, and spectrophotometry (eppendorf) in 260 nm( ). .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany).

Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents
Article Snippet: PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche). .. PCR conditions were an initial denaturation of 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 52–64 °C for 30 s, and 72 °C for 30 s. PCR products were purified with the Qiaquick PCR purification kit (Qiagen) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems).

Sequencing:

Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression
Article Snippet: All fragments are confirmed by Sanger sequencing and can be distinguished on a 2% non-denaturing agarose gel containing 0.5 g/ml ethidium bromide. .. One l 1∶100 diluted cDNA was mixed with 10x PCR buffer (Roche) (10x concentrate; 100 mM Tris-HCl, 500 mM KCl; pH 8.3), 200 M dNTPs, 2 units Taq polymerase (Roche), 400 nM of primers NvTra_poly_F1 and NvTra_poly_R1 and supplemented with milliQ water to 25 l. PCR profile was as follows: initial denaturing step of 95 C for 3 min, followed by 20 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s, ending with a final extension step of 72 C for 7 min. As negative control one l of milliQ water was used.

Article Title: Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups ▿
Article Snippet: Paragraph title: DNA isolation, PCR amplification, and sequencing. ... Each amplification reaction mixture contained ∼5 ng/μl DNA, 0.3 μM of each primer, 250 μM deoxynucleoside triphosphates (dNTPs; Fermantas, Nunningen, Switzerland), 0.04 U/μl Taq DNA polymerase (Roche Molecular Biochemicals, Manheim, Germany), and PCR buffer with MgCl2 (Roche).

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: Detection of antigenic region Since the most prevalent VacA genotype of H. pylori among Iranian strains is s1m2 , in order to find out antigenic region, the sequence of VacA gene with s1m2 genotype (4243 base pair, 1323 amino acids) which encodes the 142.973 Kilodaltons (KDa) protein, from a reference strain (NCBI GenBank, Accession number: U95971, protein id: AAC25911.1) was submitted to ABCpred, Bcepred and Emboss Antigenic web servers ( ). .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany).

Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents
Article Snippet: The exons of the subunits of SDH complex were sequenced on tumor and normal tissue using the Sanger sequencing method on ABI 310 Genetic Analyzer (Applied Biosystems). .. PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche).

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. Forward primer was used to construct a continuous sequence of the inserted DNA.

Lysis:

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: .. Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche). .. Samples were incubated at 56°C for 1 h before proteinase K was inactivated at 95°C for 10 min, samples were then stored at –20°C.

Nested PCR:

Article Title: Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor
Article Snippet: Paragraph title: HPV Detection Using Nested PCR ... The DNA sample and primer mixture was complemented with an optimal concentration of PCR Buffer containing 2 mM MgCl2 , PCR grade deoxyribonucleoside triphosphates / dNTP mix (10 mM of each nucleotide), 2U of FastStart Taq DNA Polymerase (Roche, Germany), and nuclease free water.

Article Title: Small Variant STEVOR Antigen Is Uniquely Located within Maurer's Clefts in Plasmodium falciparum-Infected Red Blood Cells
Article Snippet: The PCR mixture used with primers smkf1 and smkr1 contained 1 μM each primer, 5 μl of PCR buffer (100 mM Tris-HCl, 500 mM KCl), 2 mM MgCl2 (Roche), 1 mM each deoxynucleoside triphosphate (Amersham Pharmacia Biotech), and 5 U of Ampli Taq polymerase (Roche) in a volume of 50 μl. .. A 2-μl aliquot from the first reaction was directly transferred to the nested PCR mixture, which contained 0.5 μM each RepF1 and RepF2, 1 μM RepR, 2.5 μl of PCR buffer, 3 mM MgCl2 , 1 mM each deoxynucleoside triphosphate, and 2.5 U of Ampli Taq polymerase.

Article Title: Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †
Article Snippet: Briefly, each 25-μl PCR mixture used for bacterial PCR-denaturing gradient gel electrophoresis (DGGE) analysis contained 5 μl PCR buffer [60 mM Tris-HCl, 15 mM (NH4 )2 SO4 , 5.5 mM MgCl2 ; pH 9.0], 0.5 μl formamide, 0.5 μg T4 gene 32 protein (Roche, Almere, Netherlands), 10 nmol of each deoxyribonucleoside triphosphate, 10 pmol of each primer (GC-341F and 518R) , and 5 U of AmpliTaq DNA polymerase (Stoffel fragment; Applied Biosystems, Foster City, CA) in pure water. .. A nested PCR approach was used for amplification of the betaproteobacterial community DNA.

Plasmid Preparation:

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template. .. The gene-specific PCR products were originally generated from plasmid clones or from genomic DNA by two cycles of PCR.

