Structured Review

Promega pcr master mix
Amplification pattern by <t>RT-PCR</t> with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers <t>inF-F</t> and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.
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Images

1) Product Images from "Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications"

Article Title: Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications

Journal: BMC Microbiology

doi: 10.1186/1471-2180-11-94

Amplification pattern by RT-PCR with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers inF-F and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.
Figure Legend Snippet: Amplification pattern by RT-PCR with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers inF-F and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.

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

2) Product Images from "The Presumptive Magnetosome Protein Mms16 Is a Poly(3-Hydroxybutyrate) Granule-Bound Protein (Phasin) in Magnetospirillum gryphiswaldense"

Article Title: The Presumptive Magnetosome Protein Mms16 Is a Poly(3-Hydroxybutyrate) Granule-Bound Protein (Phasin) in Magnetospirillum gryphiswaldense

Journal: Journal of Bacteriology

doi: 10.1128/JB.187.7.2416-2425.2005

Schematic representation of the insertion deletion. (A) mms16 wild-type gene and different truncated fragments (I to III). Sizes are as indicated. (B) Molecular organization of the mms16 locus before and after insertion-duplication mutagenesis with a truncated fragment. Different fill patterns are used to mark the origins of different parts of the gene after a single crossover. Parts encoding the N and C termini of the corresponding gene products are indicated. (C) Characterization of the Da127 mutant by RT-PCR. The following primer combinations were applied in PCRs: mmpF1 plus mmpB1 (lanes 1 and 2); mmpF1 plus mmpB2 (lanes 3 and 4). A 271-bp truncated fragment was used for mutant construction. Positions of primers are indicated in panel A. cDNA obtained from the wild type (lanes 1 and 3) or the mutant strain Da127 (lanes 2 and 4) was used as a template. Identical reactions with reverse transcriptase omitted were used as negative controls (data not shown).
Figure Legend Snippet: Schematic representation of the insertion deletion. (A) mms16 wild-type gene and different truncated fragments (I to III). Sizes are as indicated. (B) Molecular organization of the mms16 locus before and after insertion-duplication mutagenesis with a truncated fragment. Different fill patterns are used to mark the origins of different parts of the gene after a single crossover. Parts encoding the N and C termini of the corresponding gene products are indicated. (C) Characterization of the Da127 mutant by RT-PCR. The following primer combinations were applied in PCRs: mmpF1 plus mmpB1 (lanes 1 and 2); mmpF1 plus mmpB2 (lanes 3 and 4). A 271-bp truncated fragment was used for mutant construction. Positions of primers are indicated in panel A. cDNA obtained from the wild type (lanes 1 and 3) or the mutant strain Da127 (lanes 2 and 4) was used as a template. Identical reactions with reverse transcriptase omitted were used as negative controls (data not shown).

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction

3) Product Images from "Microarray Generation of Thousand-Member Oligonucleotide Libraries"

Article Title: Microarray Generation of Thousand-Member Oligonucleotide Libraries

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024906

The generation of DNA templates from microarrays and parallel analysis. A ssDNA microarray was incubated with a primer (16 h) followed by elongation using Taq polymerase (16 h) producing as dsDNA microarray. The newly synthesized DNA strands were used as templates for solution phase PCR carried out over the microarray leading to amplification of the ssDNA displayed on the microarray. The dsDNA was amplified by PCR to produce fluorescently labeled ssDNA analogous to the ssDNA printed on the microarray. The fluorescently labeled ssDNA was hybridized to a complementary microarray or submitted to Solexa sequencing to allow decoding of the amplified ssDNA. FAM = 5(6)-carboxyfluorescein.
Figure Legend Snippet: The generation of DNA templates from microarrays and parallel analysis. A ssDNA microarray was incubated with a primer (16 h) followed by elongation using Taq polymerase (16 h) producing as dsDNA microarray. The newly synthesized DNA strands were used as templates for solution phase PCR carried out over the microarray leading to amplification of the ssDNA displayed on the microarray. The dsDNA was amplified by PCR to produce fluorescently labeled ssDNA analogous to the ssDNA printed on the microarray. The fluorescently labeled ssDNA was hybridized to a complementary microarray or submitted to Solexa sequencing to allow decoding of the amplified ssDNA. FAM = 5(6)-carboxyfluorescein.

Techniques Used: Microarray, Incubation, Synthesized, Polymerase Chain Reaction, Amplification, Labeling, Sequencing

4) Product Images from "Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China"

Article Title: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053483

The kinetics of infection in mice with TgCtwh6 isolates. Kinetics of infection in blood and organs after oral infection with 50 cysts of TgCtwh6 isolate (genotype Chinese 1) in mice. I: the number of DNA copies in blood and tissues of the TgCtwh6 isolate post infection at various intervals by qPCR. Each point represents the mean value of the T. gondii DNA copies for five mice ± SD. II: detection of PCR products of T. gondii 529 bp fragments extracted from brain or ascitic fluid in the recipient mice. Abbreviations: A ; blood, B ; heart, C ; liver, D ; brain, and E ; lymph node. b, n, and p represent blank, negative and positive control. * P
Figure Legend Snippet: The kinetics of infection in mice with TgCtwh6 isolates. Kinetics of infection in blood and organs after oral infection with 50 cysts of TgCtwh6 isolate (genotype Chinese 1) in mice. I: the number of DNA copies in blood and tissues of the TgCtwh6 isolate post infection at various intervals by qPCR. Each point represents the mean value of the T. gondii DNA copies for five mice ± SD. II: detection of PCR products of T. gondii 529 bp fragments extracted from brain or ascitic fluid in the recipient mice. Abbreviations: A ; blood, B ; heart, C ; liver, D ; brain, and E ; lymph node. b, n, and p represent blank, negative and positive control. * P

Techniques Used: Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control

5) Product Images from "Microarray Generation of Thousand-Member Oligonucleotide Libraries"

Article Title: Microarray Generation of Thousand-Member Oligonucleotide Libraries

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024906

The generation of DNA templates from microarrays and parallel analysis. A ssDNA microarray was incubated with a primer (16 h) followed by elongation using Taq polymerase (16 h) producing as dsDNA microarray. The newly synthesized DNA strands were used as templates for solution phase PCR carried out over the microarray leading to amplification of the ssDNA displayed on the microarray. The dsDNA was amplified by PCR to produce fluorescently labeled ssDNA analogous to the ssDNA printed on the microarray. The fluorescently labeled ssDNA was hybridized to a complementary microarray or submitted to Solexa sequencing to allow decoding of the amplified ssDNA. FAM = 5(6)-carboxyfluorescein.
Figure Legend Snippet: The generation of DNA templates from microarrays and parallel analysis. A ssDNA microarray was incubated with a primer (16 h) followed by elongation using Taq polymerase (16 h) producing as dsDNA microarray. The newly synthesized DNA strands were used as templates for solution phase PCR carried out over the microarray leading to amplification of the ssDNA displayed on the microarray. The dsDNA was amplified by PCR to produce fluorescently labeled ssDNA analogous to the ssDNA printed on the microarray. The fluorescently labeled ssDNA was hybridized to a complementary microarray or submitted to Solexa sequencing to allow decoding of the amplified ssDNA. FAM = 5(6)-carboxyfluorescein.

Techniques Used: Microarray, Incubation, Synthesized, Polymerase Chain Reaction, Amplification, Labeling, Sequencing

6) Product Images from "Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection"

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx113

In vivo transposition in prokaryotic cells. ( A ) Scheme of the experimental method. The donor plasmid DNA, carrying a gene of interest (kanamycin resistance cassette, Kan R ) flanked with (IRs), contains a conditional origin of replication, oriR6K, which prevents replication in the recipient strain. Donor plasmid DNA and purified recombinant transposase (Mos1 or Mboumar-9) are co-transfected into bacterial cells, resulting in integration of the kanamycin cassette into the genomic DNA. ( B ) Kanamycin resistant colonies were obtained only if purified transposase was included in the transfection reaction. White colonies on dark background. ( C ) Relative efficiency of transposition observed after treatment of the reactions, prior to transfection, with proteinase K, phenol, no treatment and the control (no transposase added). Two technical repeats were performed for two biological repeats. ( D ) Agarose gel of the products of colony polymerase chain reaction (PCR) to detect the presence of the donor plasmid in the kanamycin resistant colonies. Clone 1 (Lane 4) has traces of the donor plasmid backbone detected. Positive control—a colony of Escherichia coli S17 λ pir carrying donor plasmid; negative control—a colony of the recipient strain E. coli DH10B. ( E ) Five analyzed clones are resistant to kanamycin, but sensitive to chloramphenicol—the plasmid backbone resistance. Controls as in (D). ( F ) Southern Blotting analysis of the digested genomic DNA from the kanamycin resistant clones hybridized with fluorescently labeled IR DNA. Negative control: genomic DNA of the recipient strain E. coli DH10B. ( G ) WebLogo alignment of 40 bp around the TA target nucleotides duplication of the 14 integration sites by Mos1 in the bacterial genome.
Figure Legend Snippet: In vivo transposition in prokaryotic cells. ( A ) Scheme of the experimental method. The donor plasmid DNA, carrying a gene of interest (kanamycin resistance cassette, Kan R ) flanked with (IRs), contains a conditional origin of replication, oriR6K, which prevents replication in the recipient strain. Donor plasmid DNA and purified recombinant transposase (Mos1 or Mboumar-9) are co-transfected into bacterial cells, resulting in integration of the kanamycin cassette into the genomic DNA. ( B ) Kanamycin resistant colonies were obtained only if purified transposase was included in the transfection reaction. White colonies on dark background. ( C ) Relative efficiency of transposition observed after treatment of the reactions, prior to transfection, with proteinase K, phenol, no treatment and the control (no transposase added). Two technical repeats were performed for two biological repeats. ( D ) Agarose gel of the products of colony polymerase chain reaction (PCR) to detect the presence of the donor plasmid in the kanamycin resistant colonies. Clone 1 (Lane 4) has traces of the donor plasmid backbone detected. Positive control—a colony of Escherichia coli S17 λ pir carrying donor plasmid; negative control—a colony of the recipient strain E. coli DH10B. ( E ) Five analyzed clones are resistant to kanamycin, but sensitive to chloramphenicol—the plasmid backbone resistance. Controls as in (D). ( F ) Southern Blotting analysis of the digested genomic DNA from the kanamycin resistant clones hybridized with fluorescently labeled IR DNA. Negative control: genomic DNA of the recipient strain E. coli DH10B. ( G ) WebLogo alignment of 40 bp around the TA target nucleotides duplication of the 14 integration sites by Mos1 in the bacterial genome.

Techniques Used: In Vivo, Plasmid Preparation, Purification, Recombinant, Transfection, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Positive Control, Negative Control, Clone Assay, Southern Blot, Labeling

7) Product Images from "Importation of exotic ticks and tick-borne spotted fever group rickettsiae into the United States by migrating songbirds"

Article Title: Importation of exotic ticks and tick-borne spotted fever group rickettsiae into the United States by migrating songbirds

Journal: Ticks and tick-borne diseases

doi: 10.1016/j.ttbdis.2013.09.009

(A) PCR amplification of spotted fever group Rickettsia spp. in exotic ticks collected from migratory birds. Lane 1: low DNA mass ladder; lanes 2, 4, 6: empty lanes; lane 3: no-template control; lane 5: Rickettsia ompA -positive control; lanes 7–19: rickettsial ompA gene amplified from extracted tick DNA using gene-specific primers in a nested PCR assay. (B) PCR amplification of spotted fever group Rickettsia spp. in migratory songbird blood samples. Lane 1: low DNA mass ladder; lanes 2, 4: empty wells; lane 3: no-template control; lane 5: Rickettsia ompA- positive control, lanes 6–19: rickettsial ompA gene fragment amplified from extract bird blood DNA using gene-specific primers in a nested PCR assay.
Figure Legend Snippet: (A) PCR amplification of spotted fever group Rickettsia spp. in exotic ticks collected from migratory birds. Lane 1: low DNA mass ladder; lanes 2, 4, 6: empty lanes; lane 3: no-template control; lane 5: Rickettsia ompA -positive control; lanes 7–19: rickettsial ompA gene amplified from extracted tick DNA using gene-specific primers in a nested PCR assay. (B) PCR amplification of spotted fever group Rickettsia spp. in migratory songbird blood samples. Lane 1: low DNA mass ladder; lanes 2, 4: empty wells; lane 3: no-template control; lane 5: Rickettsia ompA- positive control, lanes 6–19: rickettsial ompA gene fragment amplified from extract bird blood DNA using gene-specific primers in a nested PCR assay.

