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Specificity and sensitivity of <t>PMAxx-qPCR</t> assay in detecting viable cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ). Viable or heat-killed Cmm cells at various concentrations were treated with 20 μM PMAxx, followed by <t>DNA</t> extraction and qPCR detection. Ct: threshold cycle of qPCR. CFU: colony forming unit. PMA: propidium monoazide. Columns and bars represent mean values and standard deviations. Means followed by different letters are significantly different ( P
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1) Product Images from "Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR"

Article Title: Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR

Journal: PLoS ONE

doi: 10.1371/journal.pone.0196525

Specificity and sensitivity of PMAxx-qPCR assay in detecting viable cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ). Viable or heat-killed Cmm cells at various concentrations were treated with 20 μM PMAxx, followed by DNA extraction and qPCR detection. Ct: threshold cycle of qPCR. CFU: colony forming unit. PMA: propidium monoazide. Columns and bars represent mean values and standard deviations. Means followed by different letters are significantly different ( P
Figure Legend Snippet: Specificity and sensitivity of PMAxx-qPCR assay in detecting viable cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ). Viable or heat-killed Cmm cells at various concentrations were treated with 20 μM PMAxx, followed by DNA extraction and qPCR detection. Ct: threshold cycle of qPCR. CFU: colony forming unit. PMA: propidium monoazide. Columns and bars represent mean values and standard deviations. Means followed by different letters are significantly different ( P

Techniques Used: Real-time Polymerase Chain Reaction, DNA Extraction

Detection of culturable, viable but non-culturable (VBNC) and dead cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ) by PMAxx-qPCR from artificially inoculated tomato seed. Ten tomato seeds were soaked in log phase (A), copper-induced VBNC (B) and heat-killed (C) Cmm cells suspension (10 8 CFU mL -1 ) by vacuum infiltration. After inoculation, the seed was broken by a ball mill and diluted 20-fold with 0.85% NaCl solution, followed by treatment with or without PMAxx at a final concentration of 20 μM. DNA was extracted after PMAxx treatment and used for qPCR assay. Cycle threshold (Ct) of qPCR was separated using multiple range test, and means labeled with different letters were significantly different ( P
Figure Legend Snippet: Detection of culturable, viable but non-culturable (VBNC) and dead cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ) by PMAxx-qPCR from artificially inoculated tomato seed. Ten tomato seeds were soaked in log phase (A), copper-induced VBNC (B) and heat-killed (C) Cmm cells suspension (10 8 CFU mL -1 ) by vacuum infiltration. After inoculation, the seed was broken by a ball mill and diluted 20-fold with 0.85% NaCl solution, followed by treatment with or without PMAxx at a final concentration of 20 μM. DNA was extracted after PMAxx treatment and used for qPCR assay. Cycle threshold (Ct) of qPCR was separated using multiple range test, and means labeled with different letters were significantly different ( P

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

2) Product Images from "Genetic, Epigenetic, and HPLC Fingerprint Differentiation between Natural and Ex Situ Populations of Rhodiola sachalinensis from Changbai Mountain, China"

Article Title: Genetic, Epigenetic, and HPLC Fingerprint Differentiation between Natural and Ex Situ Populations of Rhodiola sachalinensis from Changbai Mountain, China

Journal: PLoS ONE

doi: 10.1371/journal.pone.0112869

DNA methylation patterns in different populations of R. sachalinensis. EH and EM represent Eco R I/ Hpa II and Eco R I/ Msp I digestion combinations, respectively. Type 1/1 bands were present in both Eco R I/ Hpa II and Eco R I/ Msp I digestion combinations. Type 1/0 bands were present only in Eco R I/ Hpa II combinations, and Type 0/1 bands were present only in Eco R I/ Msp I digestions.
Figure Legend Snippet: DNA methylation patterns in different populations of R. sachalinensis. EH and EM represent Eco R I/ Hpa II and Eco R I/ Msp I digestion combinations, respectively. Type 1/1 bands were present in both Eco R I/ Hpa II and Eco R I/ Msp I digestion combinations. Type 1/0 bands were present only in Eco R I/ Hpa II combinations, and Type 0/1 bands were present only in Eco R I/ Msp I digestions.

Techniques Used: DNA Methylation Assay

3) Product Images from "Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR"

Article Title: Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR

Journal: PLoS ONE

doi: 10.1371/journal.pone.0196525

Specificity and sensitivity of PMAxx-qPCR assay in detecting viable cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ). Viable or heat-killed Cmm cells at various concentrations were treated with 20 μM PMAxx, followed by DNA extraction and qPCR detection. Ct: threshold cycle of qPCR. CFU: colony forming unit. PMA: propidium monoazide. Columns and bars represent mean values and standard deviations. Means followed by different letters are significantly different ( P
Figure Legend Snippet: Specificity and sensitivity of PMAxx-qPCR assay in detecting viable cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ). Viable or heat-killed Cmm cells at various concentrations were treated with 20 μM PMAxx, followed by DNA extraction and qPCR detection. Ct: threshold cycle of qPCR. CFU: colony forming unit. PMA: propidium monoazide. Columns and bars represent mean values and standard deviations. Means followed by different letters are significantly different ( P

Techniques Used: Real-time Polymerase Chain Reaction, DNA Extraction

Detection of culturable, viable but non-culturable (VBNC) and dead cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ) by PMAxx-qPCR from artificially inoculated tomato seed. Ten tomato seeds were soaked in log phase (A), copper-induced VBNC (B) and heat-killed (C) Cmm cells suspension (10 8 CFU mL -1 ) by vacuum infiltration. After inoculation, the seed was broken by a ball mill and diluted 20-fold with 0.85% NaCl solution, followed by treatment with or without PMAxx at a final concentration of 20 μM. DNA was extracted after PMAxx treatment and used for qPCR assay. Cycle threshold (Ct) of qPCR was separated using multiple range test, and means labeled with different letters were significantly different ( P
Figure Legend Snippet: Detection of culturable, viable but non-culturable (VBNC) and dead cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ) by PMAxx-qPCR from artificially inoculated tomato seed. Ten tomato seeds were soaked in log phase (A), copper-induced VBNC (B) and heat-killed (C) Cmm cells suspension (10 8 CFU mL -1 ) by vacuum infiltration. After inoculation, the seed was broken by a ball mill and diluted 20-fold with 0.85% NaCl solution, followed by treatment with or without PMAxx at a final concentration of 20 μM. DNA was extracted after PMAxx treatment and used for qPCR assay. Cycle threshold (Ct) of qPCR was separated using multiple range test, and means labeled with different letters were significantly different ( P

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

4) Product Images from "Stepwise partially overlapping primer-based PCR for genome walking"

Article Title: Stepwise partially overlapping primer-based PCR for genome walking

Journal: AMB Express

doi: 10.1186/s13568-018-0610-7

Genome walking of the gadA locus of Lactobacillus brevis NCL912 and hyg of rice. Each walking experiment contained four sets of PCR reactions that respectively utilized the four SWPOP primer sets, SWPOP1 (I), SWPOP2 (II), SWPOP3 (III) and SWPOP4 (IV), paired with a specific primer set. For each set of PCR reactions, the results of primary PCR (P), secondary PCR (S) and tertiary PCR (T) are presented. White arrows indicate target bands. M: DL2000 DNA marker
Figure Legend Snippet: Genome walking of the gadA locus of Lactobacillus brevis NCL912 and hyg of rice. Each walking experiment contained four sets of PCR reactions that respectively utilized the four SWPOP primer sets, SWPOP1 (I), SWPOP2 (II), SWPOP3 (III) and SWPOP4 (IV), paired with a specific primer set. For each set of PCR reactions, the results of primary PCR (P), secondary PCR (S) and tertiary PCR (T) are presented. White arrows indicate target bands. M: DL2000 DNA marker

Techniques Used: Polymerase Chain Reaction, Marker

5) Product Images from "Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene"

Article Title: Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

Journal: Cell Research

doi: 10.1038/cr.2013.86

Exo-endo trans-splicing of the asAmp and human ACAT1 transcripts. (A) Schematic representation of constructed plasmids (numbers 25-28) containing asAmp or ACAT1 1-1786 cDNA fused to Flag or Myc tags and empty vectors (numbers 24 and 29) with (pcDNA3) or without (pVk) the Amp r . (B , C) Analysis of trans-spliced RNA products and their translated proteins in intact cells. (B) AC29 cells were transfected with the indicated plasmids. RT-PCR was performed with primer sets asAmp-F/asAmp-R, asAmp-F/A1-R1, A1-F1/A1-R1 to detect the expression of asAmp, trans-spliced asAmp-ACAT1 and ACAT1, respectively. (C) Anti-Flag or anti-Myc antibodies were used in western blot analyses. Calculated MWs of various fusion proteins are shown. Arrows indicate fusion proteins identified by both anti-Flag and anti-Myc antibodies. (D) Schematic representation of in vitro -prepared transcripts as 5′-donor (D1) and 3′-acceptors (A1 and A2). (E) Analysis of trans-spliced RNA in the cell-free system. The cell-free trans-splicing reaction was performed using HeLa cell nuclear extract (NE) and in vitro -prepared 5′-donor and 3′-acceptors depicted in D . Specific primer set asAmp-F/A1-R2 was used in the RT-PCR assay. asAmp-F is the same as in C ; A1-R2 is complementary to the sequence of ACAT1 cDNA (upstream of A1-R1); NC, negative-control without templates. (F) DNA sequencing of the RT-PCR product (lane 2) from E . (G) Splicing efficiency of the in vitro trans-splicing between asAmp and ACAT1 RNAs was determined by qRT-PCR assays. The amount of spliced and unspliced RNA products were determined with specific primer sets asAmp-F/A1-R2 (as that used in E ) and A1-F2/A1-R2 (complementary to the sequence of ACAT1 cDNA). The percentage of spliced RNAs was calculated by dividing the amount of spliced RNAs by the total RNAs. Means and SD are shown ( n = 3). (H) Schematic representation of the exo-endo trans-splicing between asAmp and ACAT1 RNAs and the production of ACAT1 56-kDa/50-kDa isoforms. Human ACAT1 56-kDa isoform is the product of the exo-endo trans-splicing of exogenous asAmp and endogenous ACAT1 transcripts, and the ACAT1 50-kDa isoform is translated by using AUG 1397-1399 as the start codon. Underlined characters, AUG start codon; bold characters, AGA and GGC codons at the junction site; italic characters, splice-site signals (5′ SS and 3′ SS).
Figure Legend Snippet: Exo-endo trans-splicing of the asAmp and human ACAT1 transcripts. (A) Schematic representation of constructed plasmids (numbers 25-28) containing asAmp or ACAT1 1-1786 cDNA fused to Flag or Myc tags and empty vectors (numbers 24 and 29) with (pcDNA3) or without (pVk) the Amp r . (B , C) Analysis of trans-spliced RNA products and their translated proteins in intact cells. (B) AC29 cells were transfected with the indicated plasmids. RT-PCR was performed with primer sets asAmp-F/asAmp-R, asAmp-F/A1-R1, A1-F1/A1-R1 to detect the expression of asAmp, trans-spliced asAmp-ACAT1 and ACAT1, respectively. (C) Anti-Flag or anti-Myc antibodies were used in western blot analyses. Calculated MWs of various fusion proteins are shown. Arrows indicate fusion proteins identified by both anti-Flag and anti-Myc antibodies. (D) Schematic representation of in vitro -prepared transcripts as 5′-donor (D1) and 3′-acceptors (A1 and A2). (E) Analysis of trans-spliced RNA in the cell-free system. The cell-free trans-splicing reaction was performed using HeLa cell nuclear extract (NE) and in vitro -prepared 5′-donor and 3′-acceptors depicted in D . Specific primer set asAmp-F/A1-R2 was used in the RT-PCR assay. asAmp-F is the same as in C ; A1-R2 is complementary to the sequence of ACAT1 cDNA (upstream of A1-R1); NC, negative-control without templates. (F) DNA sequencing of the RT-PCR product (lane 2) from E . (G) Splicing efficiency of the in vitro trans-splicing between asAmp and ACAT1 RNAs was determined by qRT-PCR assays. The amount of spliced and unspliced RNA products were determined with specific primer sets asAmp-F/A1-R2 (as that used in E ) and A1-F2/A1-R2 (complementary to the sequence of ACAT1 cDNA). The percentage of spliced RNAs was calculated by dividing the amount of spliced RNAs by the total RNAs. Means and SD are shown ( n = 3). (H) Schematic representation of the exo-endo trans-splicing between asAmp and ACAT1 RNAs and the production of ACAT1 56-kDa/50-kDa isoforms. Human ACAT1 56-kDa isoform is the product of the exo-endo trans-splicing of exogenous asAmp and endogenous ACAT1 transcripts, and the ACAT1 50-kDa isoform is translated by using AUG 1397-1399 as the start codon. Underlined characters, AUG start codon; bold characters, AGA and GGC codons at the junction site; italic characters, splice-site signals (5′ SS and 3′ SS).