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: A heat-inactivated virus control (2 h at 56°C) was used to confirm the level of any residual plasmid DNA for each sample. .. Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche).

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. Cloning of VacA gene in bacterial expression vector and transformation The PCR product was purified from the agarose gel by high pure PCR product purification kit (Roche, Germany) according to the manufacturer guideline.

Software:

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: Gene amplification Two specific PCR primers were designed with Oligo5 software. .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany).

SYBR Green Assay:

Article Title: CD8+ Th17 Mediate Costimulation Blockade Resistant Allograft Rejection in T-bet Deficient Mice 1
Article Snippet: Five μg of total RNA were reverse transcribed using 10× PCR buffer (Roche), 10 mM dNTPs, Oligo (dT), M-MLV-RT (all from Invitrogen), and RNAsin (Promega). .. Primer binding to DNA was detected by SYBR Green I™ dye (Roche, Indianapolis, IN).

Article Title: Primary T-lymphocytes rescue the replication of HIV-1 DIS RNA mutants in part by facilitating reverse transcription
Article Snippet: Twenty-four hours postinfection cells were pelleted and lysed in PCR lysis buffer containing 1× PCR buffer (Roche) with 0.5% vol/vol Triton-X100, 0.5% vol/vol NP-40 and 75 µg/ml proteinase K (Roche). .. Each PCR reaction contained 1× SYBR Green I Master mix (BioRad), 400 nM each primer and 2.5 µl cell lysate in a 25 µl reaction volume.

Negative Control:

Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression
Article Snippet: .. One l 1∶100 diluted cDNA was mixed with 10x PCR buffer (Roche) (10x concentrate; 100 mM Tris-HCl, 500 mM KCl; pH 8.3), 200 M dNTPs, 2 units Taq polymerase (Roche), 400 nM of primers NvTra_poly_F1 and NvTra_poly_R1 and supplemented with milliQ water to 25 l. PCR profile was as follows: initial denaturing step of 95 C for 3 min, followed by 20 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s, ending with a final extension step of 72 C for 7 min. As negative control one l of milliQ water was used. .. Since the amount of product was too low to visualize on gel, all six replicates for each sample class were pooled and one l of this was used in a similar PCR regime as above but with 40 cycles.

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. DNA was extracted from macro-cysts of S. moulei isolated from infected goat sample used as positive control and water were used as negative control.

Denaturing Gradient Gel Electrophoresis:

Article Title: Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields ▿ †
Article Snippet: .. Briefly, each 25-μl PCR mixture used for bacterial PCR-denaturing gradient gel electrophoresis (DGGE) analysis contained 5 μl PCR buffer [60 mM Tris-HCl, 15 mM (NH4 )2 SO4 , 5.5 mM MgCl2 ; pH 9.0], 0.5 μl formamide, 0.5 μg T4 gene 32 protein (Roche, Almere, Netherlands), 10 nmol of each deoxyribonucleoside triphosphate, 10 pmol of each primer (GC-341F and 518R) , and 5 U of AmpliTaq DNA polymerase (Stoffel fragment; Applied Biosystems, Foster City, CA) in pure water. .. After addition of about 5 ng of template DNA, the mixtures were placed in a GeneAmp PCR system 9700 cycler (Applied Biosystems, Foster City, CA), and thermal cycling was performed as follows: initial denaturation for 5 min at 94°C, followed by 35 cycles consisting of 1 min at 94°C, 1 min at 57°C, and 3 min at 72°C and then extension for 30 min at 72°C.

Agarose Gel Electrophoresis:

Article Title: A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression
Article Snippet: All fragments are confirmed by Sanger sequencing and can be distinguished on a 2% non-denaturing agarose gel containing 0.5 g/ml ethidium bromide. .. One l 1∶100 diluted cDNA was mixed with 10x PCR buffer (Roche) (10x concentrate; 100 mM Tris-HCl, 500 mM KCl; pH 8.3), 200 M dNTPs, 2 units Taq polymerase (Roche), 400 nM of primers NvTra_poly_F1 and NvTra_poly_R1 and supplemented with milliQ water to 25 l. PCR profile was as follows: initial denaturing step of 95 C for 3 min, followed by 20 cycles of 95 C for 15 s, 57 C for 30 s and 72 C for 30 s, ending with a final extension step of 72 C for 7 min. As negative control one l of milliQ water was used.

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: Quality and quantity of the purified genomic DNA was assessed by 0.8% horizontal agarose gel electrophoresis in 1X TBE buffer (10X TBE: 890 mM Tris-base, 890 mM Boric acid, 25 mM EDTA) and visualized by ethidium bromide (1 µg/ml) staining on UV transilluminator, and spectrophotometry (eppendorf) in 260 nm( ). .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany).