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

8) Product Images from "Bacterial Community Dynamics During the Application of a Myxococcus xanthus-Inoculated Culture Medium Used for Consolidation of Ornamental Limestone"

Article Title: Bacterial Community Dynamics During the Application of a Myxococcus xanthus-Inoculated Culture Medium Used for Consolidation of Ornamental Limestone

Journal: Microbial Ecology

doi: 10.1007/s00248-010-9661-2

PCR-DGGE profiles representing the succession in the bacterial community structure, as well as the monitoring during the time course experiment of M. xanthus on decayed calcarenite. DNA was directly extracted from non-treated ( D ) and treated ( TD ) stone slabs, as well as from aliquots taken from the culture medium of treated samples during the time course experiment, and subsequently amplified with the 16S rRNA primers pair 341f/907r. The description for lane numbers and for b are as indicated for Fig. 1
Figure Legend Snippet: PCR-DGGE profiles representing the succession in the bacterial community structure, as well as the monitoring during the time course experiment of M. xanthus on decayed calcarenite. DNA was directly extracted from non-treated ( D ) and treated ( TD ) stone slabs, as well as from aliquots taken from the culture medium of treated samples during the time course experiment, and subsequently amplified with the 16S rRNA primers pair 341f/907r. The description for lane numbers and for b are as indicated for Fig. 1

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

PCR-DGGE profiles representing the succession in the bacterial community structure, as well as the monitoring during the time course experiment of M. xanthus on quarry calcarenite. DNA was directly extracted from non-treated ( Q ) and treated ( TQ ) stone slabs, as well as from aliquots taken from the culture medium of treated samples during the time course experiment, and subsequently amplified with the 16S rRNA primers pair 341f/907r. Numbers of lanes indicate sampling days. Lane M , marker of M. xanthus . DGGE-bands identified from the 16S rDNA clone libraries are numbered and are indicated by arrowheads . These bands are described in the b . Closest relative, as determined by comparative sequence analysis, level of identity with this relative, clone designation, and accession number for each band are summarized in b
Figure Legend Snippet: PCR-DGGE profiles representing the succession in the bacterial community structure, as well as the monitoring during the time course experiment of M. xanthus on quarry calcarenite. DNA was directly extracted from non-treated ( Q ) and treated ( TQ ) stone slabs, as well as from aliquots taken from the culture medium of treated samples during the time course experiment, and subsequently amplified with the 16S rRNA primers pair 341f/907r. Numbers of lanes indicate sampling days. Lane M , marker of M. xanthus . DGGE-bands identified from the 16S rDNA clone libraries are numbered and are indicated by arrowheads . These bands are described in the b . Closest relative, as determined by comparative sequence analysis, level of identity with this relative, clone designation, and accession number for each band are summarized in b

Techniques Used: Polymerase Chain Reaction, Denaturing Gradient Gel Electrophoresis, Amplification, Sampling, Marker, Sequencing

9) Product Images from "Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus"

Article Title: Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.71.6.3192-3198.2005

Disruption of the moxY gene via double-crossover recombination. (A) To disrupt the moxY gene, gene disruption vector pMOXY-DD was constructed as described in the text. The vector was linearized and then transformed into the wild-type strain SYS-4. The double-crossover recombination resulted in the replacement of most of the internal section of the target gene moxY with the ptrA gene. (B) PCR analysis of the moxY disruptant was conducted with genomic DNA of MOXY-DD-69 and the recipient strain SYS-4 by using different combinations of primers. Lanes: a, the moxY disruptant MOXY-DD-69; b, the recipient strain SYS-4; M, 1-kb molecular marker. The expected lengths of the PCR products are shown at the bottom of panel B.
Figure Legend Snippet: Disruption of the moxY gene via double-crossover recombination. (A) To disrupt the moxY gene, gene disruption vector pMOXY-DD was constructed as described in the text. The vector was linearized and then transformed into the wild-type strain SYS-4. The double-crossover recombination resulted in the replacement of most of the internal section of the target gene moxY with the ptrA gene. (B) PCR analysis of the moxY disruptant was conducted with genomic DNA of MOXY-DD-69 and the recipient strain SYS-4 by using different combinations of primers. Lanes: a, the moxY disruptant MOXY-DD-69; b, the recipient strain SYS-4; M, 1-kb molecular marker. The expected lengths of the PCR products are shown at the bottom of panel B.

Techniques Used: Plasmid Preparation, Construct, Transformation Assay, Polymerase Chain Reaction, Marker

10) Product Images from "An Insight Into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae)"

Article Title: An Insight Into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae)

Journal: Journal of medical entomology

doi:

Molecular detection of SFGR in field-collected A. maculatum. Tick tissues were tested for the presence of SFGR using the ompA -nested PCR assay. (A) 1: DNA ladder; 2: no-template control; 3: no-primer control; 4: positive control; lanes 5–15: male
Figure Legend Snippet: Molecular detection of SFGR in field-collected A. maculatum. Tick tissues were tested for the presence of SFGR using the ompA -nested PCR assay. (A) 1: DNA ladder; 2: no-template control; 3: no-primer control; 4: positive control; lanes 5–15: male

Techniques Used: Nested PCR, Positive Control

11) Product Images from "Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection *"

Article Title: Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.602649

Analysis of changes in TS during MNV1 infection. A , semiquantitative RT-PCR analysis of GAPDH and MNV1 mRNA among total RNA isolated from the polysomal fractions of mock- or MNV1-infected cells. Total RNAs were isolated from the pooled translationally active polysomal fraction and inactive free fraction of mock- or MNV1-infected cell lysates and subjected to reverse transcription using the Transcriptor first strand cDNA synthesis kit (Roche Applied Science). PCR amplification was carried out using PCR Master Mix (Promega). B , status of polysomal and nonpolysomal abundances of mRNAs upon MNV1 infection. Total RNAs were isolated from the translationally active polysomal fraction and inactive free fraction of mock- or MNV1-infected cell lysates and subjected to reverse transcription using a Transcriptor first strand cDNA synthesis kit (Roche Applied Science). PCR amplification was carried out using MESA BLUE qPCR MasterMix Plus for SYBR (Eurogentec) and an Mx3005 qPCR system (Stratagene). Shown are qPCR results from three independent biological isolates of both mock- and MNV1-infected cells. The bars represent the mean, and the error bars show the S.E. The change in TS has been calculated using the formula, (mock/MNV1) poly /(mock/MNV1) mono , where (mock/MNV1) poly and (mock/MNV1) mono represent the changes of individual mRNAs in polysomal and monosomal RNA, respectively.
Figure Legend Snippet: Analysis of changes in TS during MNV1 infection. A , semiquantitative RT-PCR analysis of GAPDH and MNV1 mRNA among total RNA isolated from the polysomal fractions of mock- or MNV1-infected cells. Total RNAs were isolated from the pooled translationally active polysomal fraction and inactive free fraction of mock- or MNV1-infected cell lysates and subjected to reverse transcription using the Transcriptor first strand cDNA synthesis kit (Roche Applied Science). PCR amplification was carried out using PCR Master Mix (Promega). B , status of polysomal and nonpolysomal abundances of mRNAs upon MNV1 infection. Total RNAs were isolated from the translationally active polysomal fraction and inactive free fraction of mock- or MNV1-infected cell lysates and subjected to reverse transcription using a Transcriptor first strand cDNA synthesis kit (Roche Applied Science). PCR amplification was carried out using MESA BLUE qPCR MasterMix Plus for SYBR (Eurogentec) and an Mx3005 qPCR system (Stratagene). Shown are qPCR results from three independent biological isolates of both mock- and MNV1-infected cells. The bars represent the mean, and the error bars show the S.E. The change in TS has been calculated using the formula, (mock/MNV1) poly /(mock/MNV1) mono , where (mock/MNV1) poly and (mock/MNV1) mono represent the changes of individual mRNAs in polysomal and monosomal RNA, respectively.

Techniques Used: Infection, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

12) Product Images from "Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan"

Article Title: Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00116

Phylogenetic tree based upon the Neighbor-Joining of 16S rDNA sequences ( E. coli 84 to 1437) derived by PCR with the primer 27F and 1492R .
Figure Legend Snippet: Phylogenetic tree based upon the Neighbor-Joining of 16S rDNA sequences ( E. coli 84 to 1437) derived by PCR with the primer 27F and 1492R .

Techniques Used: Derivative Assay, Polymerase Chain Reaction

13) Product Images from "Expression and ecdysteroid responsiveness of the nuclear receptors HR3 and E75 in the crustacean Daphnia magna"

Article Title: Expression and ecdysteroid responsiveness of the nuclear receptors HR3 and E75 in the crustacean Daphnia magna

Journal: Molecular and cellular endocrinology

doi: 10.1016/j.mce.2009.07.013

Nucleotide (lower case) and deduced amino acid (upper case) sequences of the D. magna HR3 open reading frame cDNA. Shaded regions correspond to the location of primers used for Real Time RT-PCR.
Figure Legend Snippet: Nucleotide (lower case) and deduced amino acid (upper case) sequences of the D. magna HR3 open reading frame cDNA. Shaded regions correspond to the location of primers used for Real Time RT-PCR.

Techniques Used: Quantitative RT-PCR

Nucleotide (lower case) and deduced amino acid (upper case) sequences of the D. magna E75 cDNA. Shaded regions correspond to the location of primers used for Real Time RT-PCR.
Figure Legend Snippet: Nucleotide (lower case) and deduced amino acid (upper case) sequences of the D. magna E75 cDNA. Shaded regions correspond to the location of primers used for Real Time RT-PCR.

Techniques Used: Quantitative RT-PCR

14) Product Images from "LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling"

Article Title: LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling

Journal: Scientific Reports

doi: 10.1038/s41598-018-26421-5

The expression of MALAT1, MyD88, IRAK1 and TRAF6 in DM-I/R rats at 24 and 72 hours after cerebral I/R injury. The mRNA expressions of (A) CD68 and (B) Emr1, which were the makers of the microglial cells in the peri-infarct cortical tissue of rats, were determined by qRT-PCR. (C) The expression of MALAT1. (D) Representative Western blot analysis of MyD88, IRAK1, TRAF6 protein. The relative expressions of (E) MyD88, (F) IRAK1, (G) TRAF6 protein were measured. *P
Figure Legend Snippet: The expression of MALAT1, MyD88, IRAK1 and TRAF6 in DM-I/R rats at 24 and 72 hours after cerebral I/R injury. The mRNA expressions of (A) CD68 and (B) Emr1, which were the makers of the microglial cells in the peri-infarct cortical tissue of rats, were determined by qRT-PCR. (C) The expression of MALAT1. (D) Representative Western blot analysis of MyD88, IRAK1, TRAF6 protein. The relative expressions of (E) MyD88, (F) IRAK1, (G) TRAF6 protein were measured. *P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

15) Product Images from "Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity"

Article Title: Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068648

DGGE EM profiling using 16S rRNA gene PCR amplicons. (A) Profiles of individual EM type strains obtained using the Mycobacterium genus specific primer set JSY16S. L is the reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–12 are, respectively: M. smegmatis , M. aichiense , M. aurum , M. gilvum , M. phlei , M. agri , M. peregrinum , M. duvalii, M. abscessus , M. fortuitum, M. vaccae and a mixture of equimolar quantities of the above listed EM species. (B) Profiles obtained using the slow mycobacteria specific primer set APTK16S. L is a reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–7 are, respectively: M. intracellulare, M. marinum , M. kansasii , M. xenopi , M. aviumparatuberculosis , M. bovis BCG and a mixture of equimolar quantities of the above listed EM species. (C) EM soil community profiling using the Mycobacterium genus specific primers (JSY16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1 – 4 are the four Ethiopian soils (1108, 1109,1110, 1111) and 5 is Cryfield. The arrows (1A–1I) indicate the bands that were excised and sequenced ( Table 4 ). (D) EM soil community profiling using the slow grower mycobacteria specific 16S rRNA gene specific primers (APTK16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1–4 are the four Ethiopian soils (1108, 1109, 1110, 1111) and 5 is Cryfield. The arrows (2A–2I) represent the bands that were excised and sequenced ( Table 4 ).
Figure Legend Snippet: DGGE EM profiling using 16S rRNA gene PCR amplicons. (A) Profiles of individual EM type strains obtained using the Mycobacterium genus specific primer set JSY16S. L is the reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–12 are, respectively: M. smegmatis , M. aichiense , M. aurum , M. gilvum , M. phlei , M. agri , M. peregrinum , M. duvalii, M. abscessus , M. fortuitum, M. vaccae and a mixture of equimolar quantities of the above listed EM species. (B) Profiles obtained using the slow mycobacteria specific primer set APTK16S. L is a reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–7 are, respectively: M. intracellulare, M. marinum , M. kansasii , M. xenopi , M. aviumparatuberculosis , M. bovis BCG and a mixture of equimolar quantities of the above listed EM species. (C) EM soil community profiling using the Mycobacterium genus specific primers (JSY16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1 – 4 are the four Ethiopian soils (1108, 1109,1110, 1111) and 5 is Cryfield. The arrows (1A–1I) indicate the bands that were excised and sequenced ( Table 4 ). (D) EM soil community profiling using the slow grower mycobacteria specific 16S rRNA gene specific primers (APTK16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1–4 are the four Ethiopian soils (1108, 1109, 1110, 1111) and 5 is Cryfield. The arrows (2A–2I) represent the bands that were excised and sequenced ( Table 4 ).

Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction

16) Product Images from "Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity"

Article Title: Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068648

DGGE EM profiling using 16S rRNA gene PCR amplicons. (A) Profiles of individual EM type strains obtained using the Mycobacterium genus specific primer set JSY16S. L is the reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–12 are, respectively: M. smegmatis , M. aichiense , M. aurum , M. gilvum , M. phlei , M. agri , M. peregrinum , M. duvalii, M. abscessus , M. fortuitum, M. vaccae and a mixture of equimolar quantities of the above listed EM species. (B) Profiles obtained using the slow mycobacteria specific primer set APTK16S. L is a reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–7 are, respectively: M. intracellulare, M. marinum , M. kansasii , M. xenopi , M. aviumparatuberculosis , M. bovis BCG and a mixture of equimolar quantities of the above listed EM species. (C) EM soil community profiling using the Mycobacterium genus specific primers (JSY16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1 – 4 are the four Ethiopian soils (1108, 1109,1110, 1111) and 5 is Cryfield. The arrows (1A–1I) indicate the bands that were excised and sequenced ( Table 4 ). (D) EM soil community profiling using the slow grower mycobacteria specific 16S rRNA gene specific primers (APTK16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1–4 are the four Ethiopian soils (1108, 1109, 1110, 1111) and 5 is Cryfield. The arrows (2A–2I) represent the bands that were excised and sequenced ( Table 4 ).
Figure Legend Snippet: DGGE EM profiling using 16S rRNA gene PCR amplicons. (A) Profiles of individual EM type strains obtained using the Mycobacterium genus specific primer set JSY16S. L is the reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–12 are, respectively: M. smegmatis , M. aichiense , M. aurum , M. gilvum , M. phlei , M. agri , M. peregrinum , M. duvalii, M. abscessus , M. fortuitum, M. vaccae and a mixture of equimolar quantities of the above listed EM species. (B) Profiles obtained using the slow mycobacteria specific primer set APTK16S. L is a reference ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), lanes 1–7 are, respectively: M. intracellulare, M. marinum , M. kansasii , M. xenopi , M. aviumparatuberculosis , M. bovis BCG and a mixture of equimolar quantities of the above listed EM species. (C) EM soil community profiling using the Mycobacterium genus specific primers (JSY16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1 – 4 are the four Ethiopian soils (1108, 1109,1110, 1111) and 5 is Cryfield. The arrows (1A–1I) indicate the bands that were excised and sequenced ( Table 4 ). (D) EM soil community profiling using the slow grower mycobacteria specific 16S rRNA gene specific primers (APTK16S). L is a reference sizing ladder (TrackIt™ 50 bp DNA Ladder, Invitrogen, Ltd., Paisley, UK), C is the negative PCR control, samples 1–4 are the four Ethiopian soils (1108, 1109, 1110, 1111) and 5 is Cryfield. The arrows (2A–2I) represent the bands that were excised and sequenced ( Table 4 ).

Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction

17) Product Images from "Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods"

Article Title: Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

Journal: Malaria Journal

doi: 10.1186/1475-2875-6-111

Examples of AS-PCR products for kdr -e and kdr -w genotyping . Gels A to D show examples of AS-PCR results using four different protocols, A [29], B [11], C [12], D [20]. Gels E to G show the result of using different DNA polymerases on the AS-PCR method described by [20], E: Promega PCR master mix, F: Qiagen HotStar Taq G:Finzymes Dynazyme II Gel H is an example of results of using the protocol of [20] with the Promega PCR master mix to genotype samples using the kdr -e assay from the 96 reference plate and gel I for the kdr -w assay. The same DNA templates were used in PCRs shown in gels A to G and from left to right were 100 bp DNA Ladder (Fermentas), homozygous wildtype, homozygous wildtype, heterozygous, heterozygous, homozygous mutant, homozygous mutant.
Figure Legend Snippet: Examples of AS-PCR products for kdr -e and kdr -w genotyping . Gels A to D show examples of AS-PCR results using four different protocols, A [29], B [11], C [12], D [20]. Gels E to G show the result of using different DNA polymerases on the AS-PCR method described by [20], E: Promega PCR master mix, F: Qiagen HotStar Taq G:Finzymes Dynazyme II Gel H is an example of results of using the protocol of [20] with the Promega PCR master mix to genotype samples using the kdr -e assay from the 96 reference plate and gel I for the kdr -w assay. The same DNA templates were used in PCRs shown in gels A to G and from left to right were 100 bp DNA Ladder (Fermentas), homozygous wildtype, homozygous wildtype, heterozygous, heterozygous, homozygous mutant, homozygous mutant.

Techniques Used: Polymerase Chain Reaction, Mutagenesis

18) Product Images from "Infectious necrotic hepatitis caused by Clostridiumnovyi type B in a horse: case report and review of the literature"

Article Title: Infectious necrotic hepatitis caused by Clostridiumnovyi type B in a horse: case report and review of the literature

Journal: Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

doi: 10.1177/1040638717737125

A. Electrophoretic analysis of multiplex PCR products targeting fliC gene of histotoxic clostridia. Lane M: molecular size marker; lane 1: Clostridium septicum positive control (294 bp); lane 2: Clostridium novyi type A positive control (343 bp); lane 3: C. novyi type B positive control (427 bp); lane 4: Clostridium chauvoei positive control (535 bp); lane 5: Clostridium haemolyticum positive control (694 bp); lane 6: DNA extracted from liver samples; lane 7: negative control. B. Electrophoretic analysis of 342-bp products obtained by conventional PCR targeting C. novyi type B alpha toxin gene. Lane M: molecular size marker; lane 1: C. novyi type B positive control; lane 2: DNA extracted from liver samples; lane 3 = negative control.
Figure Legend Snippet: A. Electrophoretic analysis of multiplex PCR products targeting fliC gene of histotoxic clostridia. Lane M: molecular size marker; lane 1: Clostridium septicum positive control (294 bp); lane 2: Clostridium novyi type A positive control (343 bp); lane 3: C. novyi type B positive control (427 bp); lane 4: Clostridium chauvoei positive control (535 bp); lane 5: Clostridium haemolyticum positive control (694 bp); lane 6: DNA extracted from liver samples; lane 7: negative control. B. Electrophoretic analysis of 342-bp products obtained by conventional PCR targeting C. novyi type B alpha toxin gene. Lane M: molecular size marker; lane 1: C. novyi type B positive control; lane 2: DNA extracted from liver samples; lane 3 = negative control.

Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Marker, Positive Control, Negative Control

19) Product Images from "Sympathetic Hyperactivity and Age Affect Segregation and Expression of Neurotransmitters"

Article Title: Sympathetic Hyperactivity and Age Affect Segregation and Expression of Neurotransmitters

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00411

Cold-stress increased expression of tyrosine hydroxylase (TH) mRNA and protein in adrenal medulla (AM). (A) Conventional RT-PCR analysis performed on total RNA obtained from AM showing an increase (17.5%) in TH mRNA after 5 days of cold-stress. RT-PCR assay was performed for the amplification of TH mRNA fragment of 646 bp. (B) Western blot (WB) analysis of TH protein (56 kDa) expression showing an increase (1.6-fold) after cold-stress. Graphs show quantification of TH relative to actin, mean ± SEM and the significance level. Actin was used as the housekeeping gene for RT-PCR and as the loading control for WB assays. * P
Figure Legend Snippet: Cold-stress increased expression of tyrosine hydroxylase (TH) mRNA and protein in adrenal medulla (AM). (A) Conventional RT-PCR analysis performed on total RNA obtained from AM showing an increase (17.5%) in TH mRNA after 5 days of cold-stress. RT-PCR assay was performed for the amplification of TH mRNA fragment of 646 bp. (B) Western blot (WB) analysis of TH protein (56 kDa) expression showing an increase (1.6-fold) after cold-stress. Graphs show quantification of TH relative to actin, mean ± SEM and the significance level. Actin was used as the housekeeping gene for RT-PCR and as the loading control for WB assays. * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot

20) Product Images from "Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay"

Article Title: Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0085-2

The detection rate of T. pallidum DNA in blood samples from all patients by Tpp47 -Tp-PCR and polA -Tp-PCR. a The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR among all patients ( n = 262). b The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the primary syphilis patients ( n = 84). c The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the secondary syphilis patients ( n = 97). d The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the latent syphilis patients ( n = 81)
Figure Legend Snippet: The detection rate of T. pallidum DNA in blood samples from all patients by Tpp47 -Tp-PCR and polA -Tp-PCR. a The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR among all patients ( n = 262). b The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the primary syphilis patients ( n = 84). c The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the secondary syphilis patients ( n = 97). d The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the latent syphilis patients ( n = 81)

Techniques Used: Polymerase Chain Reaction

21) Product Images from "Diverse genotypes of Kaposi's sarcoma associated herpesvirus (KSHV) identified in infant blood infections in African childhood-KS and HIV/AIDS endemic region"

Article Title: Diverse genotypes of Kaposi's sarcoma associated herpesvirus (KSHV) identified in infant blood infections in African childhood-KS and HIV/AIDS endemic region

Journal: Journal of Medical Virology

doi: 10.1002/jmv.20952

Sequence analyses of K1 variable region, VRI loop, identifies genotype diversity in childhood KSHV from African endemic region. The K1 region was PCR amplified from blood DNA, sequenced and aligned against published representatives of K1 genotypes [ Zong et al., 1999 ]. Dashes indicated identity.
Figure Legend Snippet: Sequence analyses of K1 variable region, VRI loop, identifies genotype diversity in childhood KSHV from African endemic region. The K1 region was PCR amplified from blood DNA, sequenced and aligned against published representatives of K1 genotypes [ Zong et al., 1999 ]. Dashes indicated identity.