Techniques Used: Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, In Vitro, Sequencing, Negative Control, DNA Sequencing, Quantitative RT-PCR

Existence of asAmp transcripts in higher eukaryotic cells. (A) PCR analysis of genomic DNAs from various human cell lines. Primer sets asAmp-F/asAmp-R, RP-F/asAmp-R, A1C7-F/A1C7-R and Kan r -F/Kan r -R are the same as those in Figure 2E . Primer set gyrB-F/gyrB-R is complementary to the sequence of bacterial gyrB cDNA. NC, negative-control without DNA templates. (B) RT-PCR analysis of asAmp transcripts in human cell lines HEK293T and THP-1. Primer set asAmp-F/asAmp-R was used. NC, without RNA templates. (C) The PCR products from HEK293T cells (B) were subjected to DNA sequencing. The sequence matching the 267-bp asAmp ORF is shown. (D , E) Alignment analysis of the asAmp ORF in EST database. The 267-bp asAmp ORF sequence is entered as query sequence by using BLASTN. (D) Results are shown by taxonomy tree with hit numbers in each species indicated in brackets. (E) The species containing the 267-bp asAmp ORF (gray bar) with 100% identity. Branch lengths are not proportional to phylogeny time.
Figure Legend Snippet: Existence of asAmp transcripts in higher eukaryotic cells. (A) PCR analysis of genomic DNAs from various human cell lines. Primer sets asAmp-F/asAmp-R, RP-F/asAmp-R, A1C7-F/A1C7-R and Kan r -F/Kan r -R are the same as those in Figure 2E . Primer set gyrB-F/gyrB-R is complementary to the sequence of bacterial gyrB cDNA. NC, negative-control without DNA templates. (B) RT-PCR analysis of asAmp transcripts in human cell lines HEK293T and THP-1. Primer set asAmp-F/asAmp-R was used. NC, without RNA templates. (C) The PCR products from HEK293T cells (B) were subjected to DNA sequencing. The sequence matching the 267-bp asAmp ORF is shown. (D , E) Alignment analysis of the asAmp ORF in EST database. The 267-bp asAmp ORF sequence is entered as query sequence by using BLASTN. (D) Results are shown by taxonomy tree with hit numbers in each species indicated in brackets. (E) The species containing the 267-bp asAmp ORF (gray bar) with 100% identity. Branch lengths are not proportional to phylogeny time.

Techniques Used: Polymerase Chain Reaction, Sequencing, Negative Control, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing

Interchromosomal region of human ACAT1 chimeric mRNA is required for the exo-endo trans-splicing. (A) Schematic representation of bicistrons with or without a stable hairpin. Rluc cistron and Fluc cistron are linked by the ACAT1 interchromosomal region (ACAT1 1243-1396 , derived from both chromosomes 7 and 1). A stable hairpin (Δ G = −57 kcal/mol) is included upstream or downstream of the Rluc cistron. Mutated nucleotides at the 3′ SS are underlined. (B) Western blot of bicistronic proteins using anti-Fluc or anti-Rluc antibodies. (C) RT-PCR analysis of bicistronic RNAs. Specific primer sets A1-F2/FL-R and asAmp-F/FL-R were used in the RT-PCR analysis. All the RT-PCR products were further analyzed by DNA sequencing. Mutated nucleotides at the 3′ SS are indicated by underlines (right and top panel). The trans-splicing junction (dashed line), partial sequences of exon Xa, mini-exon Xb and exon 1 are shown (right and bottom panel). (D) The human ACAT1 interchromosomal region is required for the exo-endo trans-splicing event. Number 69 construct contains the ACAT1 interchromosomal region (ACAT1 1243-1396 ). Constructs of number 70 (1-1279+d790) and 71 (u45+1290-1396) contain partial regions of human ACAT1cDNA exclusively derived from chromosome 7 and 1, respectively. RT-PCR assays were performed by using specific primer sets asAmp-F/A1-R2 and asAmp-F/A1-R3 with amplification cycles 25, 29 and 33, synchronously. Reverse primer A1-R3 is complementary to d790. d790 or u45, 790 bp downstream or 45 bp upstream of the exonic sequences. (E) Splicing efficiency of the trans-splicing between asAmp and ACAT1 RNAs in transfected cells was determined by qRT-PCR assays. Spliced and unspliced RNAs were determined with specific primer sets asAmp-F/A1-R2 and A1-F2/A1-R2 as those used in Figure 4G . The percentage of spliced RNAs was calculated by dividing the amount of spliced RNA products by the total RNAs. Means and SD are shown ( n = 3). (F) Model of the exo-endo trans-splicing (bold arrow) between the recombinant plasmid-derived asAmp transcript (donor) and the interchromosomal trans-spliced human ACAT1 mRNA (acceptor). The pre-mRNA from either chromosome 7 or 1 is not acceptor of this exo-endo trans-splicing (dashed arrow). The region and orientation of the recombinant plasmid-derived fragment and recombined cryptic promoter are diagramed. Bold characters, AGA and GGC codons at junction site; italic characters, splice-site signals.
Figure Legend Snippet: Interchromosomal region of human ACAT1 chimeric mRNA is required for the exo-endo trans-splicing. (A) Schematic representation of bicistrons with or without a stable hairpin. Rluc cistron and Fluc cistron are linked by the ACAT1 interchromosomal region (ACAT1 1243-1396 , derived from both chromosomes 7 and 1). A stable hairpin (Δ G = −57 kcal/mol) is included upstream or downstream of the Rluc cistron. Mutated nucleotides at the 3′ SS are underlined. (B) Western blot of bicistronic proteins using anti-Fluc or anti-Rluc antibodies. (C) RT-PCR analysis of bicistronic RNAs. Specific primer sets A1-F2/FL-R and asAmp-F/FL-R were used in the RT-PCR analysis. All the RT-PCR products were further analyzed by DNA sequencing. Mutated nucleotides at the 3′ SS are indicated by underlines (right and top panel). The trans-splicing junction (dashed line), partial sequences of exon Xa, mini-exon Xb and exon 1 are shown (right and bottom panel). (D) The human ACAT1 interchromosomal region is required for the exo-endo trans-splicing event. Number 69 construct contains the ACAT1 interchromosomal region (ACAT1 1243-1396 ). Constructs of number 70 (1-1279+d790) and 71 (u45+1290-1396) contain partial regions of human ACAT1cDNA exclusively derived from chromosome 7 and 1, respectively. RT-PCR assays were performed by using specific primer sets asAmp-F/A1-R2 and asAmp-F/A1-R3 with amplification cycles 25, 29 and 33, synchronously. Reverse primer A1-R3 is complementary to d790. d790 or u45, 790 bp downstream or 45 bp upstream of the exonic sequences. (E) Splicing efficiency of the trans-splicing between asAmp and ACAT1 RNAs in transfected cells was determined by qRT-PCR assays. Spliced and unspliced RNAs were determined with specific primer sets asAmp-F/A1-R2 and A1-F2/A1-R2 as those used in Figure 4G . The percentage of spliced RNAs was calculated by dividing the amount of spliced RNA products by the total RNAs. Means and SD are shown ( n = 3). (F) Model of the exo-endo trans-splicing (bold arrow) between the recombinant plasmid-derived asAmp transcript (donor) and the interchromosomal trans-spliced human ACAT1 mRNA (acceptor). The pre-mRNA from either chromosome 7 or 1 is not acceptor of this exo-endo trans-splicing (dashed arrow). The region and orientation of the recombinant plasmid-derived fragment and recombined cryptic promoter are diagramed. Bold characters, AGA and GGC codons at junction site; italic characters, splice-site signals.

Techniques Used: Derivative Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Construct, Amplification, Transfection, Quantitative RT-PCR, Recombinant, Plasmid Preparation

6) Product Images from "Method To Detect Only Live Bacteria during PCR Amplification "

Article Title: Method To Detect Only Live Bacteria during PCR Amplification

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.02171-07

(Left) Scheme for PCR suppression by EMA, psoralen, and a methylisopsoralen derivative (4′-AMDMIP) as a DNA cross-linking agent (current scheme). (Right) Scheme for PCR suppression through DNA cleavage by a new function of EMA and T-poisons containing
Figure Legend Snippet: (Left) Scheme for PCR suppression by EMA, psoralen, and a methylisopsoralen derivative (4′-AMDMIP) as a DNA cross-linking agent (current scheme). (Right) Scheme for PCR suppression through DNA cleavage by a new function of EMA and T-poisons containing

Techniques Used: Polymerase Chain Reaction

7) Product Images from "BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles"

Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1038/mtna.2013.28

Overview of EV-BEAMing. EVs from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting DNA-coated beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.
Figure Legend Snippet: Overview of EV-BEAMing. EVs from serum or CSF samples were pelleted at 100,000 g for 80 minutes and processed as follows: (1) RNA was extracted and (2) analyzed for total yield and quality using the Bioanalyzer (Agilent). (3) RNA was then reverse transcribed into cDNA and 1/2 of the sample was used to determine the IDH1 cDNA copy number inside EVs by (4) qPCR analysis. (5) The remaining sample was preamplified (14 cycles) and used as input for BEAMing PCR. The resulting DNA-coated beads were interrogated with sequence-specific fluorescent probes to produce beads with wild-type (green) and mutant (red) profiles. (6) The percentage of beads with mutant DNA was determined by FACS and used in conjunction with the qPCR data to determine the minimum number of copies present to allow reliable detection of the mutant message.

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Mutagenesis, FACS

8) Product Images from "Detection of JC Polyomavirus tumor antigen in gastric carcinoma: a report from Iran"

Article Title: Detection of JC Polyomavirus tumor antigen in gastric carcinoma: a report from Iran

Journal: Iranian Journal of Microbiology

doi:

Standard curve analysis. Synthetic DNA standard curves for absolute quantification of JC-T-Ag by real-time PCR. The JCV-Mad-1 T-Ag sequence that cloned in a pBHA plasmid vector was serially diluted (from 100 to 10 8 copies of positive control DNA) and served as a standard reference. High amplification efficiency has also been shown by a good linear relationship among each concentration.
Figure Legend Snippet: Standard curve analysis. Synthetic DNA standard curves for absolute quantification of JC-T-Ag by real-time PCR. The JCV-Mad-1 T-Ag sequence that cloned in a pBHA plasmid vector was serially diluted (from 100 to 10 8 copies of positive control DNA) and served as a standard reference. High amplification efficiency has also been shown by a good linear relationship among each concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Sequencing, Clone Assay, Plasmid Preparation, Positive Control, Amplification, Concentration Assay

9) Product Images from "Structural analysis of the full-length gene encoding a fibronectin-binding-like protein (CadF) and its adjacent genetic loci within Campylobacter lari"

Article Title: Structural analysis of the full-length gene encoding a fibronectin-binding-like protein (CadF) and its adjacent genetic loci within Campylobacter lari

Journal: BMC Microbiology

doi: 10.1186/1471-2180-9-192

Northern blot hybridization (A) and RT-PCR (B) analyses of the cadF (-like) and Cla_0387 structural gene transcripts expressed in the C. lari isolates . Lane M, 100 bp DNA ladder; Lane 1, C. lari JCM2530 T with the reverse transcriptase (RTase); lane 2, C. lari JCM2530 T without the RTase.; lane 3, UPTC CF89-12 with the RTase; lane 4, UPTC CF89-12 without the RTase. Primer extension analysis (C) of the cadF (-like) and Cla_0387 mRNA transcript in the C. lari JCM2530 T isolate cells. The arrow indicates the transcription initiation site.
Figure Legend Snippet: Northern blot hybridization (A) and RT-PCR (B) analyses of the cadF (-like) and Cla_0387 structural gene transcripts expressed in the C. lari isolates . Lane M, 100 bp DNA ladder; Lane 1, C. lari JCM2530 T with the reverse transcriptase (RTase); lane 2, C. lari JCM2530 T without the RTase.; lane 3, UPTC CF89-12 with the RTase; lane 4, UPTC CF89-12 without the RTase. Primer extension analysis (C) of the cadF (-like) and Cla_0387 mRNA transcript in the C. lari JCM2530 T isolate cells. The arrow indicates the transcription initiation site.