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. The PCR reactions were performed using Thermal Cycler (Bio-Rad, USA) using the following PCR protocol: initial hot start at 93 °C for 2 min, followed by 93 °C for 45 s, 55 °C for 45 s, 72 °C for 2 min; and the final extension at 72 °C for 10 min. PCR products were visualized by UV after electrophoresis on 1% agarose gel stained with Ethidium bromide.

Electrophoresis:

Article Title: Control of Photosynthetic and High-Light-Responsive Genes by the Histidine Kinase DspA: Negative and Positive Regulation and Interactions between Signal Transduction Pathways
Article Snippet: For RNA blot hybridizations, equal amounts of RNA (determined spectroscopically) were resolved by electrophoresis in formaldehyde gels; ethidium bromide was included in the loading buffer, allowing for visualization of the rRNA bands and confirmation of equal loading of RNA samples. .. The DNA probes used for hybridization were prepared by PCR in a 15-μl reaction mixture containing PCR buffer, 2 U of Taq DNA polymerase (Roche, Palo Alto, Calif.), 0.1 μg each of the two PCR primers, 30 μCi of [32 P]dCTP (3,000 Ci/mmol, 10 μCi/μl; NEN-DuPont, Boston, Mass.), and ∼10 ng of gene-specific PCR products as a template.

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl. .. The PCR reactions were performed using Thermal Cycler (Bio-Rad, USA) using the following PCR protocol: initial hot start at 93 °C for 2 min, followed by 93 °C for 45 s, 55 °C for 45 s, 72 °C for 2 min; and the final extension at 72 °C for 10 min. PCR products were visualized by UV after electrophoresis on 1% agarose gel stained with Ethidium bromide.

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl. .. The 82-bp PCR products were separated by electrophoresis through a 9% acrylamide gel, stained with ethidium bromide.

Spectrophotometry:

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: Quality and quantity of the purified genomic DNA was assessed by 0.8% horizontal agarose gel electrophoresis in 1X TBE buffer (10X TBE: 890 mM Tris-base, 890 mM Boric acid, 25 mM EDTA) and visualized by ethidium bromide (1 µg/ml) staining on UV transilluminator, and spectrophotometry (eppendorf) in 260 nm( ). .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany).

Concentration Assay:

Article Title: Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor
Article Snippet: .. The DNA sample and primer mixture was complemented with an optimal concentration of PCR Buffer containing 2 mM MgCl2 , PCR grade deoxyribonucleoside triphosphates / dNTP mix (10 mM of each nucleotide), 2U of FastStart Taq DNA Polymerase (Roche, Germany), and nuclease free water. .. Amplification was performed with an Applied Biosystems® Veriti® 96-Well Thermal Cycler (USA).

DNA Purification:

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). .. MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl.

Staining:

Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
Article Snippet: Quality and quantity of the purified genomic DNA was assessed by 0.8% horizontal agarose gel electrophoresis in 1X TBE buffer (10X TBE: 890 mM Tris-base, 890 mM Boric acid, 25 mM EDTA) and visualized by ethidium bromide (1 µg/ml) staining on UV transilluminator, and spectrophotometry (eppendorf) in 260 nm( ). .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany).

Article Title: Sarcocystis cruzi: First Molecular Identification from Cattle in Iran
Article Snippet: Histology Meat sample fixed in 10% formalin were processed as usual, sectioned at 7 µm and stained with haematoxylin and eosin (HE). .. One reaction mixture contained 3 µl of the DNA solution, 20 µl of dH2 O, 3 µl of 10x PCR buffer (Roche, Germany), 0.5 µl of dNTP (Roche, Germany), 0.5 µl of Tag polymrase (Roche, Germany), 0.5 µl of each primer (20 pmol), 0.5 µl of MgCl 2 and RNase-free water to make a final volume of 30 µl.

Article Title: Galanin Has Tumor Suppressor Activity and Is Frequently Inactivated by Aberrant Promoter Methylation in Head and Neck Cancer 1
Article Snippet: MSP/UMSP was performed with 25 ng of DNA solution containing 1x PCR buffer, 10x Mg, 10 mM deoxyribonucleotide triphosphate (dNTP), 3% DMSO, 1U Fast Start Polymerase (Roche, Penzberg, Germany), and 10 µM of each primer for a total volume of 25 µl. .. The 82-bp PCR products were separated by electrophoresis through a 9% acrylamide gel, stained with ethidium bromide.