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

22) Product Images from "Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease"

Article Title: Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease

Journal: Gut Pathogens

doi: 10.1186/s13099-018-0278-1

Validation of multiplex PCR using individual and combined oligonucleotide primer sets and DNA extracts from mixed bacterial culture. Multiplex PCR was performed on DNA extracts from mixed bacterial cultures. DNA template was extracted using the modified DNAzol ® . (1) Non-pathogenic E. coli strain K-12 18s primers were used (171 bp); (2) MAP UCF4 IS900 AV1/AV2 primers were used (298 bp); (3) K. pneumoniae 23s primers were used (493 bp); (4) Mycobacterium avium complex (MAC) IS1311 primers were used (534 bp); (5) a cocktail of the 4 primer sets mentioned above were used. M: DNA molecular weight marker
Figure Legend Snippet: Validation of multiplex PCR using individual and combined oligonucleotide primer sets and DNA extracts from mixed bacterial culture. Multiplex PCR was performed on DNA extracts from mixed bacterial cultures. DNA template was extracted using the modified DNAzol ® . (1) Non-pathogenic E. coli strain K-12 18s primers were used (171 bp); (2) MAP UCF4 IS900 AV1/AV2 primers were used (298 bp); (3) K. pneumoniae 23s primers were used (493 bp); (4) Mycobacterium avium complex (MAC) IS1311 primers were used (534 bp); (5) a cocktail of the 4 primer sets mentioned above were used. M: DNA molecular weight marker

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

Validation of multiplex PCR using individual and combined oligonucleotide primer sets and DNA extracts from IBD tissue. Multiplex PCR was performed on DNA extracts from intestinal tissue samples. DNA template was extracted using the modified DNAzol ® . RS1: ulcerative colitis (UC) patient; RS2: Crohn’s disease (CD) patient; (1) Non-pathogenic E. coli strain K-12 18s primers were used (171 bp); (2) MAP UCF4 IS900 AV1/AV2 primers were used (298 bp); (3) AIEC strain LF82 g ipA primers were used (357 bp); (4) K. pneumoniae 23s primers were used (493 bp); (5) Mycobacterium avium complex (MAC) IS1311 primers were used (534 bp); (6) a cocktail of the 5 primer sets mentioned above were used. M: DNA molecular weight marker
Figure Legend Snippet: Validation of multiplex PCR using individual and combined oligonucleotide primer sets and DNA extracts from IBD tissue. Multiplex PCR was performed on DNA extracts from intestinal tissue samples. DNA template was extracted using the modified DNAzol ® . RS1: ulcerative colitis (UC) patient; RS2: Crohn’s disease (CD) patient; (1) Non-pathogenic E. coli strain K-12 18s primers were used (171 bp); (2) MAP UCF4 IS900 AV1/AV2 primers were used (298 bp); (3) AIEC strain LF82 g ipA primers were used (357 bp); (4) K. pneumoniae 23s primers were used (493 bp); (5) Mycobacterium avium complex (MAC) IS1311 primers were used (534 bp); (6) a cocktail of the 5 primer sets mentioned above were used. M: DNA molecular weight marker

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

23) Product Images from "NFkB is essential for activin-induced colorectal cancer migration via upregulation of PI3K-MDM2 pathway"

Article Title: NFkB is essential for activin-induced colorectal cancer migration via upregulation of PI3K-MDM2 pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.16343

Activin increases MDM2 expression via recruitment of NFkB p65 to the MDM2 promoter (A) FET colon cancer cells were stimulated with activin (25ng/ml) for increasing time followed by mRNA analysis of MDM2 by semi-quantitative RT-PCR with detection of L19 as an internal control. (B) FET colon cancer cells pretreated with two concentrations of wtNBD peptide or mutant NBD (mNBD) peptide for 45 min were stimulated with activin (25ng/ml) for 6h under serum-free condition followed by monitoring of mRNA expression of MDM2 by qRT-PCR. (C) Map of DNA sequence of the human MDM2 promoter region indicates five potential NFkB binding sequences. (D) (Upper panel). FET colon cancer cells were treated with activin (25ng/ml) or TGFB (10ng/ml) for 3h under serum-free conditions and then subjected to ChIP analysis by immunoprecipitation of the chromatin fragments with anti p65 or non-specific IgG antibodies. ChIP assay by semi-quantitative PCR show that in the presence of activin treatment p65 binds to two regions in the MDM2 promoter: base pairs -1505 to -1520 and -3095 to -3109 from the transcription start site (TSS) while p65 binds to only one region: base pair -1505 to -1520 in response to TGFB treatment. Immunoprecipitation with non-specific IgG followed by PCR showed almost undetectable bands in semi-quantitative PCR and the total fragmented DNA showed uniform signal, indicating uniformity and specificity of the results. (D) (Lower panel). The same reactions for two transcripts NFkB2 and NFkB4 were performed and analyzed by Real-time PCR system. Data is presented as relative expression of p65 signal in treated samples versus the untreated anti p65 antibody using the comparative CT method. The bars presented the expression data for two of the transcripts NFkB2 and NFkB4. M; DNA ladder. All the experiments were repeated at least three times under same conditions. *p indicates versus control. *p
Figure Legend Snippet: Activin increases MDM2 expression via recruitment of NFkB p65 to the MDM2 promoter (A) FET colon cancer cells were stimulated with activin (25ng/ml) for increasing time followed by mRNA analysis of MDM2 by semi-quantitative RT-PCR with detection of L19 as an internal control. (B) FET colon cancer cells pretreated with two concentrations of wtNBD peptide or mutant NBD (mNBD) peptide for 45 min were stimulated with activin (25ng/ml) for 6h under serum-free condition followed by monitoring of mRNA expression of MDM2 by qRT-PCR. (C) Map of DNA sequence of the human MDM2 promoter region indicates five potential NFkB binding sequences. (D) (Upper panel). FET colon cancer cells were treated with activin (25ng/ml) or TGFB (10ng/ml) for 3h under serum-free conditions and then subjected to ChIP analysis by immunoprecipitation of the chromatin fragments with anti p65 or non-specific IgG antibodies. ChIP assay by semi-quantitative PCR show that in the presence of activin treatment p65 binds to two regions in the MDM2 promoter: base pairs -1505 to -1520 and -3095 to -3109 from the transcription start site (TSS) while p65 binds to only one region: base pair -1505 to -1520 in response to TGFB treatment. Immunoprecipitation with non-specific IgG followed by PCR showed almost undetectable bands in semi-quantitative PCR and the total fragmented DNA showed uniform signal, indicating uniformity and specificity of the results. (D) (Lower panel). The same reactions for two transcripts NFkB2 and NFkB4 were performed and analyzed by Real-time PCR system. Data is presented as relative expression of p65 signal in treated samples versus the untreated anti p65 antibody using the comparative CT method. The bars presented the expression data for two of the transcripts NFkB2 and NFkB4. M; DNA ladder. All the experiments were repeated at least three times under same conditions. *p indicates versus control. *p

Techniques Used: Expressing, Quantitative RT-PCR, Mutagenesis, Sequencing, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

24) Product Images from "Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay"

Article Title: Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0085-2

The detection rate of T. pallidum DNA in blood samples from all patients by Tpp47 -Tp-PCR and polA -Tp-PCR. a The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR among all patients ( n = 262). b The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the primary syphilis patients ( n = 84). c The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the secondary syphilis patients ( n = 97). d The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the latent syphilis patients ( n = 81)
Figure Legend Snippet: The detection rate of T. pallidum DNA in blood samples from all patients by Tpp47 -Tp-PCR and polA -Tp-PCR. a The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR among all patients ( n = 262). b The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the primary syphilis patients ( n = 84). c The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the secondary syphilis patients ( n = 97). d The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the latent syphilis patients ( n = 81)

Techniques Used: Polymerase Chain Reaction

25) Product Images from "Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining"

Article Title: Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining

Journal: eLife

doi: 10.7554/eLife.13740

Supporting information for Polθ mediated alt-EJ in vitro. ( A ) Schematic of alt-EJ reaction and subsequent procedures used for amplification and sequencing of end-joining products. Control alt-EJ reactions were performed with 10 mM Mg 2+ and 1 mM Mn 2+ . ( B ) Non-denaturing gels showing the products of PCR reactions containing either purified DNA from alt-EJ reactions performed in the presence of Polθ and Lig3 (top left), Polθ alone (top middle), and in the absence of Polθ and Lig3 (top right), or no DNA with primers only (bottom middle). Products in the top middle and top right gels are due to primer-dimer events as shown in the primers only control (bottom middle gel). Lanes 1-8 represent PCR reactions performed at the following respective temperatures: 61°C, 60.8°C, 60.4°C, 59.9°C, 59.2°C, 58.6°C, 58.2°C, 58°C. Lanes 9–13 represent PCR reactions performed in the absence of PCR primers RP435 and RP431 and at the following respective temperatures: 61°C, 60.4°C, 59.9°C, 59.2°C, 58.2°C. The absence of PCR products in lanes 9–13 show that Taq polymerase cannot amplify original pssDNA templates via end-joining or other mechanisms. ( C ) Plot showing percent of end-joining products observed in cloning vectors following end-joining reactions containing the indicated proteins. Red, end-joining products with insertions; grey, end-joining products without insertions. n = 64 (+Polθ, +Lig3), n = 72 (+Polθ, –Lig3), n =12 (–Polθ, –Lig3). End-joining products in the absence of Polθ and Lig3 are likely due to infrequent byproducts of PCR. DOI: http://dx.doi.org/10.7554/eLife.13740.015
Figure Legend Snippet: Supporting information for Polθ mediated alt-EJ in vitro. ( A ) Schematic of alt-EJ reaction and subsequent procedures used for amplification and sequencing of end-joining products. Control alt-EJ reactions were performed with 10 mM Mg 2+ and 1 mM Mn 2+ . ( B ) Non-denaturing gels showing the products of PCR reactions containing either purified DNA from alt-EJ reactions performed in the presence of Polθ and Lig3 (top left), Polθ alone (top middle), and in the absence of Polθ and Lig3 (top right), or no DNA with primers only (bottom middle). Products in the top middle and top right gels are due to primer-dimer events as shown in the primers only control (bottom middle gel). Lanes 1-8 represent PCR reactions performed at the following respective temperatures: 61°C, 60.8°C, 60.4°C, 59.9°C, 59.2°C, 58.6°C, 58.2°C, 58°C. Lanes 9–13 represent PCR reactions performed in the absence of PCR primers RP435 and RP431 and at the following respective temperatures: 61°C, 60.4°C, 59.9°C, 59.2°C, 58.2°C. The absence of PCR products in lanes 9–13 show that Taq polymerase cannot amplify original pssDNA templates via end-joining or other mechanisms. ( C ) Plot showing percent of end-joining products observed in cloning vectors following end-joining reactions containing the indicated proteins. Red, end-joining products with insertions; grey, end-joining products without insertions. n = 64 (+Polθ, +Lig3), n = 72 (+Polθ, –Lig3), n =12 (–Polθ, –Lig3). End-joining products in the absence of Polθ and Lig3 are likely due to infrequent byproducts of PCR. DOI: http://dx.doi.org/10.7554/eLife.13740.015

Techniques Used: In Vitro, Amplification, Sequencing, Polymerase Chain Reaction, Purification, Clone Assay

26) Product Images from "Type IV collagen drives alveolar epithelial–endothelial association and the morphogenetic movements of septation"

Article Title: Type IV collagen drives alveolar epithelial–endothelial association and the morphogenetic movements of septation

Journal: BMC Biology

doi: 10.1186/s12915-016-0281-2

Abnormal development of alveolar myofibroblast in Col4a mutants. a Myofibroblast progenitors positive for PDGFRα are normally scattered in the alveolar walls and at the tips of primitive septa at E18.5 (arrows). b In Col4a1 +/Δex41 lungs, PDGFRα myofibroblast progenitors cluster in a patchy distribution (arrow). c , d In P6 Col4a1 +/Δex41 , the number of PDGFRα + is reduced. e , f α-SMA shows a decrease of differentiated alveolar myofibroblasts at the primary septa and a patchy distribution in Col4a1 +/Δex41 (arrow) when compared with normal lungs at E18.5 (arrow). Arrowheads in c point to red blood cells. g At P6, some α-SMA + cells normally localize at the tips of developing septa (arrow). h Col4a1 +/Δex41 mutants display a decrease in septal and interstitial α-SMA + cells. i Real-time PCR at E18.5 shows statistically significant decrease in Pdgfrα mRNA (Wilcoxon ran-sum test P
Figure Legend Snippet: Abnormal development of alveolar myofibroblast in Col4a mutants. a Myofibroblast progenitors positive for PDGFRα are normally scattered in the alveolar walls and at the tips of primitive septa at E18.5 (arrows). b In Col4a1 +/Δex41 lungs, PDGFRα myofibroblast progenitors cluster in a patchy distribution (arrow). c , d In P6 Col4a1 +/Δex41 , the number of PDGFRα + is reduced. e , f α-SMA shows a decrease of differentiated alveolar myofibroblasts at the primary septa and a patchy distribution in Col4a1 +/Δex41 (arrow) when compared with normal lungs at E18.5 (arrow). Arrowheads in c point to red blood cells. g At P6, some α-SMA + cells normally localize at the tips of developing septa (arrow). h Col4a1 +/Δex41 mutants display a decrease in septal and interstitial α-SMA + cells. i Real-time PCR at E18.5 shows statistically significant decrease in Pdgfrα mRNA (Wilcoxon ran-sum test P