Techniques Used: Northern Blot, Hybridization, Reverse Transcription Polymerase Chain Reaction

10) Product Images from "Two Different Panton-Valentine Leukocidin Phage Lineages Predominate in Japan ▿"

Article Title: Two Different Panton-Valentine Leukocidin Phage Lineages Predominate in Japan ▿

Journal:

doi: 10.1128/JCM.00136-08

Representative results of seven multiplex PCRs (M-PCRs) for identifying PVL phages. A 1-kb molecular weight marker (MWM) was run on both sides of the gel. Lanes: 1, PCR-1 using chromosomal DNA of 81/108; 2, PCR-2 using chromosomal DNA of JCSC2958; 3,
Figure Legend Snippet: Representative results of seven multiplex PCRs (M-PCRs) for identifying PVL phages. A 1-kb molecular weight marker (MWM) was run on both sides of the gel. Lanes: 1, PCR-1 using chromosomal DNA of 81/108; 2, PCR-2 using chromosomal DNA of JCSC2958; 3,

Techniques Used: Multiplex Assay, Molecular Weight, Marker, Polymerase Chain Reaction

11) Product Images from "Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams"

Article Title: Loss of genes related to Nucleotide Excision Repair (NER) and implications for reductive genome evolution in symbionts of deep-sea vesicomyid clams

Journal: PLoS ONE

doi: 10.1371/journal.pone.0171274

Electrophorograms of the RT-PCR products of NER genes in Ca . Vesicomyosocius okutanii (Vok) and the symbiont of C . pacifica (Cpac_S). A, Cpac_S; B, Vok. Expression of NER-related genes was analyzed by RT-PCR. Lane 1, uvrA ; lane 2, uvrB or corresponding DNA region; lane 3, uvrC or corresponding DNA region; lane 4, uvrD ; lane 5, uvrDp ; lane 6, recA ; lane 7, mfd ; lane 8, 16S rRNA gene; lane 9, 16S rRNA gene negative control (with RNase treatment before RT PCR). M, molecular markers. Primers and the predicted lengths of amplicons are shown in S3 Table . Red arrowheads indicate the position of a band where no signal was detected.
Figure Legend Snippet: Electrophorograms of the RT-PCR products of NER genes in Ca . Vesicomyosocius okutanii (Vok) and the symbiont of C . pacifica (Cpac_S). A, Cpac_S; B, Vok. Expression of NER-related genes was analyzed by RT-PCR. Lane 1, uvrA ; lane 2, uvrB or corresponding DNA region; lane 3, uvrC or corresponding DNA region; lane 4, uvrD ; lane 5, uvrDp ; lane 6, recA ; lane 7, mfd ; lane 8, 16S rRNA gene; lane 9, 16S rRNA gene negative control (with RNase treatment before RT PCR). M, molecular markers. Primers and the predicted lengths of amplicons are shown in S3 Table . Red arrowheads indicate the position of a band where no signal was detected.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

12) Product Images from "Organization of the Plasmid cpe Locus in Clostridium perfringens Type A Isolates "

Article Title: Organization of the Plasmid cpe Locus in Clostridium perfringens Type A Isolates

Journal: Infection and Immunity

doi: 10.1128/IAI.70.8.4261-4272.2002

Southern blotting analysis of dcm sequences in C. perfringens type A food poisoning isolates. Note that the probe, generated by PCR with primers to F4969 dcm sequences and F4969 sequences ∼400 bp downstream of the dcm sequences, does not hybridize with DNA from any type A food poisoning isolates tested. However, this probe does react with DNA isolated from F4969.
Figure Legend Snippet: Southern blotting analysis of dcm sequences in C. perfringens type A food poisoning isolates. Note that the probe, generated by PCR with primers to F4969 dcm sequences and F4969 sequences ∼400 bp downstream of the dcm sequences, does not hybridize with DNA from any type A food poisoning isolates tested. However, this probe does react with DNA isolated from F4969.

Techniques Used: Southern Blot, Generated, Polymerase Chain Reaction, Isolation

PCR detection of IS 1151 sequences in C. perfringens type A isolates. (A) PCR results obtained with primers specific for IS 1151 sequences which amplified (arrow at right) the expected ∼0.6-kbp product with total DNA from the previously sequenced IS 1151 ). Numbers on the left indicate migration of molecular weight markers. (B) PCR results obtained with a primer specific for cpe sequences and a reverse primer specific for IS 1151 sequences. Sequencing of the ∼1.2-kbp product obtained with F4013 DNA confirmed the inclusion of sequences spanning from cpe to IS 1151 . (C) Southern blot results obtained by hybridizing Xba I-digested DNA from type A isolates carrying a cpe plasmid with an internal IS 1151 -like probe.
Figure Legend Snippet: PCR detection of IS 1151 sequences in C. perfringens type A isolates. (A) PCR results obtained with primers specific for IS 1151 sequences which amplified (arrow at right) the expected ∼0.6-kbp product with total DNA from the previously sequenced IS 1151 ). Numbers on the left indicate migration of molecular weight markers. (B) PCR results obtained with a primer specific for cpe sequences and a reverse primer specific for IS 1151 sequences. Sequencing of the ∼1.2-kbp product obtained with F4013 DNA confirmed the inclusion of sequences spanning from cpe to IS 1151 . (C) Southern blot results obtained by hybridizing Xba I-digested DNA from type A isolates carrying a cpe plasmid with an internal IS 1151 -like probe.

Techniques Used: Polymerase Chain Reaction, Amplification, Migration, Molecular Weight, Sequencing, Southern Blot, Plasmid Preparation

PCR analyses of sequences upstream of the plasmid cpe gene of type A isolates. (A) Results of a dcm -specific PCR assay (expected ∼0.4-kbp product marked by arrow) with primers internal to F4969 dcm sequences. (B) Results of a PCR assay performed with primers designed to amplify a product from the DNA region between dcm sequences and sequences ∼400 bp downstream of them (based upon sequencing of the cpe -containing Xba I fragment of F4969). In this PCR assay, F4969 DNA yields an ∼0.9-kbp PCR product (small arrow), which is consistent with sequencing results for the F4969 plasmid cpe locus; note that one cpe -negative type A isolate, 297442, gave an ∼2.0-kbp product (large arrow). (C) PCR analysis of the association between dcm gene and IS 1469 genes. An ∼2.1-kbp product (arrow) was detected in all type A isolates carrying a plasmid cpe gene. (D) PCR analysis of the association between dcm sequences and the cpe gene, which is consistent with sequencing results for the plasmid cpe locus of F4969. An ∼3.5-kbp product (arrow) was detected in all type A isolates carrying a plasmid cpe gene, which is consistent with sequencing results for the plasmid cpe locus of F4969. Numbers on the left indicate migration of molecular size markers.
Figure Legend Snippet: PCR analyses of sequences upstream of the plasmid cpe gene of type A isolates. (A) Results of a dcm -specific PCR assay (expected ∼0.4-kbp product marked by arrow) with primers internal to F4969 dcm sequences. (B) Results of a PCR assay performed with primers designed to amplify a product from the DNA region between dcm sequences and sequences ∼400 bp downstream of them (based upon sequencing of the cpe -containing Xba I fragment of F4969). In this PCR assay, F4969 DNA yields an ∼0.9-kbp PCR product (small arrow), which is consistent with sequencing results for the F4969 plasmid cpe locus; note that one cpe -negative type A isolate, 297442, gave an ∼2.0-kbp product (large arrow). (C) PCR analysis of the association between dcm gene and IS 1469 genes. An ∼2.1-kbp product (arrow) was detected in all type A isolates carrying a plasmid cpe gene. (D) PCR analysis of the association between dcm sequences and the cpe gene, which is consistent with sequencing results for the plasmid cpe locus of F4969. An ∼3.5-kbp product (arrow) was detected in all type A isolates carrying a plasmid cpe gene, which is consistent with sequencing results for the plasmid cpe locus of F4969. Numbers on the left indicate migration of molecular size markers.

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Migration

Southern blot analyses of cpe -positive C. perfringens type A isolates. Results shown are for type A isolates carrying a cpe plasmid hybridized with a cpe probe, a dcm probe, or an IS 1470 -like probe. The uppermost bands in panel C lie in the gel wells. The F5603 lane shows hybridization of the probes to incompletely digested DNA. Numbers on the left indicate migration of molecular size markers.
Figure Legend Snippet: Southern blot analyses of cpe -positive C. perfringens type A isolates. Results shown are for type A isolates carrying a cpe plasmid hybridized with a cpe probe, a dcm probe, or an IS 1470 -like probe. The uppermost bands in panel C lie in the gel wells. The F5603 lane shows hybridization of the probes to incompletely digested DNA. Numbers on the left indicate migration of molecular size markers.

Techniques Used: Southern Blot, Plasmid Preparation, Hybridization, Migration

PCR analyses of the region between the cpe gene and downstream IS 1470 -like sequences. (A) PCR product amplified from specified type A isolates using primers for cpe gene sequences and sequences present in IS 1470 -like sequences of F4969, which generate an ∼2.2-kbp product (arrow at right) with DNA from F4969. Numbers on the left indicate migration of molecular size markers. (B) PCR products amplified from specified type A isolates using primers specific for internal IS 1470 -like sequences. This reaction generated the expected 0.9-kbp product from F4969 DNA (arrow). Numbers on the left indicate migration of molecular size markers.
Figure Legend Snippet: PCR analyses of the region between the cpe gene and downstream IS 1470 -like sequences. (A) PCR product amplified from specified type A isolates using primers for cpe gene sequences and sequences present in IS 1470 -like sequences of F4969, which generate an ∼2.2-kbp product (arrow at right) with DNA from F4969. Numbers on the left indicate migration of molecular size markers. (B) PCR products amplified from specified type A isolates using primers specific for internal IS 1470 -like sequences. This reaction generated the expected 0.9-kbp product from F4969 DNA (arrow). Numbers on the left indicate migration of molecular size markers.

Techniques Used: Polymerase Chain Reaction, Amplification, Migration, Generated

13) Product Images from "Microbial community and metabolomic comparison of irritable bowel syndrome faeces"

Article Title: Microbial community and metabolomic comparison of irritable bowel syndrome faeces

Journal: Journal of Medical Microbiology

doi: 10.1099/jmm.0.028126-0

(a) Dendrogram showing the DGGE profiles of the IBS and nIBS faecal bacterial communities. Community DNA was detected by PCR amplification with universal bacterial 16S rRNA gene (V3–V5 regions) primers followed by DGGE. The dendrogram was constructed using D sc and the UPGMA algorithm. The arrow indicates the eluted band of Eubacterium biforme . (b) PCA of DGGE fingerprints of the 16S rRNA gene of dominant bacteria in IBS (□) and nIBS (▴) samples.
Figure Legend Snippet: (a) Dendrogram showing the DGGE profiles of the IBS and nIBS faecal bacterial communities. Community DNA was detected by PCR amplification with universal bacterial 16S rRNA gene (V3–V5 regions) primers followed by DGGE. The dendrogram was constructed using D sc and the UPGMA algorithm. The arrow indicates the eluted band of Eubacterium biforme . (b) PCA of DGGE fingerprints of the 16S rRNA gene of dominant bacteria in IBS (□) and nIBS (▴) samples.

Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Construct

14) Product Images from "Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA"

Article Title: Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00862-16

PCR product restriction fragment length polymorphism profiles of cloned genes. (A) Lane 1, PCR product from pooled genomic DNA and cry_F/cry_R primers; lane 2, PCR product from pooled genomic DNA and cry_F/cry_R and REcry9Da_F/REcry9Ea/b_F primers; lane
Figure Legend Snippet: PCR product restriction fragment length polymorphism profiles of cloned genes. (A) Lane 1, PCR product from pooled genomic DNA and cry_F/cry_R primers; lane 2, PCR product from pooled genomic DNA and cry_F/cry_R and REcry9Da_F/REcry9Ea/b_F primers; lane

Techniques Used: Polymerase Chain Reaction, Clone Assay

15) Product Images from "An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species"

Article Title: An Insulator Element Located at the Cyclin B1 Interacting Protein 1 Gene Locus Is Highly Conserved among Mammalian Species

Journal: PLoS ONE

doi: 10.1371/journal.pone.0131204

Exon-intron structure of the Ccnb1ip1 gene in the JF1 and C57BL/6 genomes. RT-PCR products from exon 1 to exon 5 were detected in an E9.5 embryo of JF1. Exon 1 of Ccnb1ip1 was located in the vicinity of the Ccnb1ip1 insulator (red box) and enhancer (blue box) in the JF1 genome. The sequences corresponding to exon 2 and exon 4 of the Ccnb1ip1 gene were found to be present, but those corresponding to exon 1 and exon 3 to be absent (deleted) in the B6 genome. The horizon bars (1 to 7) indicate genomic regions for which DNA methylation statuses were assessed ( Fig 1 ).
Figure Legend Snippet: Exon-intron structure of the Ccnb1ip1 gene in the JF1 and C57BL/6 genomes. RT-PCR products from exon 1 to exon 5 were detected in an E9.5 embryo of JF1. Exon 1 of Ccnb1ip1 was located in the vicinity of the Ccnb1ip1 insulator (red box) and enhancer (blue box) in the JF1 genome. The sequences corresponding to exon 2 and exon 4 of the Ccnb1ip1 gene were found to be present, but those corresponding to exon 1 and exon 3 to be absent (deleted) in the B6 genome. The horizon bars (1 to 7) indicate genomic regions for which DNA methylation statuses were assessed ( Fig 1 ).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, DNA Methylation Assay

16) Product Images from "Characterization of a new member of Iridoviridae, Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp (Litopenaeus vannamei)"

Article Title: Characterization of a new member of Iridoviridae, Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp (Litopenaeus vannamei)

Journal: Scientific Reports

doi: 10.1038/s41598-017-10738-8

Detection of the shrimp hemocyte iridescent virus with the nested PCR method. ( a ) Specificity analysis of the nested PCR method. M, DL500 molecular mass marker; Lanes 1–6: PCR amplified products with DNA template samples extracted from health L. vannamei and L. vannamei infected with WSSV, IHHNV, HPV, AHPND, and EHP, respectively; lane 7: PCR amplified products with DNA template sample extracted from shrimp infected with the iridescent virus; and lane 8: negative control. ( b ) Sensitivity test of the nested PCR method. M, DL1000 molecular mass marker; Lane 1: negative control; Lines 2–10: PCR amplified products with the diluted DNA solutions (10 0 –10 −8 ) of L. vannamei naturally infected with the shrimp iridescent virus.
Figure Legend Snippet: Detection of the shrimp hemocyte iridescent virus with the nested PCR method. ( a ) Specificity analysis of the nested PCR method. M, DL500 molecular mass marker; Lanes 1–6: PCR amplified products with DNA template samples extracted from health L. vannamei and L. vannamei infected with WSSV, IHHNV, HPV, AHPND, and EHP, respectively; lane 7: PCR amplified products with DNA template sample extracted from shrimp infected with the iridescent virus; and lane 8: negative control. ( b ) Sensitivity test of the nested PCR method. M, DL1000 molecular mass marker; Lane 1: negative control; Lines 2–10: PCR amplified products with the diluted DNA solutions (10 0 –10 −8 ) of L. vannamei naturally infected with the shrimp iridescent virus.