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    Roche pcr buffer
    Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: <t>HPV16</t> (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control
    Pcr Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: HPV16 (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control

    Journal: Journal of Mid-Life Health

    Article Title: Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India

    doi: 10.4103/0976-7800.127786

    Figure Lengend Snippet: Human papillomavirus (HPV)16 type-specific polymerase chain reaction products. Lane 4: 50 bp ladder; lanes 3, 5-8: HPV16 (+) ve samples showing specific band size at 116 bp; lane 9: HPV16 (–)ve sample; lane 2: Positive control; lane 1: Negative control

    Article Snippet: HPV TS-PCR The HPV16-specific PCR mixture (20 μl) contained ×10 PCR buffer, 2 mM MgCl2 , 200 μM dNTP, 4 ng primers, 0.5 U FS Taq and 100 ng DNA.

    Techniques: Polymerase Chain Reaction, Positive Control, Negative Control

    Flow chart depicting study design. TS-PCR: Type-specific polymerase chain reaction, HPV: Human papillomavirus, H and E: Hematoxylin and eosin, PAS: Periodic Acid-Schiff

    Journal: Journal of Mid-Life Health

    Article Title: Polymerase chain reaction and deoxyribonucleic acid-sequencing based study on distribution of human papillomavirus 16/18 among histopathological types of cervical intra-epithelial neoplasia and primary invasive cervical carcinoma: A scenario in North Bengal, India

    doi: 10.4103/0976-7800.127786

    Figure Lengend Snippet: Flow chart depicting study design. TS-PCR: Type-specific polymerase chain reaction, HPV: Human papillomavirus, H and E: Hematoxylin and eosin, PAS: Periodic Acid-Schiff

    Article Snippet: HPV TS-PCR The HPV16-specific PCR mixture (20 μl) contained ×10 PCR buffer, 2 mM MgCl2 , 200 μM dNTP, 4 ng primers, 0.5 U FS Taq and 100 ng DNA.

    Techniques: Flow Cytometry, Polymerase Chain Reaction

    The linear range of the real-time PCR method when detecting purified DNA from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample

    Journal: Applied and Environmental Microbiology

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters

    doi: 10.1128/AEM.70.6.3588-3592.2004

    Figure Lengend Snippet: The linear range of the real-time PCR method when detecting purified DNA from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample

    Article Snippet: The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample.

    Techniques: Real-time Polymerase Chain Reaction, Purification

    Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Journal: Clinical Epigenetics

    Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents

    doi: 10.1186/s13148-018-0594-9

    Figure Lengend Snippet: Methylation status of MGMT promoter gene (MS-PCR) in four examples of GIST. Lanes containing PCR products derived from unmethylated and methylated alleles are marked U and M, respectively (with a length of 93 and 81 bp, in that order). GISTs 41 and 44 show a hypermethylated MGMT promoter gene; the same gene is unmethylated in GISTs 10 and 17 (the bands present in the M lanes of these two GISTs and in both lanes of UC are primer dimers). UC untreated control DNA, PC positive control DNA, MW molecular weight marks (100-bp ladder)

    Article Snippet: PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche).

    Techniques: Methylation, Mass Spectrometry, Polymerase Chain Reaction, Derivative Assay, Positive Control, Molecular Weight

    v-miRs modulate expression of cellular miRNAs in human oral keratinocytes (HOK). Heat maps showing differentially expressed cellular miRNAs in (A) miR-H1, (B) miR-K12-3-3p, and (C) miR-UL70-3p transfected HOK. (D) Venn diagram showing the distribution of unique and overlapping altered cellular miRNAs in v-miR-transfected HOK. (E) Validation of microarray by quantitative RT-PCR for miR-7, miR-21-3p, and miR-29b (marked by black arrows in the heat map) in another separate cohort of transfected HOK. RNU6 was used as endogenous control. Data are expressed as mean ± SEM of four independent transfections. Student’s t -test was conducted to calculate p -values (* p

    Journal: Frontiers in Immunology

    Article Title: Viral miRNAs Alter Host Cell miRNA Profiles and Modulate Innate Immune Responses

    doi: 10.3389/fimmu.2018.00433

    Figure Lengend Snippet: v-miRs modulate expression of cellular miRNAs in human oral keratinocytes (HOK). Heat maps showing differentially expressed cellular miRNAs in (A) miR-H1, (B) miR-K12-3-3p, and (C) miR-UL70-3p transfected HOK. (D) Venn diagram showing the distribution of unique and overlapping altered cellular miRNAs in v-miR-transfected HOK. (E) Validation of microarray by quantitative RT-PCR for miR-7, miR-21-3p, and miR-29b (marked by black arrows in the heat map) in another separate cohort of transfected HOK. RNU6 was used as endogenous control. Data are expressed as mean ± SEM of four independent transfections. Student’s t -test was conducted to calculate p -values (* p

    Article Snippet: The reactions were run using miRNA specific primer and universal primer (both from Qiagen) in the PCR mix buffer containing SYBR Green (Roche, Indianapolis, IN, USA).

    Techniques: Expressing, Transfection, Microarray, Quantitative RT-PCR