Techniques Used: Real-time Polymerase Chain Reaction

Abnormal alveolar tropoelastin and elastin fiber accumulation. a At E18.5, tropoelastin expression in the developing septa in normal lungs is detected at the tips (arrow) and throughout the interstitium (arrowhead). c By contrast, in Col4a1 +/Δex41 lungs, tropoelastin expression shows a patchy interstitial accumulation (arrowhead) with abnormal expression in the maldeveloped primary septa (arrow). b Hart’s staining marks the lung elastin fibers at E18.5. Control lungs show well-defined thin elastin fibers (black) in the saccule spaces (arrowhead) and at the tip of developing primary septa (arrow). d Saccule elastin fibers in Col4a1 +/Δex41 mutants are interstitial and tortuous or fragmented at E18.5 (arrows). e Real-time PCR for Tropoelastin . The expression of Tropoelastin is decreased in Col4a1 +/Δex41 (Wilcoxon rank-sum test P
Figure Legend Snippet: Abnormal alveolar tropoelastin and elastin fiber accumulation. a At E18.5, tropoelastin expression in the developing septa in normal lungs is detected at the tips (arrow) and throughout the interstitium (arrowhead). c By contrast, in Col4a1 +/Δex41 lungs, tropoelastin expression shows a patchy interstitial accumulation (arrowhead) with abnormal expression in the maldeveloped primary septa (arrow). b Hart’s staining marks the lung elastin fibers at E18.5. Control lungs show well-defined thin elastin fibers (black) in the saccule spaces (arrowhead) and at the tip of developing primary septa (arrow). d Saccule elastin fibers in Col4a1 +/Δex41 mutants are interstitial and tortuous or fragmented at E18.5 (arrows). e Real-time PCR for Tropoelastin . The expression of Tropoelastin is decreased in Col4a1 +/Δex41 (Wilcoxon rank-sum test P

Techniques Used: Expressing, Staining, Real-time Polymerase Chain Reaction

Lung development timeline and type IV collagen expression in the chicken and the mouse. a Mouse and chicken lung development comparative timeline. b Microarray analysis shows that vascular related genes, among which are Col4a1 and Col4a2 , show the highest significance in late chick lung development. c Real-time PCR shows differential expression of Col4a1 (blue) and Col4a2 (green) between E16 and E18 in chick lungs. Col4a1 and Col4a2 expression increases at E16 and E18, and is statistically significant (Wilcoxon rank-sum test P
Figure Legend Snippet: Lung development timeline and type IV collagen expression in the chicken and the mouse. a Mouse and chicken lung development comparative timeline. b Microarray analysis shows that vascular related genes, among which are Col4a1 and Col4a2 , show the highest significance in late chick lung development. c Real-time PCR shows differential expression of Col4a1 (blue) and Col4a2 (green) between E16 and E18 in chick lungs. Col4a1 and Col4a2 expression increases at E16 and E18, and is statistically significant (Wilcoxon rank-sum test P

Techniques Used: Expressing, Microarray, Real-time Polymerase Chain Reaction

Decreased epithelial progenitors and increased type II pneumocytes in Col4a1 +/Δex41 . a – j Epithelial proliferation and differentiation were evaluated by staining with Ki67, NKX2.1, SOX9, and pSPC. a , b Overall proliferation evaluated by Ki67 is increased in Col4a1 +/ Δex41 lungs. c – h Double immunohistochemistry for Ki67 and NKX2.1 shows slightly active epithelial proliferation in NKX2.1 cells (arrows) in normal ( c – e ) or Col4a1 +/Δex41 lungs at E18.5 ( f – h ). l The bar charts show the percentage NKX2.1, SOX9 progenitors and pSPC + cells over the total distal area of the lung of Col4a1 and Col4a2 mutants and wild type mice. Col4a1 +/Δex41 mutants have a statistically significant decrease of SOX9 cells and an increase of pSPC type II pneumocytes, while NKX2.1 + cells are unchanged compared with normal lungs at E18.5. j Real-time PCR of Nkx2.1 , Sox9 and pSpc . Only Sox9 mRNA expression is decreased in Col4a1 +/Δex41 . Gapdh was used as a normalizer. k The number of pSPC + cells at P30 is increased in mutant lungs. l , o NKX2.1, m , p SOX9 and n , q pSPC localization in normal lungs displays a scattered pattern around the saccular walls (arrows), while in Col4a1 +/Δex41 mutants NKX2.1 and pSPC are clustered together (arrows). Scale bars = 200 μm in a , b , 50 μm in c – h , and 200 μm in l – q
Figure Legend Snippet: Decreased epithelial progenitors and increased type II pneumocytes in Col4a1 +/Δex41 . a – j Epithelial proliferation and differentiation were evaluated by staining with Ki67, NKX2.1, SOX9, and pSPC. a , b Overall proliferation evaluated by Ki67 is increased in Col4a1 +/ Δex41 lungs. c – h Double immunohistochemistry for Ki67 and NKX2.1 shows slightly active epithelial proliferation in NKX2.1 cells (arrows) in normal ( c – e ) or Col4a1 +/Δex41 lungs at E18.5 ( f – h ). l The bar charts show the percentage NKX2.1, SOX9 progenitors and pSPC + cells over the total distal area of the lung of Col4a1 and Col4a2 mutants and wild type mice. Col4a1 +/Δex41 mutants have a statistically significant decrease of SOX9 cells and an increase of pSPC type II pneumocytes, while NKX2.1 + cells are unchanged compared with normal lungs at E18.5. j Real-time PCR of Nkx2.1 , Sox9 and pSpc . Only Sox9 mRNA expression is decreased in Col4a1 +/Δex41 . Gapdh was used as a normalizer. k The number of pSPC + cells at P30 is increased in mutant lungs. l , o NKX2.1, m , p SOX9 and n , q pSPC localization in normal lungs displays a scattered pattern around the saccular walls (arrows), while in Col4a1 +/Δex41 mutants NKX2.1 and pSPC are clustered together (arrows). Scale bars = 200 μm in a , b , 50 μm in c – h , and 200 μm in l – q

Techniques Used: Staining, Immunohistochemistry, Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Mutagenesis

27) Product Images from "During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems"

Article Title: During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems

Journal: Cellular Microbiology

doi: 10.1111/j.1462-5822.2007.01099.x

RT-PCR confirmation of transcriptomic data. RNA was extracted from Salmonella cells released from infected epithelial cells at 2 and 6 h p.i. and from mid-exponential LB cultures, reverse transcribed to cDNA and used as template for RT-PCR amplification of entB (A), invF (B), prgH (C), sifA (D), ssaG (E), flgI (F), fliC (G), fliF (H), fljB (I), gapA (J), pgi (K), zwf (L), nuoB (M) and nusG (N) cDNAs using specific primers pairs ( Table 5 and Experimental procedures ). Each panel shows the expression levels observed from the transcriptomic data (graph on the left) and the RT-PCR analyses (graph on the right). Black bars show expression levels determined from LB culture. Purple and magenta bars show expression levels obtained inside epithelial cells at 2 and 6 h p.i. respectively.
Figure Legend Snippet: RT-PCR confirmation of transcriptomic data. RNA was extracted from Salmonella cells released from infected epithelial cells at 2 and 6 h p.i. and from mid-exponential LB cultures, reverse transcribed to cDNA and used as template for RT-PCR amplification of entB (A), invF (B), prgH (C), sifA (D), ssaG (E), flgI (F), fliC (G), fliF (H), fljB (I), gapA (J), pgi (K), zwf (L), nuoB (M) and nusG (N) cDNAs using specific primers pairs ( Table 5 and Experimental procedures ). Each panel shows the expression levels observed from the transcriptomic data (graph on the left) and the RT-PCR analyses (graph on the right). Black bars show expression levels determined from LB culture. Purple and magenta bars show expression levels obtained inside epithelial cells at 2 and 6 h p.i. respectively.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection, Amplification, Expressing

28) Product Images from "Sympathetic Hyperactivity and Age Affect Segregation and Expression of Neurotransmitters"

Article Title: Sympathetic Hyperactivity and Age Affect Segregation and Expression of Neurotransmitters

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00411

Cold-stress increased expression of tyrosine hydroxylase (TH) mRNA and protein in adrenal medulla (AM). (A) Conventional RT-PCR analysis performed on total RNA obtained from AM showing an increase (17.5%) in TH mRNA after 5 days of cold-stress. RT-PCR assay was performed for the amplification of TH mRNA fragment of 646 bp. (B) Western blot (WB) analysis of TH protein (56 kDa) expression showing an increase (1.6-fold) after cold-stress. Graphs show quantification of TH relative to actin, mean ± SEM and the significance level. Actin was used as the housekeeping gene for RT-PCR and as the loading control for WB assays. * P
Figure Legend Snippet: Cold-stress increased expression of tyrosine hydroxylase (TH) mRNA and protein in adrenal medulla (AM). (A) Conventional RT-PCR analysis performed on total RNA obtained from AM showing an increase (17.5%) in TH mRNA after 5 days of cold-stress. RT-PCR assay was performed for the amplification of TH mRNA fragment of 646 bp. (B) Western blot (WB) analysis of TH protein (56 kDa) expression showing an increase (1.6-fold) after cold-stress. Graphs show quantification of TH relative to actin, mean ± SEM and the significance level. Actin was used as the housekeeping gene for RT-PCR and as the loading control for WB assays. * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot

29) Product Images from "Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF"

Article Title: Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp524

( A ) Native electrophoresis of hACF remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of hACF complex (lane 6: 8 nM; lane 7: 24 nM; lanes 8 and 9: 72 nM) in the presence (lanes 6–8) or absence (lane 9) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–E ) Schematic representation of individual DNA molecules remodeled by hACF. Bisulfite-converted DNAs from gel slices (black frames, lanes 6–9) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( F ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel E (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–D (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.
Figure Legend Snippet: ( A ) Native electrophoresis of hACF remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of hACF complex (lane 6: 8 nM; lane 7: 24 nM; lanes 8 and 9: 72 nM) in the presence (lanes 6–8) or absence (lane 9) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–E ) Schematic representation of individual DNA molecules remodeled by hACF. Bisulfite-converted DNAs from gel slices (black frames, lanes 6–9) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( F ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel E (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–D (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

Techniques Used: Electrophoresis, Incubation, Amplification, Polymerase Chain Reaction, Clone Assay, Methylation

( A ) Native electrophoresis of BRG1 remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of BRG1 (lane 10: 55 nM; lane 11: 170 nM; lanes 12 and 13: 500 nM) in the presence (lanes 10–12) or absence (lane 13) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–F ) Schematic representation of individual DNA molecules remodeled by BRG1. Bisulfite-converted DNAs from gel slices (black frames, lanes 10–13) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.
Figure Legend Snippet: ( A ) Native electrophoresis of BRG1 remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of BRG1 (lane 10: 55 nM; lane 11: 170 nM; lanes 12 and 13: 500 nM) in the presence (lanes 10–12) or absence (lane 13) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–F ) Schematic representation of individual DNA molecules remodeled by BRG1. Bisulfite-converted DNAs from gel slices (black frames, lanes 10–13) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

Techniques Used: Electrophoresis, Incubation, Amplification, Polymerase Chain Reaction, Clone Assay, Methylation

( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.
Figure Legend Snippet: ( A ) Native electrophoresis of SNF2H remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of SNF2H (lane 2: 6 nM; lane 3: 19 nM; lanes 4 and 5: 57 nM) in the presence (lanes 2–4) or absence (lane 5) of ATP (1 mM) as indicated on top, for 1 h at 30°C. Reactions were stopped by addition of ADP (10 mM) and incubation on ice for 10 min. Methylation of the remodeled products were performed as follow. The reactions were incubated for 15 min at 37°C after addition of M.SssI (5 U) and S-adenosylmethionine (160 μM). The remodeling reactions were separated by native gel electrophoresis after addition of competitor plasmid DNA and visualized by ethidium bromide staining. Gel areas excised and used for analysis are delimited by a black frame. Back frames are connected by dashed lines when gel slices were combined. ( B–F ) Schematic representation of individual DNA molecules remodeled by SNF2H. Bisulfite-converted DNAs from gel slices (black frames, lanes 3–5) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 2 D. The number of remodeled molecules shown is proportional to the average intensity of the bands generated after remodeling at enzyme concentration allowing maximal remodeling in three independent experiments. ( G ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel F (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–E (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

Techniques Used: Electrophoresis, Incubation, Methylation, Nucleic Acid Electrophoresis, Plasmid Preparation, Staining, Amplification, Polymerase Chain Reaction, Clone Assay, Generated, Concentration Assay

( A ) Proteins used in the nucleosome remodeling assays were separated by SDS-PAGE and visualized by Coomassie Blue staining. Molecular masses are indicated on the left and enzymes indicated on top. ( B ) Analysis of remodeling activities in the presence of M.SssI. The restriction enzyme-accessibility (REA) assays measured the ability of the remodeling factors to expose an MfeI restriction site at bp position 108 (31-bp away from the first protected site) in the absence or presence of M.SssI. M601 nucleosomes (50 nM) were incubated with MfeI (25 U) in the absence (blue curves) or the presence (2.5 U, purple curves; 5 U, red curves) of M.SssI. Reactions in the absence or presence of remodeling enzyme are specified by diamond or circle plots, respectively (as indicated on top of each panel). Reactions in the presence of remodeler but absence of ATP are plotted in black. Enzymes were used at the following concentrations: SNF2H: 530 nM; hACF: 160 nM; BRG1: 690 nM and hSWI/SNF 68 nM. Curve fits of the data (obtained from averaging at least two independent experiments) were achieved using first-order exponential decay using an apparent endpoint of the reactions with the KaleidaGraph software. k obs for SNF2H = 0.18 ± 0.01 min −1 without M.SssI, 0.19 ± 0.01 min −1 + 2.5 U M.SssI and 0.15 ± 0.01 min −1 + 5 U M.SssI. k obs for hACF= 0.08 ± 0.01 min −1 without M.SssI, 0.09 ± 0.01 min −1 + 2.5 U M.SssI and 0.09 ± 0.01 min −1 + 5 U M.SssI. k obs for BRG1 = 0.02 ± 0.001 min −1 without M.SssI, 0.019 ± 0.001 min −1 + 2.5 U M.SssI and 0.02 ± 0.001 min −1 + 5 U M.SssI. k obs for hSWI/SNF = 0.13 ± 0.01 min −1 without M.SssI, 0.11 ± 0.01 min −1 + 2.5 U M.SssI and 0.1 ± 0.005 min −1 + 5 U M.SssI. ( C ) Nucleosomes (50 nM) were incubated as in Figure 3 , but addition of remodeler was omitted. Nucleosomes were then methylated with of M.SssI (5 U), separated by native gel electrophoresis and visualized by ethidium bromide staining. The gel area excised and used for analysis is delimited by a black frame. ( D ) Schematic representation of individual DNA molecules. Bisulfite-converted DNA from the excised gel slice (black frame, lane 1) was amplified by PCR, cloned and sequenced. Each line represents the sequence of individual DNA clones and the circles represent CpG dinucleotides. Methylated and unmethylated CpGs are indicated by filled (black) and open circles, respectively. ( E ) The frequency of methylation was determined at given CpG sites by averaging methylation for all the DNA molecules showed in panel D and expressed as a percentage. The position of the CpGs relative to the DNA sequence is indicated on the X -axis and clone numbers are indicated on the Y -axis.
Figure Legend Snippet: ( A ) Proteins used in the nucleosome remodeling assays were separated by SDS-PAGE and visualized by Coomassie Blue staining. Molecular masses are indicated on the left and enzymes indicated on top. ( B ) Analysis of remodeling activities in the presence of M.SssI. The restriction enzyme-accessibility (REA) assays measured the ability of the remodeling factors to expose an MfeI restriction site at bp position 108 (31-bp away from the first protected site) in the absence or presence of M.SssI. M601 nucleosomes (50 nM) were incubated with MfeI (25 U) in the absence (blue curves) or the presence (2.5 U, purple curves; 5 U, red curves) of M.SssI. Reactions in the absence or presence of remodeling enzyme are specified by diamond or circle plots, respectively (as indicated on top of each panel). Reactions in the presence of remodeler but absence of ATP are plotted in black. Enzymes were used at the following concentrations: SNF2H: 530 nM; hACF: 160 nM; BRG1: 690 nM and hSWI/SNF 68 nM. Curve fits of the data (obtained from averaging at least two independent experiments) were achieved using first-order exponential decay using an apparent endpoint of the reactions with the KaleidaGraph software. k obs for SNF2H = 0.18 ± 0.01 min −1 without M.SssI, 0.19 ± 0.01 min −1 + 2.5 U M.SssI and 0.15 ± 0.01 min −1 + 5 U M.SssI. k obs for hACF= 0.08 ± 0.01 min −1 without M.SssI, 0.09 ± 0.01 min −1 + 2.5 U M.SssI and 0.09 ± 0.01 min −1 + 5 U M.SssI. k obs for BRG1 = 0.02 ± 0.001 min −1 without M.SssI, 0.019 ± 0.001 min −1 + 2.5 U M.SssI and 0.02 ± 0.001 min −1 + 5 U M.SssI. k obs for hSWI/SNF = 0.13 ± 0.01 min −1 without M.SssI, 0.11 ± 0.01 min −1 + 2.5 U M.SssI and 0.1 ± 0.005 min −1 + 5 U M.SssI. ( C ) Nucleosomes (50 nM) were incubated as in Figure 3 , but addition of remodeler was omitted. Nucleosomes were then methylated with of M.SssI (5 U), separated by native gel electrophoresis and visualized by ethidium bromide staining. The gel area excised and used for analysis is delimited by a black frame. ( D ) Schematic representation of individual DNA molecules. Bisulfite-converted DNA from the excised gel slice (black frame, lane 1) was amplified by PCR, cloned and sequenced. Each line represents the sequence of individual DNA clones and the circles represent CpG dinucleotides. Methylated and unmethylated CpGs are indicated by filled (black) and open circles, respectively. ( E ) The frequency of methylation was determined at given CpG sites by averaging methylation for all the DNA molecules showed in panel D and expressed as a percentage. The position of the CpGs relative to the DNA sequence is indicated on the X -axis and clone numbers are indicated on the Y -axis.

Techniques Used: SDS Page, Staining, Incubation, Software, Methylation, Nucleic Acid Electrophoresis, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing

( A ) Native electrophoresis of hSWI/SNF remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of hSWI/SNF complex (lane 14: 6 nM; lane 15: 19 nM; lanes 16 and 17: 56 nM) in the presence (lanes 14–16) or absence (lane 17) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–E ) Schematic representation of individual DNA molecules remodeled by hSWI/SNF. Bisulfite-converted DNAs from gel slices (black frames, lanes 14–17) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( F ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel E (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–D (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.
Figure Legend Snippet: ( A ) Native electrophoresis of hSWI/SNF remodeled products. Nucleosomes (∼100 nM) were incubated with increasing concentrations of hSWI/SNF complex (lane 14: 6 nM; lane 15: 19 nM; lanes 16 and 17: 56 nM) in the presence (lanes 14–16) or absence (lane 17) of ATP (1 mM) as indicated on top. Reactions were handled identically and in parallel to samples in Figure 3 . ( B–E ) Schematic representation of individual DNA molecules remodeled by hSWI/SNF. Bisulfite-converted DNAs from gel slices (black frames, lanes 14–17) were amplified by PCR, cloned and sequenced. Individual DNA clones are represented as described in Figure 3 . ( F ) Frequency of methylation at a given CpG site. Upper panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panel E (reaction without ATP). Lower panel: the frequency of methylation was determined by averaging methylation for all the DNA molecules showed in panels B–D (reactions with ATP). In both the upper and lower panels, frequencies obtained from the nucleosome substrate in the absence of remodeler ( Figure 2 E) are shown in grey for comparison.

Techniques Used: Electrophoresis, Incubation, Amplification, Polymerase Chain Reaction, Clone Assay, Methylation

30) Product Images from "Unravelling the RNA-Binding Properties of SAFB Proteins in Breast Cancer Cells"

Article Title: Unravelling the RNA-Binding Properties of SAFB Proteins in Breast Cancer Cells

Journal: BioMed Research International

doi: 10.1155/2015/395816

SAFB2 regulates expression of MALAT-1 . (a) Expression of MALAT-1 was measured by qRT-PCR using RNA from MCF-7 and MDA-MB-231 cells transfected with negative, SAFB1, SAFB2 or SAFB1 and SAFB2 siRNA using validated TaqMan probes specifically targeting MALAT-1. Data represents the average of three biological replicates ± SD. Repeated Measures ANOVA with Dunnett's Multiple Comparison Test statistical significance of mRNA expression was calculated using Student's t -test; P
Figure Legend Snippet: SAFB2 regulates expression of MALAT-1 . (a) Expression of MALAT-1 was measured by qRT-PCR using RNA from MCF-7 and MDA-MB-231 cells transfected with negative, SAFB1, SAFB2 or SAFB1 and SAFB2 siRNA using validated TaqMan probes specifically targeting MALAT-1. Data represents the average of three biological replicates ± SD. Repeated Measures ANOVA with Dunnett's Multiple Comparison Test statistical significance of mRNA expression was calculated using Student's t -test; P

Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Transfection

31) Product Images from "Molecular Detection and Identification of Rickettsia Species in Ixodes pacificus in California"

Article Title: Molecular Detection and Identification of Rickettsia Species in Ixodes pacificus in California

Journal: Vector Borne and Zoonotic Diseases

doi: 10.1089/vbz.2010.0077

Detection of 16S rRNA, gltA , and ompA genes by PCR amplification of Ixodes pacificus tick extracts. Ninety-six ticks were collected in the Napa Valley, California. Tick DNA was extracted, pooled, and used as template for PCR amplification. The PCR products were electrophoresed and observed by staining with ethidium bromide. kb, 1 kb DNA ladder (Promega). Ip, I. pacificus tick extract; (+), Rickettsia conorii DNA positive control; (−), no template negative control; arrow, PCR amplicons. PCR, polymerase chain reaction.
Figure Legend Snippet: Detection of 16S rRNA, gltA , and ompA genes by PCR amplification of Ixodes pacificus tick extracts. Ninety-six ticks were collected in the Napa Valley, California. Tick DNA was extracted, pooled, and used as template for PCR amplification. The PCR products were electrophoresed and observed by staining with ethidium bromide. kb, 1 kb DNA ladder (Promega). Ip, I. pacificus tick extract; (+), Rickettsia conorii DNA positive control; (−), no template negative control; arrow, PCR amplicons. PCR, polymerase chain reaction.