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

17) Product Images from "FljA-Mediated Posttranscriptional Control of Phase 1 Flagellin Expression in Flagellar Phase Variation of Salmonella enterica Serovar Typhimurium"

Article Title: FljA-Mediated Posttranscriptional Control of Phase 1 Flagellin Expression in Flagellar Phase Variation of Salmonella enterica Serovar Typhimurium

Journal: Journal of Bacteriology

doi: 10.1128/JB.188.3.958-967.2006

Effect of FljA on the fliC expression in vitro. (A) DNA-directed transcription-translation-coupled system. The fliC gene was expressed from the trc promoter. The fliC transcript encoded by pIC495-2 contains the fliC UTR, whereas that encoded by pIC495-1 contains the trc UTR. Proteins were synthesized in an E. coli S30 extract with or without His-FljA. Proteins were separated by SDS-PAGE, and FliC was detected by Western blotting with anti-FliC polyclonal antibody. (B) In vitro transcription. The DNA fragment containing the trc promoter and the fliC UTR was transcribed in vitro with E. coli RNA polymerase holoenzyme with or without His-FljA in the presence of [α- 32 P]UTP. Synthesized RNAs were separated on a polyacrylamide gel containing 6 M urea and detected by autoradiography. nt, nucleotide. (C) RNA-directed protein synthesis. An RNA template used was synthesized in vitro and contained the fliC UTR and ORF. Protein synthesis was carried out in an E. coli S30 extract with or without His-FljA in the presence of [ 35 S]methionine. Synthesized FliC protein was immunoprecipitated, separated by SDS-PAGE, and detected by fluorography. An extra band with a higher mobility might have been derived from fliC mRNA of incomplete length.
Figure Legend Snippet: Effect of FljA on the fliC expression in vitro. (A) DNA-directed transcription-translation-coupled system. The fliC gene was expressed from the trc promoter. The fliC transcript encoded by pIC495-2 contains the fliC UTR, whereas that encoded by pIC495-1 contains the trc UTR. Proteins were synthesized in an E. coli S30 extract with or without His-FljA. Proteins were separated by SDS-PAGE, and FliC was detected by Western blotting with anti-FliC polyclonal antibody. (B) In vitro transcription. The DNA fragment containing the trc promoter and the fliC UTR was transcribed in vitro with E. coli RNA polymerase holoenzyme with or without His-FljA in the presence of [α- 32 P]UTP. Synthesized RNAs were separated on a polyacrylamide gel containing 6 M urea and detected by autoradiography. nt, nucleotide. (C) RNA-directed protein synthesis. An RNA template used was synthesized in vitro and contained the fliC UTR and ORF. Protein synthesis was carried out in an E. coli S30 extract with or without His-FljA in the presence of [ 35 S]methionine. Synthesized FliC protein was immunoprecipitated, separated by SDS-PAGE, and detected by fluorography. An extra band with a higher mobility might have been derived from fliC mRNA of incomplete length.

Techniques Used: Expressing, In Vitro, Synthesized, SDS Page, Western Blot, Autoradiography, Immunoprecipitation, Derivative Assay

18) Product Images from "Lineage-Specific Distribution of Insertion Sequence Excision Enhancer in Enterotoxigenic Escherichia coli Isolated from Swine"

Article Title: Lineage-Specific Distribution of Insertion Sequence Excision Enhancer in Enterotoxigenic Escherichia coli Isolated from Swine

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03696-13

Genotyping and phylogenetic analysis of E. coli strains. A dendrogram obtained by PFGE of XbaI-digested DNA from 73 E. coli O139 and O149 strains is shown on the left side. Information about each strain, the results of the PCR screening for genes encoding
Figure Legend Snippet: Genotyping and phylogenetic analysis of E. coli strains. A dendrogram obtained by PFGE of XbaI-digested DNA from 73 E. coli O139 and O149 strains is shown on the left side. Information about each strain, the results of the PCR screening for genes encoding

Techniques Used: Polymerase Chain Reaction

19) Product Images from "Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats"

Article Title: Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats

Journal: The Plant Pathology Journal

doi: 10.5423/PPJ.OA.09.2014.0096

Gel electrophoresis of the polymerase chain reaction products formed with primer Pc-12-F/Pc-12-R and bacterial DNA of Pseudomonas coronafaciens strains. Lanes 1~13, P. coronafaciens LMG 5060, KACC 13262, KACC 12133, LMG 2170, LMG 5030, LMG 5061, LMG 5081, LMG 5380, LMG 5449, LMG 5452, LMG 5536, LMG 13190, LMG 2330; lane 14, water as a negative control.
Figure Legend Snippet: Gel electrophoresis of the polymerase chain reaction products formed with primer Pc-12-F/Pc-12-R and bacterial DNA of Pseudomonas coronafaciens strains. Lanes 1~13, P. coronafaciens LMG 5060, KACC 13262, KACC 12133, LMG 2170, LMG 5030, LMG 5061, LMG 5081, LMG 5380, LMG 5449, LMG 5452, LMG 5536, LMG 13190, LMG 2330; lane 14, water as a negative control.

Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Negative Control

Sensitivity and specificity of the TaqMan real-time PCR with primer, Pc-12-ne-F/Pc-12-ne-R and TaqMan probe, Pc-taqman. (A) The linear regression generated by ten-fold dilution of DNA of Pseudomonas coronafaciens LMG 5060 and (B) TaqMan PCR with DNAs (circle dot) of P. coronafaciens LMG 5060, and P. coronafaciens strains (square dot): P. coronafaciens KACC 13262, KACC 12133, LMG 2170, LMG 5030, LMG 5061, LMG 5081, LMG 5380, LMG 5449, LMG 5452, LMG 5536, LMG 13190, and LMG 2330.
Figure Legend Snippet: Sensitivity and specificity of the TaqMan real-time PCR with primer, Pc-12-ne-F/Pc-12-ne-R and TaqMan probe, Pc-taqman. (A) The linear regression generated by ten-fold dilution of DNA of Pseudomonas coronafaciens LMG 5060 and (B) TaqMan PCR with DNAs (circle dot) of P. coronafaciens LMG 5060, and P. coronafaciens strains (square dot): P. coronafaciens KACC 13262, KACC 12133, LMG 2170, LMG 5030, LMG 5061, LMG 5081, LMG 5380, LMG 5449, LMG 5452, LMG 5536, LMG 13190, and LMG 2330.

Techniques Used: Real-time Polymerase Chain Reaction, Generated, Polymerase Chain Reaction

20) Product Images from "Ectopic Migration of an Adult Heartworm in a Dog with Dirofilariasis"

Article Title: Ectopic Migration of an Adult Heartworm in a Dog with Dirofilariasis

Journal: The Korean Journal of Parasitology

doi: 10.3347/kjp.2008.46.3.171

PCR-based detection of D. immitis DNA in an adult worm found in the abdominal cavity of the dog. M is a 100-bp marker. P is a D. immitis DNA-positive control. S is an adult worm collected from abdominal cavity.
Figure Legend Snippet: PCR-based detection of D. immitis DNA in an adult worm found in the abdominal cavity of the dog. M is a 100-bp marker. P is a D. immitis DNA-positive control. S is an adult worm collected from abdominal cavity.

Techniques Used: Polymerase Chain Reaction, Marker, Positive Control

21) Product Images from "Regionalized GC content of template DNA as a predictor of PCR success"

Article Title: Regionalized GC content of template DNA as a predictor of PCR success

Journal: Nucleic Acids Research

doi:

F -values for a one-way ANOVA computed from a learning set of 370 DNA target templates. Large F -values represent a significant difference between the failed and successful PCR. P = 0.05 for F = 3.87 and P = 0.001 for F = 11.
Figure Legend Snippet: F -values for a one-way ANOVA computed from a learning set of 370 DNA target templates. Large F -values represent a significant difference between the failed and successful PCR. P = 0.05 for F = 3.87 and P = 0.001 for F = 11.

Techniques Used: Polymerase Chain Reaction

22) Product Images from "The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection"

Article Title: The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection

Journal: Archives of Virology

doi: 10.1007/s00705-017-3563-2

( A ) PCR amplification of the env gene from DF-1/K cells. (M) DNA marker; (DF-1/K): Genomic DNA extracted from DF-1/K cells; (DF-1): Genomic DNA extracted from DF-1 cells. ( B ) Verification of the stability of the ALV-K env gene in DF-1/K cells during passage. (M) DNAMarker; Lanes 1-5 and 7-10: ALV-K env gene cell-culture passage levels 5, 15, 25, 30, 35, 40, 45, 50, 60, respectively; Lane 6: DF-1 cells (negative control)
Figure Legend Snippet: ( A ) PCR amplification of the env gene from DF-1/K cells. (M) DNA marker; (DF-1/K): Genomic DNA extracted from DF-1/K cells; (DF-1): Genomic DNA extracted from DF-1 cells. ( B ) Verification of the stability of the ALV-K env gene in DF-1/K cells during passage. (M) DNAMarker; Lanes 1-5 and 7-10: ALV-K env gene cell-culture passage levels 5, 15, 25, 30, 35, 40, 45, 50, 60, respectively; Lane 6: DF-1 cells (negative control)

Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Cell Culture, Negative Control

A) . PCR amplification of the ALV-K env gene in DF-1/K cells. (M) DNA marker; (1): RNA extracted from DF-1/K cells; (2): Genomic DNA extracted from DF-1/K cells; (3) RNA extracted from DF-1 cells. B . Levels of ALV-K env gene transcription in DF-1/K cells were determined by real-time RT-PCR with gene specific primers. DF-1 cells served as a negative control. Data are representative of two independent experiments, both performed in triplicate
Figure Legend Snippet: A) . PCR amplification of the ALV-K env gene in DF-1/K cells. (M) DNA marker; (1): RNA extracted from DF-1/K cells; (2): Genomic DNA extracted from DF-1/K cells; (3) RNA extracted from DF-1 cells. B . Levels of ALV-K env gene transcription in DF-1/K cells were determined by real-time RT-PCR with gene specific primers. DF-1 cells served as a negative control. Data are representative of two independent experiments, both performed in triplicate

Techniques Used: Polymerase Chain Reaction, Amplification, Marker, Quantitative RT-PCR, Negative Control

23) Product Images from "Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China"

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China

Journal: Viruses

doi: 10.3390/v10040194

PCR amplification of the complete genomes of 3 isolates. M: DNA marker 3; 1–3: GD150509, GD160403, and GD160607; A, B, C, D: 4 DNA fragments of the complete genome.
Figure Legend Snippet: PCR amplification of the complete genomes of 3 isolates. M: DNA marker 3; 1–3: GD150509, GD160403, and GD160607; A, B, C, D: 4 DNA fragments of the complete genome.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

PCR amplification of the complete genomes of 3 isolates. M: DNA marker DL5000; 1–3: GDFX0601, GDFX0602, and GDFX0603; A, B, C, D: 4 DNA fragments of the complete genome.
Figure Legend Snippet: PCR amplification of the complete genomes of 3 isolates. M: DNA marker DL5000; 1–3: GDFX0601, GDFX0602, and GDFX0603; A, B, C, D: 4 DNA fragments of the complete genome.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

24) Product Images from "Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing: the story for Liuwei Dihuang Wan"

Article Title: Biological ingredient analysis of traditional Chinese medicine preparation based on high-throughput sequencing: the story for Liuwei Dihuang Wan

Journal: Scientific Reports

doi: 10.1038/srep05147

Prescribed species analysis results for LDW samples. (a) Identification results based on ITS2. (b) Identification results based on trnL . Notice that P. cocos was not discovered in any LDW samples based on HTS results with universal ITS2 primers, but its existence was confirmed by specific primers on selected samples. In addition, the relative abundance was not only related to the amount of biological ingredients, but also the quality and concentration of DNA during experiment.
Figure Legend Snippet: Prescribed species analysis results for LDW samples. (a) Identification results based on ITS2. (b) Identification results based on trnL . Notice that P. cocos was not discovered in any LDW samples based on HTS results with universal ITS2 primers, but its existence was confirmed by specific primers on selected samples. In addition, the relative abundance was not only related to the amount of biological ingredients, but also the quality and concentration of DNA during experiment.

Techniques Used: Concentration Assay

Phylogeny and relative abundance of species detected in LDW samples from different manufacturers and batches. (a) Phylogeny and relative abundance of species detected in LDW from 3 manufacturers (MH, MS and MT) based on ITS2. (b) Phylogeny and relative abundance of species detected in LDW from 3 manufacturers (MH, MS and MT) based on trnL . (c) Phylogeny and relative abundance of species detected in LDW from 3 batches (MS) based on ITS2. Species marked in red boxes are prescribed species, while others are contaminated species. Notice that the relative abundance was not only related to the amount of biological ingredients, but also the quality and concentration of DNA during experiment.
Figure Legend Snippet: Phylogeny and relative abundance of species detected in LDW samples from different manufacturers and batches. (a) Phylogeny and relative abundance of species detected in LDW from 3 manufacturers (MH, MS and MT) based on ITS2. (b) Phylogeny and relative abundance of species detected in LDW from 3 manufacturers (MH, MS and MT) based on trnL . (c) Phylogeny and relative abundance of species detected in LDW from 3 batches (MS) based on ITS2. Species marked in red boxes are prescribed species, while others are contaminated species. Notice that the relative abundance was not only related to the amount of biological ingredients, but also the quality and concentration of DNA during experiment.

Techniques Used: Mass Spectrometry, Concentration Assay

25) Product Images from "Specific function of a plastid sigma factor for ndhF gene transcription"

Article Title: Specific function of a plastid sigma factor for ndhF gene transcription

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki908

Characterization of the sig4 knock-out mutant. ( A ) Schematic presentation of the T-DNA insertion and the location of the primers. The exact position of the T-DNA insertion into the SIG4 gene (210) was determined by sequencing of the DNA fragments amplified by primer pairs 1/2 and 3/4. ( B ) The absence of SIG4 mRNA in the T-DNA insertion line was verified by RT–PCR. Total RNA prepared from 7-day-old WT and sig4 plantlets has been analysed in parallel for the presence of all six sigma factors. ( C ) WT and sig4 knock-out plants grown for 4 weeks under 16 h light/8 h dark cycle.
Figure Legend Snippet: Characterization of the sig4 knock-out mutant. ( A ) Schematic presentation of the T-DNA insertion and the location of the primers. The exact position of the T-DNA insertion into the SIG4 gene (210) was determined by sequencing of the DNA fragments amplified by primer pairs 1/2 and 3/4. ( B ) The absence of SIG4 mRNA in the T-DNA insertion line was verified by RT–PCR. Total RNA prepared from 7-day-old WT and sig4 plantlets has been analysed in parallel for the presence of all six sigma factors. ( C ) WT and sig4 knock-out plants grown for 4 weeks under 16 h light/8 h dark cycle.

Techniques Used: Knock-Out, Mutagenesis, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction

26) Product Images from "Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus ▿"

Article Title: Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus ▿

Journal:

doi: 10.1128/JCM.00027-08

Sensitivities of C-PCR (A), first-round N-PCR with external primers Tox-100 and Tox-303 (B), and second-round N-PCR with internal primers Tox-130 and Tox-200 (C) to detect V. vulnificus in plasmid DNA. Lanes: 1, 100-bp ladder marker (Bioneer); 2, negative
Figure Legend Snippet: Sensitivities of C-PCR (A), first-round N-PCR with external primers Tox-100 and Tox-303 (B), and second-round N-PCR with internal primers Tox-130 and Tox-200 (C) to detect V. vulnificus in plasmid DNA. Lanes: 1, 100-bp ladder marker (Bioneer); 2, negative

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Marker

Standard curves (5 × 10 8 to 5 × 10 0 copies/μl) from the Q-PCR assay. Plasmid DNA was used as the template. Circled numerals: 1, negative control (sterile distilled water); 2 to 10, plasmid DNA serially diluted from 5 ×
Figure Legend Snippet: Standard curves (5 × 10 8 to 5 × 10 0 copies/μl) from the Q-PCR assay. Plasmid DNA was used as the template. Circled numerals: 1, negative control (sterile distilled water); 2 to 10, plasmid DNA serially diluted from 5 ×

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Negative Control

27) Product Images from "Prevalence of JC Virus in Chinese Patients with Colorectal Cancer"

Article Title: Prevalence of JC Virus in Chinese Patients with Colorectal Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035900

Detection of the JCV T-antigen sequence in samples from CRC patients. (A) The 110 bp fragment was amplified from DNA isolated from matched samples of colorectal cancer (upper) and normal tumor adjacent tissues (lower) with nested PCR. M: DL 2,000 DNA Marker (TaKaRa); P: positive control; N1: first-round negative control; N2: second-round negative control. (B) Images from JCV nested-PCR product arrays. Nested-PCR products of tumor tissues (left) and normal tumor adjacent tissues (right) were spotted onto surfaces of aminosilane slides as three replicates, hybridized with TAMRA-labeled oligonucleotide probe and finally visualized by AXON scanner. (C) The numbers of JCV-positive samples in 137 matched pairs of tumor tissue, non-cancerous adjacent tumor tissue and PB samples from CRC patients.
Figure Legend Snippet: Detection of the JCV T-antigen sequence in samples from CRC patients. (A) The 110 bp fragment was amplified from DNA isolated from matched samples of colorectal cancer (upper) and normal tumor adjacent tissues (lower) with nested PCR. M: DL 2,000 DNA Marker (TaKaRa); P: positive control; N1: first-round negative control; N2: second-round negative control. (B) Images from JCV nested-PCR product arrays. Nested-PCR products of tumor tissues (left) and normal tumor adjacent tissues (right) were spotted onto surfaces of aminosilane slides as three replicates, hybridized with TAMRA-labeled oligonucleotide probe and finally visualized by AXON scanner. (C) The numbers of JCV-positive samples in 137 matched pairs of tumor tissue, non-cancerous adjacent tumor tissue and PB samples from CRC patients.

Techniques Used: Sequencing, Amplification, Isolation, Nested PCR, Marker, Positive Control, Negative Control, Labeling

JCV viral load was higher in tumor tissues than in non-cancerous tumor adjacent tissues and PB samples. JCV positive samples determined by nested PCR were analyzed using qRT-PCR. Blots represent averaged copy numbers (3 replicate wells) of JCV DNA for tumor tissue, non-cancerous tissue and PB, respectively, and bars represent calculated medians. P -values were calculated using the Wilcoxon's test.
Figure Legend Snippet: JCV viral load was higher in tumor tissues than in non-cancerous tumor adjacent tissues and PB samples. JCV positive samples determined by nested PCR were analyzed using qRT-PCR. Blots represent averaged copy numbers (3 replicate wells) of JCV DNA for tumor tissue, non-cancerous tissue and PB, respectively, and bars represent calculated medians. P -values were calculated using the Wilcoxon's test.

Techniques Used: Nested PCR, Quantitative RT-PCR

28) Product Images from "Association of Genotyping of Bacillus cereus with Clinical Features of Post-Traumatic Endophthalmitis"

Article Title: Association of Genotyping of Bacillus cereus with Clinical Features of Post-Traumatic Endophthalmitis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0147878

PCR amplification of the vrrA gene among clinical B . cereus isolates. Lane1-14: Bc1-Bc14; lane16-25: Bc16-Bc25; Lane26: B . cereus ATCC14579; Lane27: B . thuringiensis CTCC22945; Lane28: Bacillus subtilis ATCC9372; Lane29: ddH2O; Lane15 and Lane30: DNA marker. The predicted size of the product was approximately 430bp. A total of twenty–four B . cereus strains were confirmed by the PCR, and non- B . cereus strains did not yield a PCR product.
Figure Legend Snippet: PCR amplification of the vrrA gene among clinical B . cereus isolates. Lane1-14: Bc1-Bc14; lane16-25: Bc16-Bc25; Lane26: B . cereus ATCC14579; Lane27: B . thuringiensis CTCC22945; Lane28: Bacillus subtilis ATCC9372; Lane29: ddH2O; Lane15 and Lane30: DNA marker. The predicted size of the product was approximately 430bp. A total of twenty–four B . cereus strains were confirmed by the PCR, and non- B . cereus strains did not yield a PCR product.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

29) Product Images from "Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China"

Article Title: Phylogenetic Analysis and Pathogenicity Assessment of the Emerging Recombinant Subgroup K of Avian Leukosis Virus in South China

Journal: Viruses

doi: 10.3390/v10040194

PCR amplification of the complete genomes of 3 isolates. M: DNA marker 3; 1–3: GD150509, GD160403, and GD160607; A, B, C, D: 4 DNA fragments of the complete genome.
Figure Legend Snippet: PCR amplification of the complete genomes of 3 isolates. M: DNA marker 3; 1–3: GD150509, GD160403, and GD160607; A, B, C, D: 4 DNA fragments of the complete genome.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

PCR amplification of the complete genomes of 3 isolates. M: DNA marker DL5000; 1–3: GDFX0601, GDFX0602, and GDFX0603; A, B, C, D: 4 DNA fragments of the complete genome.
Figure Legend Snippet: PCR amplification of the complete genomes of 3 isolates. M: DNA marker DL5000; 1–3: GDFX0601, GDFX0602, and GDFX0603; A, B, C, D: 4 DNA fragments of the complete genome.

Techniques Used: Polymerase Chain Reaction, Amplification, Marker

30) Product Images from "Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow ▿"

Article Title: Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow ▿

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00202-11

Schematic genome structure of ClorDNAV. Primers used in the PCR experiments are shown as open arrows (see the text and ). Gray arrows indicate the locations for ORFs VP1 to VP4, and the gray colored box is the partially double-stranded DNA region.
Figure Legend Snippet: Schematic genome structure of ClorDNAV. Primers used in the PCR experiments are shown as open arrows (see the text and ). Gray arrows indicate the locations for ORFs VP1 to VP4, and the gray colored box is the partially double-stranded DNA region.

Techniques Used: Polymerase Chain Reaction

31) Product Images from "Stepwise partially overlapping primer-based PCR for genome walking"

Article Title: Stepwise partially overlapping primer-based PCR for genome walking

Journal: AMB Express

doi: 10.1186/s13568-018-0610-7

Genome walking of the gadA locus of Lactobacillus brevis NCL912 and hyg of rice. Each walking experiment contained four sets of PCR reactions that respectively utilized the four SWPOP primer sets, SWPOP1 (I), SWPOP2 (II), SWPOP3 (III) and SWPOP4 (IV), paired with a specific primer set. For each set of PCR reactions, the results of primary PCR (P), secondary PCR (S) and tertiary PCR (T) are presented. White arrows indicate target bands. M: DL2000 DNA marker
Figure Legend Snippet: Genome walking of the gadA locus of Lactobacillus brevis NCL912 and hyg of rice. Each walking experiment contained four sets of PCR reactions that respectively utilized the four SWPOP primer sets, SWPOP1 (I), SWPOP2 (II), SWPOP3 (III) and SWPOP4 (IV), paired with a specific primer set. For each set of PCR reactions, the results of primary PCR (P), secondary PCR (S) and tertiary PCR (T) are presented. White arrows indicate target bands. M: DL2000 DNA marker

Techniques Used: Polymerase Chain Reaction, Marker

32) Product Images from "Assessment of pathogenesis of infective endocarditis by plasma IgG antibody titer test against periodontal bacteria"

Article Title: Assessment of pathogenesis of infective endocarditis by plasma IgG antibody titer test against periodontal bacteria

Journal: Clinical Case Reports

doi: 10.1002/ccr3.1066

Detection of bacterial DNA by PCR . Amplified DNA was electrophoresed on 0.7% agarose gel and visualized by ethidium bromide staining. Bacterial DNA in cardiac valve ( CV ) was detected. Square with red dashed line shows detected bacteria from CV samples. DNA bands in numbered lanes (1–10) were extracted and used for DNA sequencing for homology search (Table 3 ). M1: DNA marker (100‐bp ladder); M2: DNA marker ( λ DNA digested by Hin d III ); N: negative control without bacterial DNA ; Aa ( Aggregatibacter actinomycetemcomitans ), Pg ( Porphyromonas gingivalis ), Pi ( Prevotella intermedia ), Ec ( Eichenerra corrodens ), Sm ( Streptococcus mutans ), Fn ( Fusobacterium nucleatum ), and Sa ( Staphylococcus aureus ): positive controls with appropriate bacterial DNA ; CV : test DNA sample extracted from cardiac valve obtained at her first cardiovascular surgery.
Figure Legend Snippet: Detection of bacterial DNA by PCR . Amplified DNA was electrophoresed on 0.7% agarose gel and visualized by ethidium bromide staining. Bacterial DNA in cardiac valve ( CV ) was detected. Square with red dashed line shows detected bacteria from CV samples. DNA bands in numbered lanes (1–10) were extracted and used for DNA sequencing for homology search (Table 3 ). M1: DNA marker (100‐bp ladder); M2: DNA marker ( λ DNA digested by Hin d III ); N: negative control without bacterial DNA ; Aa ( Aggregatibacter actinomycetemcomitans ), Pg ( Porphyromonas gingivalis ), Pi ( Prevotella intermedia ), Ec ( Eichenerra corrodens ), Sm ( Streptococcus mutans ), Fn ( Fusobacterium nucleatum ), and Sa ( Staphylococcus aureus ): positive controls with appropriate bacterial DNA ; CV : test DNA sample extracted from cardiac valve obtained at her first cardiovascular surgery.

Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, DNA Sequencing, Marker, Negative Control

33) Product Images from "Development of specific PCR assays for the detection of Cryptocaryon irritans"

Article Title: Development of specific PCR assays for the detection of Cryptocaryon irritans

Journal: Parasitology Research

doi: 10.1007/s00436-008-0993-5

Agarose gel electrophoresis of PCR products amplified from mixed genomic DNA of C. irritans and I. multifiliis . Lanes 1 and 7 represent a DNA size marker. Lane 2 presents the mixture of C. irritans and I. multifiliis DNA amplified using the primer set P1/S15 and lane 3 represents the genomic DNA of I. multifiliis . Lane 4 represents the mixed genomic DNA of C. irritans and I. multifiliis amplified using the primer set S01–S02. Lane 5 represents the genomic DNA of C. irritans using the primer set S01–S02. Lane 6 represents no-DNA control
Figure Legend Snippet: Agarose gel electrophoresis of PCR products amplified from mixed genomic DNA of C. irritans and I. multifiliis . Lanes 1 and 7 represent a DNA size marker. Lane 2 presents the mixture of C. irritans and I. multifiliis DNA amplified using the primer set P1/S15 and lane 3 represents the genomic DNA of I. multifiliis . Lane 4 represents the mixed genomic DNA of C. irritans and I. multifiliis amplified using the primer set S01–S02. Lane 5 represents the genomic DNA of C. irritans using the primer set S01–S02. Lane 6 represents no-DNA control

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker

Agarose gel electrophoresis of PCR products amplified using conserved primer set P1/NC2 ( a ), Cryptocaryon irritans -specific primer set P1/S15 ( b ), and Ichthyophthirius multifiliis -specific primer set S01/S02 ( c ). Lanes 1 and 11 represent a DNA size marker. Lanes 2 to 9 represent C. irritans , Pseudokeroronpsis rubra , Pseudokeroronpsis carnae , Euplotes sp. 1, I. multifiliis , Pseudourostyla cristata , Paramecium caudaium (cf. Table 1 ), and host (fish) DNA, respectively. Lane 10 represents no-DNA control
Figure Legend Snippet: Agarose gel electrophoresis of PCR products amplified using conserved primer set P1/NC2 ( a ), Cryptocaryon irritans -specific primer set P1/S15 ( b ), and Ichthyophthirius multifiliis -specific primer set S01/S02 ( c ). Lanes 1 and 11 represent a DNA size marker. Lanes 2 to 9 represent C. irritans , Pseudokeroronpsis rubra , Pseudokeroronpsis carnae , Euplotes sp. 1, I. multifiliis , Pseudourostyla cristata , Paramecium caudaium (cf. Table 1 ), and host (fish) DNA, respectively. Lane 10 represents no-DNA control

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Fluorescence In Situ Hybridization

Agarose gel electrophoresis of PCR products amplified from the simulated water body using Cryptocaryon irritans -specific primer set P1/S15. Lanes 1 and 10 represent a DNA size marker, lanes 2 to 7 represent C. irritans from the infected water, lane 8 represents the uninfected water, and lane 9 represents no-DNA control
Figure Legend Snippet: Agarose gel electrophoresis of PCR products amplified from the simulated water body using Cryptocaryon irritans -specific primer set P1/S15. Lanes 1 and 10 represent a DNA size marker, lanes 2 to 7 represent C. irritans from the infected water, lane 8 represents the uninfected water, and lane 9 represents no-DNA control

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Infection

Evaluation of the sensitivity of the specific PCR assay for Cryptocaryon irritans (primer set P1/S15) by agarose gel electrophoresis. Lanes 1 and 13 represent a DNA size marker. Lane 2 represents DNA undiluted (450 ng/µl), and lanes 3 to 11 represent C. irritans DNA diluted for 1:50 (9 ng/µl), 1:100 (4.5 ng/µl), 1:200 (2.25 ng/µl), 1:400 (1.13 ng/µl), 1:800 (0.56 ng/µl), 1:10 3 (0.45 ng/µl), 1:10 4 (45 pg/µl), 1:10 5 (4.5 pg/µl), and 1:10 6 (0.45 pg/µl), respectively. Lane 12 represents no-DNA control
Figure Legend Snippet: Evaluation of the sensitivity of the specific PCR assay for Cryptocaryon irritans (primer set P1/S15) by agarose gel electrophoresis. Lanes 1 and 13 represent a DNA size marker. Lane 2 represents DNA undiluted (450 ng/µl), and lanes 3 to 11 represent C. irritans DNA diluted for 1:50 (9 ng/µl), 1:100 (4.5 ng/µl), 1:200 (2.25 ng/µl), 1:400 (1.13 ng/µl), 1:800 (0.56 ng/µl), 1:10 3 (0.45 ng/µl), 1:10 4 (45 pg/µl), 1:10 5 (4.5 pg/µl), and 1:10 6 (0.45 pg/µl), respectively. Lane 12 represents no-DNA control

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

34) Product Images from "Complete Genome Sequence of the First Chinese Virulent Infectious Laryngotracheitis Virus"

Article Title: Complete Genome Sequence of the First Chinese Virulent Infectious Laryngotracheitis Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070154

Ethidium bromide stained agarose gels of PCR amplified DNA. A: The PCR product amplified from the genomic DNA using the gB primers (Lane 1) and negative control (Lane 2). B: The single primer PCR products using the SP-U L -5′; C: The specificity of the product using the nested primers; D: The PCR products of IR S and TR S . Lane 1: The PCR product amplified using the U L -IR S forward and U L -IR S reverse primers; Lane 2: The PCR product amplified using the IR-US forward and IR-US reverse primers; Lane 3: The PCR product amplified using the U S -TR forward and US-TR reverse primers; and Lane 4: The PCR product amplified using the TR-UL forward and TR-UL reverse primers.
Figure Legend Snippet: Ethidium bromide stained agarose gels of PCR amplified DNA. A: The PCR product amplified from the genomic DNA using the gB primers (Lane 1) and negative control (Lane 2). B: The single primer PCR products using the SP-U L -5′; C: The specificity of the product using the nested primers; D: The PCR products of IR S and TR S . Lane 1: The PCR product amplified using the U L -IR S forward and U L -IR S reverse primers; Lane 2: The PCR product amplified using the IR-US forward and IR-US reverse primers; Lane 3: The PCR product amplified using the U S -TR forward and US-TR reverse primers; and Lane 4: The PCR product amplified using the TR-UL forward and TR-UL reverse primers.

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

35) Product Images from "Relationship between Expression of Cellular Receptor-27.8kDa and Lymphocystis Disease Virus (LCDV) Infection"

Article Title: Relationship between Expression of Cellular Receptor-27.8kDa and Lymphocystis Disease Virus (LCDV) Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0127940

Dynamics of LCDV copies in FG and HINAE cells post LCDV infection investigated by qPCR. (A) Standard curve of LCDV MCP qPCR assays. The X-axis showed the positive control plasmid copy number in Log 10 value, and the Y-axis indicated the corresponding cycle threshold (Ct) value. R 2 : coefficient of determination. (B) Changes of LCDV copies in FG and HINAE cells post LCDV infection. 0 h represented un-infected cells. Error bars represented SD. Data represented the number of LCDV copies per microgram of total DNA in the cell samples (mean ± SD; n = 3).
Figure Legend Snippet: Dynamics of LCDV copies in FG and HINAE cells post LCDV infection investigated by qPCR. (A) Standard curve of LCDV MCP qPCR assays. The X-axis showed the positive control plasmid copy number in Log 10 value, and the Y-axis indicated the corresponding cycle threshold (Ct) value. R 2 : coefficient of determination. (B) Changes of LCDV copies in FG and HINAE cells post LCDV infection. 0 h represented un-infected cells. Error bars represented SD. Data represented the number of LCDV copies per microgram of total DNA in the cell samples (mean ± SD; n = 3).

Techniques Used: Infection, Real-time Polymerase Chain Reaction, Positive Control, Plasmid Preparation

36) Product Images from "Detection of Talaromyces marneffei from Fresh Tissue of an Inhalational Murine Pulmonary Model Using Nested PCR"

Article Title: Detection of Talaromyces marneffei from Fresh Tissue of an Inhalational Murine Pulmonary Model Using Nested PCR

Journal: PLoS ONE

doi: 10.1371/journal.pone.0149634

Specificity and sensibility of nested PCR. A 600-bp PCR product were amplified from all fungal samples (a) and a 400-bp PCR product was amplified from T . marneffei samples (b). M, 100-bp-ladder DNA; Lane1 to 13, T . marneffei , Aspergillus flavus , Aspergillus fumigatus , Aspergillus niger , Cryptococcus neoformans , Candida albicans , Candida krusei , Fonsecaea pedrosoi , Fonsecaea monophora , Histoplasma capsulatum , Paracoccidioides brasiliensis , Negative control (mice DNA), Negative control (water) respectively. Sensitivity of nested PCR (c) and single PCR (d) was 8.4×10 4 and 8.4×10 7 fg/μl, respectively. M, 100-bp-ladder DNA; lanes 1 to 13, 8.4×10 9 , 8.4×10 8 , 8.4×10 7 , 8.4×10 6 , 8.4×10 5 , 8.4×10 4 , 8.4×10 3 , 8.4×10 2 , 8.4×10 1 , 8.4×10 0 , 8.4×10 −1 , 8.4×10 −2 , 8.4×10 −3 f g /μl, respectively.
Figure Legend Snippet: Specificity and sensibility of nested PCR. A 600-bp PCR product were amplified from all fungal samples (a) and a 400-bp PCR product was amplified from T . marneffei samples (b). M, 100-bp-ladder DNA; Lane1 to 13, T . marneffei , Aspergillus flavus , Aspergillus fumigatus , Aspergillus niger , Cryptococcus neoformans , Candida albicans , Candida krusei , Fonsecaea pedrosoi , Fonsecaea monophora , Histoplasma capsulatum , Paracoccidioides brasiliensis , Negative control (mice DNA), Negative control (water) respectively. Sensitivity of nested PCR (c) and single PCR (d) was 8.4×10 4 and 8.4×10 7 fg/μl, respectively. M, 100-bp-ladder DNA; lanes 1 to 13, 8.4×10 9 , 8.4×10 8 , 8.4×10 7 , 8.4×10 6 , 8.4×10 5 , 8.4×10 4 , 8.4×10 3 , 8.4×10 2 , 8.4×10 1 , 8.4×10 0 , 8.4×10 −1 , 8.4×10 −2 , 8.4×10 −3 f g /μl, respectively.

Techniques Used: Nested PCR, Polymerase Chain Reaction, Amplification, Negative Control, Mouse Assay

Nested PCR assays in fresh lung tissues (a) and BALF (b). A 400-bp specific product was amplified in most of samples. M: 100-bp-ladder DNA, P, purified genomic DNA from T . marneffei , T, lung tissue from healthy mice, B, BALF from healthy mice, N, water.
Figure Legend Snippet: Nested PCR assays in fresh lung tissues (a) and BALF (b). A 400-bp specific product was amplified in most of samples. M: 100-bp-ladder DNA, P, purified genomic DNA from T . marneffei , T, lung tissue from healthy mice, B, BALF from healthy mice, N, water.

Techniques Used: Nested PCR, Amplification, Purification, Mouse Assay

37) Product Images from "Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing"

Article Title: Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing

Journal: BMC Genomics

doi: 10.1186/s12864-015-2079-y

Mutant discovery by using HRM and indexed amplicon sequencing. DNA extracted from M2’ plants was preserved as the original DNA stock in 96-well plates. The DNA pool in a 384-well plate (four samples per pool) was used for both methods. After a mutation was detected by HRM analysis, base changes in four original DNA samples were confirmed by direct sequencing. If the mutation was found to be silent, HRM analysis and direct sequencing of other regions were performed. In indexed amplicon sequencing, 7 target gene regions (1.3–7.5 kb, 30.3 kb in total) were amplified by long-range PCR. The amplicons of four samples were further pooled. The 96 samples were indexed by using a transposome-based Nextera XT Index kit. Bulk read data for all 96 DNA pools were obtained from Miseq and mapped onto the reference sequences of target genes after classification of the DNA pool by using indices. Base changes at high frequency in many reads were treated as a mutation and were filtered by using a Glyma_189 gene annotation to exclude mutations that did not lead to amino acid substitutions. Based on the information from DNA pool classification with indices, the base change and the plant in which it occurred could be determined by direct sequencing of each of the 16 original M2’ DNA samples. Amplicon sequencing using NGS allows rapid and effective detection of DNA pools containing mutations that cause desirable functional amino acid substitutions
Figure Legend Snippet: Mutant discovery by using HRM and indexed amplicon sequencing. DNA extracted from M2’ plants was preserved as the original DNA stock in 96-well plates. The DNA pool in a 384-well plate (four samples per pool) was used for both methods. After a mutation was detected by HRM analysis, base changes in four original DNA samples were confirmed by direct sequencing. If the mutation was found to be silent, HRM analysis and direct sequencing of other regions were performed. In indexed amplicon sequencing, 7 target gene regions (1.3–7.5 kb, 30.3 kb in total) were amplified by long-range PCR. The amplicons of four samples were further pooled. The 96 samples were indexed by using a transposome-based Nextera XT Index kit. Bulk read data for all 96 DNA pools were obtained from Miseq and mapped onto the reference sequences of target genes after classification of the DNA pool by using indices. Base changes at high frequency in many reads were treated as a mutation and were filtered by using a Glyma_189 gene annotation to exclude mutations that did not lead to amino acid substitutions. Based on the information from DNA pool classification with indices, the base change and the plant in which it occurred could be determined by direct sequencing of each of the 16 original M2’ DNA samples. Amplicon sequencing using NGS allows rapid and effective detection of DNA pools containing mutations that cause desirable functional amino acid substitutions

Techniques Used: Mutagenesis, Amplification, Sequencing, Polymerase Chain Reaction, Next-Generation Sequencing, Functional Assay

38) Product Images from "Feline Immunodeficiency Virus OrfA Is Distinct from Other Lentivirus Transactivators"

Article Title: Feline Immunodeficiency Virus OrfA Is Distinct from Other Lentivirus Transactivators

Journal: Journal of Virology

doi: 10.1128/JVI.76.19.9624-9634.2002

Gal4-OrfA assay. (A) PCR strategy followed for generating the Gal4-OrfA deletion clones by PCR-ligation-PCR (see Materials and Methods for details). (B) Diagram of the deletion clones of OrfA fused in frame with the Gal4 DNA-binding domain generated by the strategy describe in panel A. (C) Autoradiogram of the various deletion constructs expressed in vitro under the T7 promoter, immunoprecipitated with anti-OrfA polyclonal serum, and resolved by SDS-10 to 20% PAGE. (D) Transactivation of the G 5 -E 1 b-CAT target construct by the different Gal4-OrfA deletion constructs and appropriate controls.
Figure Legend Snippet: Gal4-OrfA assay. (A) PCR strategy followed for generating the Gal4-OrfA deletion clones by PCR-ligation-PCR (see Materials and Methods for details). (B) Diagram of the deletion clones of OrfA fused in frame with the Gal4 DNA-binding domain generated by the strategy describe in panel A. (C) Autoradiogram of the various deletion constructs expressed in vitro under the T7 promoter, immunoprecipitated with anti-OrfA polyclonal serum, and resolved by SDS-10 to 20% PAGE. (D) Transactivation of the G 5 -E 1 b-CAT target construct by the different Gal4-OrfA deletion constructs and appropriate controls.