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

32) Product Images from "Multiple T-type Ca2+ current subtypes in electrophysiologically characterized hamster dorsal horn neurons: possible role in spinal sensory integration"

Article Title: Multiple T-type Ca2+ current subtypes in electrophysiologically characterized hamster dorsal horn neurons: possible role in spinal sensory integration

Journal: Journal of Neurophysiology

doi: 10.1152/jn.01083.2010

Detection of Ca V 3.1 subunit mRNA from a dorsal horn neuron with a “fast” I T component (see text). Amplification plots of Ca V 3.1 subunit fragments were generated from a real-time PCR experiment. Each measurement was averaged from 3 replicate reactions. The fluorescence signal was normalized to an internal passive reference dye (dRn), and plots were base-lined by Stratagene software. Amplified product from cell 080707-1 (●) and hamster spinal cord cDNA with (■, positive control 1 ) and without (▲, positive control 2 ) a first-step Ca V 3 PCR amplification are shown. For a negative control, cDNA template was replaced with water (○). The threshold fluorescence value (indicated by dashed line) was determined by the software using an amplification-based algorithm, resulting in threshold cycles (C t ) of 23, 31, and 35 for positive control 1 , amplified cell product, and positive control 2 , respectively. Inset : the decay of I T ( bottom ) recorded from the same dorsal horn neuron can be fitted with a single exponential (τ = 20 ms). The voltage command used to activate the current is shown above the trace.
Figure Legend Snippet: Detection of Ca V 3.1 subunit mRNA from a dorsal horn neuron with a “fast” I T component (see text). Amplification plots of Ca V 3.1 subunit fragments were generated from a real-time PCR experiment. Each measurement was averaged from 3 replicate reactions. The fluorescence signal was normalized to an internal passive reference dye (dRn), and plots were base-lined by Stratagene software. Amplified product from cell 080707-1 (●) and hamster spinal cord cDNA with (■, positive control 1 ) and without (▲, positive control 2 ) a first-step Ca V 3 PCR amplification are shown. For a negative control, cDNA template was replaced with water (○). The threshold fluorescence value (indicated by dashed line) was determined by the software using an amplification-based algorithm, resulting in threshold cycles (C t ) of 23, 31, and 35 for positive control 1 , amplified cell product, and positive control 2 , respectively. Inset : the decay of I T ( bottom ) recorded from the same dorsal horn neuron can be fitted with a single exponential (τ = 20 ms). The voltage command used to activate the current is shown above the trace.

Techniques Used: Amplification, Generated, Real-time Polymerase Chain Reaction, Fluorescence, Software, Positive Control, Polymerase Chain Reaction, Negative Control, Mass Spectrometry

33) Product Images from "A modifiable microarray-based universal sensor: providing sample-to-results automation"

Article Title: A modifiable microarray-based universal sensor: providing sample-to-results automation

Journal: Heliyon

doi: 10.1016/j.heliyon.2016.e00179

(A) Image of microarray showing hybridization of target amplicons generated from DNA automatically isolated and PCR amplified from Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV). The sample preparation and PCR amplification was automatically performed using the cartridge by adding 200 μl of cultured targets (1 × 10 3 copies/ml) to the sample reservoir either individually or all three mixed together. The PCR amplicons were detected using the two step hybridization method described in Materials and Methods. Each universal probe was immobilized at two concentrations (20 μM, 2 μM as described in Materials and Methods). (B) To estimate the limit of detection, the target microorganisms were diluted to 25, 50 and 75 copies per ml and 200 μl of the specimens added to the cartridge either individually or all three combined. The location and concentration of the various STI target probes as well as the Spotting Controls (SC) on the DNA array are shown in the filter key.
Figure Legend Snippet: (A) Image of microarray showing hybridization of target amplicons generated from DNA automatically isolated and PCR amplified from Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV). The sample preparation and PCR amplification was automatically performed using the cartridge by adding 200 μl of cultured targets (1 × 10 3 copies/ml) to the sample reservoir either individually or all three mixed together. The PCR amplicons were detected using the two step hybridization method described in Materials and Methods. Each universal probe was immobilized at two concentrations (20 μM, 2 μM as described in Materials and Methods). (B) To estimate the limit of detection, the target microorganisms were diluted to 25, 50 and 75 copies per ml and 200 μl of the specimens added to the cartridge either individually or all three combined. The location and concentration of the various STI target probes as well as the Spotting Controls (SC) on the DNA array are shown in the filter key.

Techniques Used: Microarray, Hybridization, Generated, Isolation, Polymerase Chain Reaction, Amplification, Sample Prep, Cell Culture, Concentration Assay, DNA Array

34) Product Images from "Molecular Diagnosis and Identification of Leishmania Species in Jordan from Saved Dry Samples"

Article Title: Molecular Diagnosis and Identification of Leishmania Species in Jordan from Saved Dry Samples

Journal: BioMed Research International

doi: 10.1155/2016/6871739

Representative pictures showing agarose gel electrophoresis (2%) of random PCR products (300–350 bp) which were extracted from the 30 positive Leishmania samples. Lane M: 100 bp DNA ladder. Lanes 1–12: PCR products randomly selected from 30 clinical samples (a) and (b) showing the digestion of amplified ITS1 regions for different Leishmania species with the restriction endonuclease Hae IIII. Lane M: 100 bp DNA ladder. Lane 1: negative control. Lane 2: healthy individual control. Lane 3: L. major positive control showing two bands (203 bp and 132 bp). Lane 4: L. tropica positive control showing three bands (185 bp, 57 bp, and 24 bp). Lanes 5, 6, 7, 8, 9, 10, 11, 13, 14, and 15: random samples for L. major detected in clinical samples. Lane 12: L. tropica detected in clinical samples.
Figure Legend Snippet: Representative pictures showing agarose gel electrophoresis (2%) of random PCR products (300–350 bp) which were extracted from the 30 positive Leishmania samples. Lane M: 100 bp DNA ladder. Lanes 1–12: PCR products randomly selected from 30 clinical samples (a) and (b) showing the digestion of amplified ITS1 regions for different Leishmania species with the restriction endonuclease Hae IIII. Lane M: 100 bp DNA ladder. Lane 1: negative control. Lane 2: healthy individual control. Lane 3: L. major positive control showing two bands (203 bp and 132 bp). Lane 4: L. tropica positive control showing three bands (185 bp, 57 bp, and 24 bp). Lanes 5, 6, 7, 8, 9, 10, 11, 13, 14, and 15: random samples for L. major detected in clinical samples. Lane 12: L. tropica detected in clinical samples.

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

35) Product Images from "Efficient Human Cytomegalovirus Reactivation Is Maturation Dependent in the Langerhans Dendritic Cell Lineage and Can Be Studied using a CD14+ Experimental Latency Model"

Article Title: Efficient Human Cytomegalovirus Reactivation Is Maturation Dependent in the Langerhans Dendritic Cell Lineage and Can Be Studied using a CD14+ Experimental Latency Model

Journal: Journal of Virology

doi: 10.1128/JVI.00598-12

Efficient reactivation of HCMV from MoLCs is maturation dependent and enhanced by IL-6. (A) CD14 + monocytes TB40/e (lanes 1 and 2) or mock (lanes 3 and 4) infected were analyzed for RNA expression at 3 days postinfection. RNA with (+) or without (−) prior RT was amplified in UL138, IE72, and actin-specific PCRs. For the IE72 PCR, an HCMV DNA PCR-positive control was included to confirm that the PCR had worked (lane 5). (B) RNA isolated from immature MoLCs either mock treated (lane 1) or treated with IL-6 (lane 2), IL-8 (lane 3), LPS (lane 4), LPS plus neutralizing IL-6 antibody (lane 5), or LPS plus neutralizing IL-8 antibody (lane 6) was (+) or was not (−) subjected to RT and then amplified in an IE72 or actin PCR. (C and D) MoLCs were cocultured with fibroblasts to assay HCMV reactivation. After 10 days, the cultures were analyzed for evidence of plaque formation (C) and HCMV reactivation by inoculating fresh monolayers of fibroblasts with 50 μl of the supernatant and staining for IE gene expression 24 h postinfection as an indicator of infectious virus in the supernatant (D).
Figure Legend Snippet: Efficient reactivation of HCMV from MoLCs is maturation dependent and enhanced by IL-6. (A) CD14 + monocytes TB40/e (lanes 1 and 2) or mock (lanes 3 and 4) infected were analyzed for RNA expression at 3 days postinfection. RNA with (+) or without (−) prior RT was amplified in UL138, IE72, and actin-specific PCRs. For the IE72 PCR, an HCMV DNA PCR-positive control was included to confirm that the PCR had worked (lane 5). (B) RNA isolated from immature MoLCs either mock treated (lane 1) or treated with IL-6 (lane 2), IL-8 (lane 3), LPS (lane 4), LPS plus neutralizing IL-6 antibody (lane 5), or LPS plus neutralizing IL-8 antibody (lane 6) was (+) or was not (−) subjected to RT and then amplified in an IE72 or actin PCR. (C and D) MoLCs were cocultured with fibroblasts to assay HCMV reactivation. After 10 days, the cultures were analyzed for evidence of plaque formation (C) and HCMV reactivation by inoculating fresh monolayers of fibroblasts with 50 μl of the supernatant and staining for IE gene expression 24 h postinfection as an indicator of infectious virus in the supernatant (D).

Techniques Used: Infection, RNA Expression, Amplification, Polymerase Chain Reaction, Positive Control, Isolation, Staining, Expressing

36) Product Images from "Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner"

Article Title: Runx Transcription Factors Repress Human and Murine c-Myc Expression in a DNA-Binding and C-Terminally Dependent Manner

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069083

Human Jurkat T cells lentivirally transduced with Runx1.d190 show increased transcription of c-Myc . (A) Schematic of the structure of Runx1 and Runx1.d190. (B) 293T. cells were transfected with empty pEGFP-N1 vector (EGFPonly, left column) as a control for cytoplasmic staining, pEGFP-N1 vector containing full-length Runx1 fused in-frame to EGFP (Runx1FL, middle column) or Runx1.d190 fused in-frame to EGFP (Runx1.d190, right column). The nuclear DNA was visualized by staining with Hoescht 33342 (Nuclear, top row). Nuclear (top row) and EGFP (middle row) fluorescence are shown in isolation and merged (Merged, bottom row). (C) Relative differences in transcription between Jurkat T cells lentivirally transduced with control empty vector or vector encoding Runx1.d190 as determined by microarray analysis are shown. A complete listing of genes whose transcription is affected by Runx1.d190 in Jurkat T cells is located at http://www.ncbi.nlm.nih.gov/geo/ . (D) ChIP analysis. Chromatin was prepared from Jurkat T cells lentivirally transduced with Runx1.d190 and immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out using primer sets amplifying Runx1-binding sites at -0.83 (i), -7.9 (ii) and -8.9 kb (iii) upstream of the human c-Myc transcriptional start site. N =3.
Figure Legend Snippet: Human Jurkat T cells lentivirally transduced with Runx1.d190 show increased transcription of c-Myc . (A) Schematic of the structure of Runx1 and Runx1.d190. (B) 293T. cells were transfected with empty pEGFP-N1 vector (EGFPonly, left column) as a control for cytoplasmic staining, pEGFP-N1 vector containing full-length Runx1 fused in-frame to EGFP (Runx1FL, middle column) or Runx1.d190 fused in-frame to EGFP (Runx1.d190, right column). The nuclear DNA was visualized by staining with Hoescht 33342 (Nuclear, top row). Nuclear (top row) and EGFP (middle row) fluorescence are shown in isolation and merged (Merged, bottom row). (C) Relative differences in transcription between Jurkat T cells lentivirally transduced with control empty vector or vector encoding Runx1.d190 as determined by microarray analysis are shown. A complete listing of genes whose transcription is affected by Runx1.d190 in Jurkat T cells is located at http://www.ncbi.nlm.nih.gov/geo/ . (D) ChIP analysis. Chromatin was prepared from Jurkat T cells lentivirally transduced with Runx1.d190 and immunoprecipitated with preimmune sera (Pre-immune) or anti-distal Runx1 (α-Runx1). PCR was carried out using primer sets amplifying Runx1-binding sites at -0.83 (i), -7.9 (ii) and -8.9 kb (iii) upstream of the human c-Myc transcriptional start site. N =3.

Techniques Used: Transduction, Transfection, Plasmid Preparation, Staining, Fluorescence, Isolation, Microarray, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Binding Assay

37) Product Images from "Molecular characterisation and disease severity of leptospirosis in Sri Lanka"

Article Title: Molecular characterisation and disease severity of leptospirosis in Sri Lanka

Journal: Memórias do Instituto Oswaldo Cruz

doi: 10.1590/0074-02760150070

: hae 111 digestion of Leptospira . Lane 1: 100 bp DNA marker; 2: undigested polymerase chain reaction (PCR) product; 3: Leptospira interrogans serovar Canicola (100 bp, 300 bp, 400 bp); 4: L. interrogans serovar Icterohaemorrhagiae (100 bp, 200 bp, 300 bp); 5: L. interrogans serovar Pyrogenes (100 bp, 300 bp, 400 bp); 6; Leptospira biflexa Patoc 1 strain; 7-13: flaB PCR positive patient samples.
Figure Legend Snippet: : hae 111 digestion of Leptospira . Lane 1: 100 bp DNA marker; 2: undigested polymerase chain reaction (PCR) product; 3: Leptospira interrogans serovar Canicola (100 bp, 300 bp, 400 bp); 4: L. interrogans serovar Icterohaemorrhagiae (100 bp, 200 bp, 300 bp); 5: L. interrogans serovar Pyrogenes (100 bp, 300 bp, 400 bp); 6; Leptospira biflexa Patoc 1 strain; 7-13: flaB PCR positive patient samples.