Techniques Used: Polymerase Chain Reaction, Clone Assay, Ligation, Binding Assay, Generated, Construct, In Vitro, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

39) Product Images from "Detection of JC Polyomavirus tumor antigen in gastric carcinoma: a report from Iran"

Article Title: Detection of JC Polyomavirus tumor antigen in gastric carcinoma: a report from Iran

Journal: Iranian Journal of Microbiology

doi:

Standard curve analysis. Synthetic DNA standard curves for absolute quantification of JC-T-Ag by real-time PCR. The JCV-Mad-1 T-Ag sequence that cloned in a pBHA plasmid vector was serially diluted (from 100 to 10 8 copies of positive control DNA) and served as a standard reference. High amplification efficiency has also been shown by a good linear relationship among each concentration.
Figure Legend Snippet: Standard curve analysis. Synthetic DNA standard curves for absolute quantification of JC-T-Ag by real-time PCR. The JCV-Mad-1 T-Ag sequence that cloned in a pBHA plasmid vector was serially diluted (from 100 to 10 8 copies of positive control DNA) and served as a standard reference. High amplification efficiency has also been shown by a good linear relationship among each concentration.

Techniques Used: Real-time Polymerase Chain Reaction, Sequencing, Clone Assay, Plasmid Preparation, Positive Control, Amplification, Concentration Assay

40) Product Images from "MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform"

Article Title: MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform

Journal: Scientific Reports

doi: 10.1038/srep16963

Construction of the MIG-seq library. ( a ) Multiple non-repetitive regions from various inter-simple sequence repeats (ISSRs) are amplified from genomic DNA by multiplexed PCR with tailed ISSR primers (1st PCR). ( b ) The 1st PCR products are subsequently used as the templates for the 2nd PCR (tailed PCR). This step enables the addition of complementary sequences for the binding sites of Illumina sequencing flow cell and indices (barcodes) for each sample to the 1st PCR products, using common forward and indexed reverse primers. ( c ) After measuring the approximate concentration of each 2nd PCR product, they are pooled in equimolar concentrations as a single mixture library. The mixture is then purified, fragments with a size range of 300–800 bp are isolated, the final concentration is measured by quantitative PCR, and is then used for Illumina paired-end sequencing (reads 1 and 2) and index reading. Sequencing of the first 17 nucleotides (primer region) of read 1, and 3 nucleotides (anchor region) of read 2 are skipped using the ‘DarkCycle’ option of the sequencer (indicated as the gray region in the arrows). ( d ) The sequence of the resulting library consists of binding sites for the P5 flow cell oligonucleotides and read 1 sequencing primer, forward ISSR primer, DNA insert, reverse ISSR primer, binding sites f or read 2 and index sequencing primers, and P7 flow cell oligonucleotides.
Figure Legend Snippet: Construction of the MIG-seq library. ( a ) Multiple non-repetitive regions from various inter-simple sequence repeats (ISSRs) are amplified from genomic DNA by multiplexed PCR with tailed ISSR primers (1st PCR). ( b ) The 1st PCR products are subsequently used as the templates for the 2nd PCR (tailed PCR). This step enables the addition of complementary sequences for the binding sites of Illumina sequencing flow cell and indices (barcodes) for each sample to the 1st PCR products, using common forward and indexed reverse primers. ( c ) After measuring the approximate concentration of each 2nd PCR product, they are pooled in equimolar concentrations as a single mixture library. The mixture is then purified, fragments with a size range of 300–800 bp are isolated, the final concentration is measured by quantitative PCR, and is then used for Illumina paired-end sequencing (reads 1 and 2) and index reading. Sequencing of the first 17 nucleotides (primer region) of read 1, and 3 nucleotides (anchor region) of read 2 are skipped using the ‘DarkCycle’ option of the sequencer (indicated as the gray region in the arrows). ( d ) The sequence of the resulting library consists of binding sites for the P5 flow cell oligonucleotides and read 1 sequencing primer, forward ISSR primer, DNA insert, reverse ISSR primer, binding sites f or read 2 and index sequencing primers, and P7 flow cell oligonucleotides.

Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Binding Assay, Flow Cytometry, Concentration Assay, Purification, Isolation, Real-time Polymerase Chain Reaction

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Article Snippet: .. Fifty microliters of PCR master mix was prepared and contained 150 ng of template DNA, 5 μl of 10× Ex- Taq buffer (Takara-Bio), 200 μM each deoxynucleoside triphosphate (Takara-Bio), 0.25 μM of 23S rRNA gene or hly gene primers (Takara-Bio), 0.4× SYBR green (BMA, Rockland, ME), and 1.25 U of Ex- Taq polymerase (Takara-Bio). .. The PCR protocol for the 23S rRNA gene of L. monocytogenes was 1 cycle at 4°C for 3 min, 1 cycle at 94°C for 30 s, and 40 cycles at 94°C for 20 s, 46°C for 30 s, and 72°C for 1 min. After PCR, the Tm (melting-point measurement) pattern analysis of PCR product was carried out with 1 cycle at 95°C for 3 min, followed by being cooled at 60°C and heated to 95°C at the rate of 0.75°C per min.

Article Title: Detection of JC Polyomavirus tumor antigen in gastric carcinoma: a report from Iran
Article Snippet: .. The PCR reaction mixture volume was 20 μL consisting of 1.5 μL of template DNA, 10 μL of Takara SYBR Premix Ex Taq II, 6.5 μL dH2O and 1 μL (10 pmol) of each primer. .. The following PCR protocol was used: initial denaturation step (95°C for 7 minutes), amplification and quantification program repeated 35 times (denaturation at 95°C for 15 seconds, followed by annealing at 60°C for 45 seconds and extension 72°C for 60 seconds with a single fluorescence measurement).

Article Title: Stepwise partially overlapping primer-based PCR for genome walking
Article Snippet: .. In the primary PCR reaction, the 50 μL reaction mixture contained 1 × LA PCR buffer II (Mg2+ plus), 400 μM of each dNTP, 0.2 μM of each primer, template DNA (10–100 ng for L. brevis NCL912 and 100–1000 ng for rice), and 2.5 U of TaKaRa LA Taq HS. .. In the secondary/tertiary PCR reaction, the 50 μL reaction mixture included 1 × LA PCR buffer II (Mg2+ plus), 400 μM of each dNTP, 0.2 μM of each primer, 1 μL of the previous round of PCR products, and 2.5 U of TaKaRa LA Taq HS.

Article Title: Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene
Article Snippet: .. Twenty-five microlitres PCR reaction consists of 1 μl template DNA or cDNA, 1 U Taq DNA polymerase (TaKaRa) and 0.1 μM of each oligonucleotide primer in nuclease-free water. qPCR was performed by using Brilliant SYBR Green qPCR Master Mix and Mx3005P™ instrument (Stratagene). .. Products of PCR and RT-PCR were identified by agarose gel electrophoresis, and cycle threshold (Ct) values of qPCR were used to calculate the quantity of each target DNA or cDNA.

Modification:

Article Title: BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles
Article Snippet: .. We set up 150 μl PCR reactions containing 10 μl 20 pmol/l template DNA, 2X TITANIUM Taq DNA Polymerase (Clontech, Mountain View, CA), 1X TITANIUM Taq buffer (Clontech), 1 mmol/l dNTP mix, 8.3 mmol/l MgCl2 (Invitrogen), 0.05 μmol/l universal forward primer (5′-tcccgcgaaattaatacgac-3′), 8 μmol/l nested reverse primer (5′-AATCAGTTGCTCTGTATTGATCC-3′), and 6 × 107 magnetic streptavidin beads (MyOne, Invitrogen) coated with a modified universal forward primer (5′-dual biotion-T-Spacer18-tcccgcgaaattaatacgac-3′). .. The aqueous reaction mixture was then combined with 600 μl of an oil/emulsifier mixture of 7% w/v ABIL WE09 (Degussa, Essen, GE), 20% v/v M3516 mineral oil (Sigma-Aldrich, St Louis, MO) and 73% v/v Tegosoft DEC (Degussa), and one 5 mm steel bead (Qiagen) in one well of a 96-deep-well plate (Abgene, Lafayette, CO).

SYBR Green Assay:

Article Title: Method To Detect Only Live Bacteria during PCR Amplification
Article Snippet: .. Fifty microliters of PCR master mix was prepared and contained 150 ng of template DNA, 5 μl of 10× Ex- Taq buffer (Takara-Bio), 200 μM each deoxynucleoside triphosphate (Takara-Bio), 0.25 μM of 23S rRNA gene or hly gene primers (Takara-Bio), 0.4× SYBR green (BMA, Rockland, ME), and 1.25 U of Ex- Taq polymerase (Takara-Bio). .. The PCR protocol for the 23S rRNA gene of L. monocytogenes was 1 cycle at 4°C for 3 min, 1 cycle at 94°C for 30 s, and 40 cycles at 94°C for 20 s, 46°C for 30 s, and 72°C for 1 min. After PCR, the Tm (melting-point measurement) pattern analysis of PCR product was carried out with 1 cycle at 95°C for 3 min, followed by being cooled at 60°C and heated to 95°C at the rate of 0.75°C per min.

Article Title: Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene
Article Snippet: .. Twenty-five microlitres PCR reaction consists of 1 μl template DNA or cDNA, 1 U Taq DNA polymerase (TaKaRa) and 0.1 μM of each oligonucleotide primer in nuclease-free water. qPCR was performed by using Brilliant SYBR Green qPCR Master Mix and Mx3005P™ instrument (Stratagene). .. Products of PCR and RT-PCR were identified by agarose gel electrophoresis, and cycle threshold (Ct) values of qPCR were used to calculate the quantity of each target DNA or cDNA.

Real-time Polymerase Chain Reaction:

Article Title: Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
Article Snippet: .. The reaction for qPCR contained 2 μL of template DNA, 10 μL of 2 × SYBR Premix Ex Taq, 0.4 μL of 50 × ROX Reference Dye II (TaKaRa, Kusatsu, Shiga, Japan), 0.4 μM of each forward and reverse primer, and ultrapure water to bring up the final reaction volume to 20 μL. .. The qPCR was performed by using the ABI 7500 Fast fluorescence system (Applied Biosystems, Carlsbad, California, USA), and the following program setting: 1 min at 95°C; 40 cycles of 10 s at 95°C, 30 s at 60°C and 34 s at 72°C.

Article Title: Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene
Article Snippet: .. Twenty-five microlitres PCR reaction consists of 1 μl template DNA or cDNA, 1 U Taq DNA polymerase (TaKaRa) and 0.1 μM of each oligonucleotide primer in nuclease-free water. qPCR was performed by using Brilliant SYBR Green qPCR Master Mix and Mx3005P™ instrument (Stratagene). .. Products of PCR and RT-PCR were identified by agarose gel electrophoresis, and cycle threshold (Ct) values of qPCR were used to calculate the quantity of each target DNA or cDNA.

Construct:

Article Title: Genetic, Epigenetic, and HPLC Fingerprint Differentiation between Natural and Ex Situ Populations of Rhodiola sachalinensis from Changbai Mountain, China
Article Snippet: .. The optimized ISSR-PCR reaction system (25 µl) for R. sachalinensis was constructed of 2 µl template DNA, 2.5 µl of 10× Taq DNA polymerase buffer, 0.25 mM dNTPs, 1.5 mM MgCl2 , 0.4 µM primer, 1.25 U of Taq polymerase (Takara, Dalian, China), and double-distilled water. .. The amplifications were carried out in a Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Germany) under the following conditions: pre-denaturation for 5 min at 94°C, followed by 40 cycles of denaturation for 30 s at 94°C, annealing for 60 s at various temperatures (see ), and elongation for 90 s at 72°C, with a final extension for 10 min at 72°C.

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  • 91
    TaKaRa template plasmid dna
    Optimization of the <t>TTcDR</t> system. A ) Optimization of each component in the TTcDR reaction. TTcDR reactions were performed at the indicated concentrations of the various components with circular <t>DNA</t> (1 ng/μl) for 12 h at 30 °C. The amount of DNA product was measured as described in Fig. 2 . The original concentrations and the optimized concentrations are indicated by black and white arrowheads, respectively. NTPs include each nucleotide triphosphate (ATP:GTP:CTP:UTP = 3.75:2.5:1.25:1.25) and the same molarity of magnesium acetate. B ) Comparison of DNA produced by TTcDR before and after optimization. The TTcDR reaction was performed with 1 ng/μl DNA for 2 h at 30 °C. The compositions before and after optimization are described in the Methods section. The error bars indicate the standard error (n = 14). Original gel images are shown in Figure S2 .
    Template Plasmid Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa template extracted dna
    T-RFLP profiling of the cyanobacterial 16S rRNA gene amplified as template total <t>DNA</t> of the gastric content of dead egrets. KS2P (A) and KS4P (B) were used for <t>PCR</t> amplification. HaeIII was used for digestion. The x-axis indicates the terminal restriction fragment length (bases) between 0 and 700 bases, and the y-axis represents the relative height of the peak. The highest peak was calculated as 1. A relative height of over 0.1 showed the T-RF length.
    Template Extracted Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa dna templates
    Composite images of ADGE microarray and regular microarray. The clones corresponding to the contiguous area of twelve spots (3 × 4) were amplified by using <t>PCR</t> with the primers having a Taq I site at the end. After cut with Taq I , the same amount of <t>DNA</t> for each clone was ligated to the CT and TT adapters. The CT and TT adapter-linked DNA fragments were mixed in ratios of 1:1 for the three clones of the first column, 1:2 for the clones of the second column, 1:3 for the clones of the third column, 1:4 for the clones of the fourth column. The top panel is the result of ADGE microarray while the bottom panel is the result of regular microarray. The ratios are represented by Cy5 (green) to Cy3 (red) and normalized with the value of clones in the first column. The detected ratios are averages of three spots in each column.
    Dna Templates, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Optimization of the TTcDR system. A ) Optimization of each component in the TTcDR reaction. TTcDR reactions were performed at the indicated concentrations of the various components with circular DNA (1 ng/μl) for 12 h at 30 °C. The amount of DNA product was measured as described in Fig. 2 . The original concentrations and the optimized concentrations are indicated by black and white arrowheads, respectively. NTPs include each nucleotide triphosphate (ATP:GTP:CTP:UTP = 3.75:2.5:1.25:1.25) and the same molarity of magnesium acetate. B ) Comparison of DNA produced by TTcDR before and after optimization. The TTcDR reaction was performed with 1 ng/μl DNA for 2 h at 30 °C. The compositions before and after optimization are described in the Methods section. The error bars indicate the standard error (n = 14). Original gel images are shown in Figure S2 .

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Optimization of the TTcDR system. A ) Optimization of each component in the TTcDR reaction. TTcDR reactions were performed at the indicated concentrations of the various components with circular DNA (1 ng/μl) for 12 h at 30 °C. The amount of DNA product was measured as described in Fig. 2 . The original concentrations and the optimized concentrations are indicated by black and white arrowheads, respectively. NTPs include each nucleotide triphosphate (ATP:GTP:CTP:UTP = 3.75:2.5:1.25:1.25) and the same molarity of magnesium acetate. B ) Comparison of DNA produced by TTcDR before and after optimization. The TTcDR reaction was performed with 1 ng/μl DNA for 2 h at 30 °C. The compositions before and after optimization are described in the Methods section. The error bars indicate the standard error (n = 14). Original gel images are shown in Figure S2 .

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Produced

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Marker, Purification

    Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Translation of phi29 DNA polymerase from newly synthesized DNA in the TTcDR reaction. A ) Experimental procedure. First, we performed the optimized TTcDR reaction without [ 35 S]-methionine in the presence or absence of dNTPs, and one-tenth of the mixture was transferred to the second reaction mixture, which contained [ 35 S]-methionine, to detect translation from the replicated DNA product in the first reaction. After incubation at 30 °C for 12 h, an aliquot was used for 10% SDS-PAGE and autoradiography. B ) Translation results. Increased translation of the DNA polymerase was detected when the first reaction contained dNTPs, indicating that the translation occurred from the DNA produced in the first reaction.

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Synthesized, Incubation, SDS Page, Autoradiography, Produced

    Characterization of the optimized TTcDR reaction. A ) Cleavage of the TTcDR product by restriction enzymes. After the TTcDR reaction was conducted for 12 h at 30 °C, the indicated restriction enzymes were added and incubated for 1 h at 37 °C. An aliquot was used for 1% agarose gel electrophoresis and autoradiography. The sample treated with Pst I was purified using a DNA column (Life Technologies) before electrophoresis. B ) Time-course data for the translation of DNA polymerase during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 35 S]-methionine at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 10% SDS-PAGE and autoradiography. The error bars indicate the standard error (n = 4). C ) Time-course data for DNA replication during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 32 P]-dCTP at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 1% agarose gel electrophoresis and autoradiography. The error bars indicate the standard error (n = 4).

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Characterization of the optimized TTcDR reaction. A ) Cleavage of the TTcDR product by restriction enzymes. After the TTcDR reaction was conducted for 12 h at 30 °C, the indicated restriction enzymes were added and incubated for 1 h at 37 °C. An aliquot was used for 1% agarose gel electrophoresis and autoradiography. The sample treated with Pst I was purified using a DNA column (Life Technologies) before electrophoresis. B ) Time-course data for the translation of DNA polymerase during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 35 S]-methionine at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 10% SDS-PAGE and autoradiography. The error bars indicate the standard error (n = 4). C ) Time-course data for DNA replication during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 32 P]-dCTP at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 1% agarose gel electrophoresis and autoradiography. The error bars indicate the standard error (n = 4).

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Incubation, Agarose Gel Electrophoresis, Autoradiography, Purification, Electrophoresis, SDS Page

    DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: DNA replication with or without random hexamers in the absence of TTcDR components. DNA replication was performed by purified phi29 DNA polymerase with or without random hexamers in the TTcDR mixtures in which some of the components (NTP, tRNA, T7 polymerase, ribosome, and translation proteins) were omitted, and the amount of replicated DNA was measured as described in the Methods section. The translation proteins contained all protein factors in the translation system (e.g., IFs, EFs, RFs, and aminoacyl-tRNA synthetases). In the experiments with random hexamers, the template plasmid was heated with the hexamers at 95 °C for 3 min and then cooled immediately.

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Purification, Plasmid Preparation

    T-RFLP profiling of the cyanobacterial 16S rRNA gene amplified as template total DNA of the gastric content of dead egrets. KS2P (A) and KS4P (B) were used for PCR amplification. HaeIII was used for digestion. The x-axis indicates the terminal restriction fragment length (bases) between 0 and 700 bases, and the y-axis represents the relative height of the peak. The highest peak was calculated as 1. A relative height of over 0.1 showed the T-RF length.

    Journal: The Open Microbiology Journal

    Article Title: Molecular Analysis of the Cyanobacterial Community in Gastric Contents of Egrets with Symptoms of Steatitis

    doi: 10.2174/1874285801509010160

    Figure Lengend Snippet: T-RFLP profiling of the cyanobacterial 16S rRNA gene amplified as template total DNA of the gastric content of dead egrets. KS2P (A) and KS4P (B) were used for PCR amplification. HaeIII was used for digestion. The x-axis indicates the terminal restriction fragment length (bases) between 0 and 700 bases, and the y-axis represents the relative height of the peak. The highest peak was calculated as 1. A relative height of over 0.1 showed the T-RF length.

    Article Snippet: The PCR mixture (30 µl) was prepared by combining 0.1-0.01 µg of template-extracted DNA, 1.0 µl of 10 pmol/µl primers, Takara Ex Taq polymerase, dNTPs, and 3.0 µl of optimized 10-fold Ex buffer (Takara Bio) in a PCR thermal cycler.

    Techniques: Amplification, Polymerase Chain Reaction

    Specificity and sensitivity of PMAxx-qPCR assay in detecting viable cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ). Viable or heat-killed Cmm cells at various concentrations were treated with 20 μM PMAxx, followed by DNA extraction and qPCR detection. Ct: threshold cycle of qPCR. CFU: colony forming unit. PMA: propidium monoazide. Columns and bars represent mean values and standard deviations. Means followed by different letters are significantly different ( P

    Journal: PLoS ONE

    Article Title: Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR

    doi: 10.1371/journal.pone.0196525

    Figure Lengend Snippet: Specificity and sensitivity of PMAxx-qPCR assay in detecting viable cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ). Viable or heat-killed Cmm cells at various concentrations were treated with 20 μM PMAxx, followed by DNA extraction and qPCR detection. Ct: threshold cycle of qPCR. CFU: colony forming unit. PMA: propidium monoazide. Columns and bars represent mean values and standard deviations. Means followed by different letters are significantly different ( P

    Article Snippet: The reaction for qPCR contained 2 μL of template DNA, 10 μL of 2 × SYBR Premix Ex Taq, 0.4 μL of 50 × ROX Reference Dye II (TaKaRa, Kusatsu, Shiga, Japan), 0.4 μM of each forward and reverse primer, and ultrapure water to bring up the final reaction volume to 20 μL.

    Techniques: Real-time Polymerase Chain Reaction, DNA Extraction

    Detection of culturable, viable but non-culturable (VBNC) and dead cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ) by PMAxx-qPCR from artificially inoculated tomato seed. Ten tomato seeds were soaked in log phase (A), copper-induced VBNC (B) and heat-killed (C) Cmm cells suspension (10 8 CFU mL -1 ) by vacuum infiltration. After inoculation, the seed was broken by a ball mill and diluted 20-fold with 0.85% NaCl solution, followed by treatment with or without PMAxx at a final concentration of 20 μM. DNA was extracted after PMAxx treatment and used for qPCR assay. Cycle threshold (Ct) of qPCR was separated using multiple range test, and means labeled with different letters were significantly different ( P

    Journal: PLoS ONE

    Article Title: Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR

    doi: 10.1371/journal.pone.0196525

    Figure Lengend Snippet: Detection of culturable, viable but non-culturable (VBNC) and dead cells of Clavibacter michiganensis subsp. michiganensis ( Cmm ) by PMAxx-qPCR from artificially inoculated tomato seed. Ten tomato seeds were soaked in log phase (A), copper-induced VBNC (B) and heat-killed (C) Cmm cells suspension (10 8 CFU mL -1 ) by vacuum infiltration. After inoculation, the seed was broken by a ball mill and diluted 20-fold with 0.85% NaCl solution, followed by treatment with or without PMAxx at a final concentration of 20 μM. DNA was extracted after PMAxx treatment and used for qPCR assay. Cycle threshold (Ct) of qPCR was separated using multiple range test, and means labeled with different letters were significantly different ( P

    Article Snippet: The reaction for qPCR contained 2 μL of template DNA, 10 μL of 2 × SYBR Premix Ex Taq, 0.4 μL of 50 × ROX Reference Dye II (TaKaRa, Kusatsu, Shiga, Japan), 0.4 μM of each forward and reverse primer, and ultrapure water to bring up the final reaction volume to 20 μL.

    Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Labeling

    Composite images of ADGE microarray and regular microarray. The clones corresponding to the contiguous area of twelve spots (3 × 4) were amplified by using PCR with the primers having a Taq I site at the end. After cut with Taq I , the same amount of DNA for each clone was ligated to the CT and TT adapters. The CT and TT adapter-linked DNA fragments were mixed in ratios of 1:1 for the three clones of the first column, 1:2 for the clones of the second column, 1:3 for the clones of the third column, 1:4 for the clones of the fourth column. The top panel is the result of ADGE microarray while the bottom panel is the result of regular microarray. The ratios are represented by Cy5 (green) to Cy3 (red) and normalized with the value of clones in the first column. The detected ratios are averages of three spots in each column.

    Journal: BMC Genomics

    Article Title: Sensitivity and fidelity of DNA microarray improved with integration of Amplified Differential Gene Expression (ADGE)

    doi: 10.1186/1471-2164-4-28

    Figure Lengend Snippet: Composite images of ADGE microarray and regular microarray. The clones corresponding to the contiguous area of twelve spots (3 × 4) were amplified by using PCR with the primers having a Taq I site at the end. After cut with Taq I , the same amount of DNA for each clone was ligated to the CT and TT adapters. The CT and TT adapter-linked DNA fragments were mixed in ratios of 1:1 for the three clones of the first column, 1:2 for the clones of the second column, 1:3 for the clones of the third column, 1:4 for the clones of the fourth column. The top panel is the result of ADGE microarray while the bottom panel is the result of regular microarray. The ratios are represented by Cy5 (green) to Cy3 (red) and normalized with the value of clones in the first column. The detected ratios are averages of three spots in each column.

    Article Snippet: The PCR labeling reaction was set up with 42 μl of DNA templates, 5 μl of 10x Clontech PCR buffer, 1 μl of 10 mM dNTPS containing 8 mM aminoallyl-dUTP and 2 mM dTTP, 1 μl of 10 μM CT primer or TT primer and 1 μl of Clontech cDNA polymerase.

    Techniques: Microarray, Clone Assay, Amplification, Polymerase Chain Reaction