Techniques Used: Marker, Polymerase Chain Reaction

: hind 111 digestion of Leptospira . Lane 1: 100 bp DNA marker; 2: undigested polymerase chain reaction (PCR) product; 3: Leptospira interrogans serovar Canicola; 4: L. interrogans serovar Icterohae-morrhagiae; 5: L. interrogans serovar Pyrogenes; 6: Leptospira biflexa Patoc 1 strain; 7-13: flaB PCR positive patient samples.
Figure Legend Snippet: : hind 111 digestion of Leptospira . Lane 1: 100 bp DNA marker; 2: undigested polymerase chain reaction (PCR) product; 3: Leptospira interrogans serovar Canicola; 4: L. interrogans serovar Icterohae-morrhagiae; 5: L. interrogans serovar Pyrogenes; 6: Leptospira biflexa Patoc 1 strain; 7-13: flaB PCR positive patient samples.

Techniques Used: Marker, Polymerase Chain Reaction

38) Product Images from "Rapid TaqMan-Based Quantification of Chlorophyll d-Containing Cyanobacteria in the Genus Acaryochloris"

Article Title: Rapid TaqMan-Based Quantification of Chlorophyll d-Containing Cyanobacteria in the Genus Acaryochloris

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00334-14

Sensitivity and amplification efficiencies of the Acaryochloris -specific TaqMan assay. Threshold cycle ( C T ) values were determined by qPCR amplification of A. marina MBIC11017 16S rRNA gene fragments within linearized plasmid vectors of known concentrations (10 0 to 10 6 copies). All measurements were performed in technical triplicates and displayed as the mean C T with standard deviations (not visible due to small deviations). Using the primer pair and TaqMan probe developed in this study, the detection limit of the assay is ∼10 1 (indicated by the dotted line). The calculated PCR amplification efficiency was 94.6%, as derived from the slope of the standard curve ( R 2 > 0.99).
Figure Legend Snippet: Sensitivity and amplification efficiencies of the Acaryochloris -specific TaqMan assay. Threshold cycle ( C T ) values were determined by qPCR amplification of A. marina MBIC11017 16S rRNA gene fragments within linearized plasmid vectors of known concentrations (10 0 to 10 6 copies). All measurements were performed in technical triplicates and displayed as the mean C T with standard deviations (not visible due to small deviations). Using the primer pair and TaqMan probe developed in this study, the detection limit of the assay is ∼10 1 (indicated by the dotted line). The calculated PCR amplification efficiency was 94.6%, as derived from the slope of the standard curve ( R 2 > 0.99).

Techniques Used: Amplification, TaqMan Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Polymerase Chain Reaction, Derivative Assay

39) Product Images from "Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay"

Article Title: Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0085-2

The detection rate of T. pallidum DNA in blood samples from all patients by Tpp47 -Tp-PCR and polA -Tp-PCR. a The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR among all patients ( n = 262). b The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the primary syphilis patients ( n = 84). c The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the secondary syphilis patients ( n = 97). d The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the latent syphilis patients ( n = 81)
Figure Legend Snippet: The detection rate of T. pallidum DNA in blood samples from all patients by Tpp47 -Tp-PCR and polA -Tp-PCR. a The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR among all patients ( n = 262). b The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the primary syphilis patients ( n = 84). c The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the secondary syphilis patients ( n = 97). d The positive specimens of polA -Tp-PCR and/or Tpp47 -Tp-PCR in the latent syphilis patients ( n = 81)

Techniques Used: Polymerase Chain Reaction

40) Product Images from "Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods"

Article Title: Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

Journal: Malaria Journal

doi: 10.1186/1475-2875-6-111

Examples of AS-PCR products for kdr -e and kdr -w genotyping . Gels A to D show examples of AS-PCR results using four different protocols, A [29], B [11], C [12], D [20]. Gels E to G show the result of using different DNA polymerases on the AS-PCR method described by [20], E: Promega PCR master mix, F: Qiagen HotStar Taq G:Finzymes Dynazyme II Gel H is an example of results of using the protocol of [20] with the Promega PCR master mix to genotype samples using the kdr -e assay from the 96 reference plate and gel I for the kdr -w assay. The same DNA templates were used in PCRs shown in gels A to G and from left to right were 100 bp DNA Ladder (Fermentas), homozygous wildtype, homozygous wildtype, heterozygous, heterozygous, homozygous mutant, homozygous mutant.
Figure Legend Snippet: Examples of AS-PCR products for kdr -e and kdr -w genotyping . Gels A to D show examples of AS-PCR results using four different protocols, A [29], B [11], C [12], D [20]. Gels E to G show the result of using different DNA polymerases on the AS-PCR method described by [20], E: Promega PCR master mix, F: Qiagen HotStar Taq G:Finzymes Dynazyme II Gel H is an example of results of using the protocol of [20] with the Promega PCR master mix to genotype samples using the kdr -e assay from the 96 reference plate and gel I for the kdr -w assay. The same DNA templates were used in PCRs shown in gels A to G and from left to right were 100 bp DNA Ladder (Fermentas), homozygous wildtype, homozygous wildtype, heterozygous, heterozygous, homozygous mutant, homozygous mutant.

Techniques Used: Polymerase Chain Reaction, Mutagenesis

Related Articles

Clone Assay:

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination
Article Snippet: .. Sμ region DNA of individual clones were re-amplified using the Sμ-specific primers (as above) and GoTaq Long PCR Master Mix. ..

Negative Control:

Article Title: Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo
Article Snippet: .. 3′-Homology Arm PCR Analysis 3′- homology arm (HA) PCR analyses were performed on the gDNAs of viable colonies using a GoTaq® Long PCR reaction system (Promega, M4021) according to the manufacturer’s manual. gDNA template from untransfected E14-Bra-GFP mESCs served as a negative control. ..

Amplification:

Article Title: The Paleo-Indian Entry into South America According to Mitogenomes
Article Snippet: .. The amplification was performed with 10–50 ng of template DNA in 50 µl of reaction mix containing 1× GoTaq LongPCR Master Mix (Promega) and 0.2 µM of each primer, according to manufacturer’s instructions. .. The PCR program included an initial denaturation step at 94 °C for 2 min, and 30 cycles with the following thermal profile: 94 °C for 30 s, 55 °C for 30 s, 65 °C for 9 min, with a final extension step at 72 °C for 10 min.

Polymerase Chain Reaction:

Article Title: Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes
Article Snippet: .. Five of these transformation events were verified using Long-Range PCR with Promega GoTaq® Long PCR Master Mix and primers LT-PCR-1 and LT-PCR-2 (see Supplementary Table ) to test for the presence of Cas9. .. Each of those five events was confirmed to express Cas9 by RT-PCR with primers RT-PCR-1 and RT-PCR-2 (see Supplementary Table ).

Article Title: Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons
Article Snippet: .. Specific primers with restriction sites were designed to amplify the coding region of European robin (er) Cry1a (GenBank accession number AY585716) and erCry1b (GenBank accession number AY585717). erCry1a sense (5'-aaagctagcatgggggtgaacgc-3') and antisense (5'-aaaggatccgtgtaatttgtgctctgtc-3') primers and erCry1b sense (5'-aaagctagcatgggggtgaacgc-3') and antisense (5'-acgtcgacccaaaatctatccatagtatt-3') primers were used to amplify the entire respective coding regions with the RT-PCR kit GoTaq® Long PCR Master Mix (Promega, Madison, WI, USA) from total RNA of European robin retina. .. To amplify the coding region of garden warbler (gw) Cry1a (GenBank accession number AJ632120), we used the vector pDIA92B-His-CRY1a as a template; similarly, the amplification of the gwCry1b transcript (GenBank accession number DQ838738) was carried out using the vector pDIA92B-HisCRY1b as template [ ].

Article Title: C11orf95-RELA fusions drive oncogenic NF-κB signaling in ependymoma
Article Snippet: .. PCR was carried out using GoTaq® Long PCR Master Mix (Promega, Madison, WI), using specific primers ( ). .. Cloning and Retroviral Production Human cDNA clones of C11ORF95, RELA and YAP1 were cloned into the pCX4-IRES-Red Fluorescence (cRFP) vector.

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination
Article Snippet: .. Sμ region DNA of individual clones were re-amplified using the Sμ-specific primers (as above) and GoTaq Long PCR Master Mix. ..

Article Title: Assessing the Effectiveness of a Far-Red Fluorescent Reporter for Tracking Stem Cells In Vivo
Article Snippet: .. 3′-Homology Arm PCR Analysis 3′- homology arm (HA) PCR analyses were performed on the gDNAs of viable colonies using a GoTaq® Long PCR reaction system (Promega, M4021) according to the manufacturer’s manual. gDNA template from untransfected E14-Bra-GFP mESCs served as a negative control. ..

Article Title: Cinnamic Acid and Sorbic acid Conversion Are Mediated by the Same Transcriptional Regulator in Aspergillus niger
Article Snippet: .. These three fragments were fused in a PCR reaction using the GoTaq Long PCR Master Mix (Promega, Madison, WI, USA). .. The fusion PCR mixture contained 0.4 μl of each amplified product, 0.6 μl of 10 μM upstream and downstream primers , 12.5 μl GoTaq Long PCR Master Mix in a total volume of 25 μl.

Article Title: Digital PCR Quantitation of Muscle Mitochondrial DNA: Age, Fiber Type, and Mutation-Induced Changes
Article Snippet: .. Long extension PCR reactions were assembled according to the manufacturer’s instructions, Go-Taq Long PCR master mix (Promega, Madison, WI). .. PCR cycling conditions were polymerase activation at 95°C for 2 minutes, denaturation at 94°C for 20 seconds, and annealing at 68°C for 10 minutes, repeated for 40 cycles.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons
Article Snippet: .. Specific primers with restriction sites were designed to amplify the coding region of European robin (er) Cry1a (GenBank accession number AY585716) and erCry1b (GenBank accession number AY585717). erCry1a sense (5'-aaagctagcatgggggtgaacgc-3') and antisense (5'-aaaggatccgtgtaatttgtgctctgtc-3') primers and erCry1b sense (5'-aaagctagcatgggggtgaacgc-3') and antisense (5'-acgtcgacccaaaatctatccatagtatt-3') primers were used to amplify the entire respective coding regions with the RT-PCR kit GoTaq® Long PCR Master Mix (Promega, Madison, WI, USA) from total RNA of European robin retina. .. To amplify the coding region of garden warbler (gw) Cry1a (GenBank accession number AJ632120), we used the vector pDIA92B-His-CRY1a as a template; similarly, the amplification of the gwCry1b transcript (GenBank accession number DQ838738) was carried out using the vector pDIA92B-HisCRY1b as template [ ].

Transformation Assay:

Article Title: Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes
Article Snippet: .. Five of these transformation events were verified using Long-Range PCR with Promega GoTaq® Long PCR Master Mix and primers LT-PCR-1 and LT-PCR-2 (see Supplementary Table ) to test for the presence of Cas9. .. Each of those five events was confirmed to express Cas9 by RT-PCR with primers RT-PCR-1 and RT-PCR-2 (see Supplementary Table ).

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    Promega pcr master mix
    Amplification pattern by <t>RT-PCR</t> with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers <t>inF-F</t> and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.
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    Amplification pattern by RT-PCR with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers inF-F and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.

    Journal: BMC Microbiology

    Article Title: Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications

    doi: 10.1186/1471-2180-11-94

    Figure Lengend Snippet: Amplification pattern by RT-PCR with the site-specific primer pairs for intron-F and G . PCR products of from cDNA amplified with the primers inF-F and inF-R are eluted in lanes 2, 3, 15 and 16, and with primers inG-F and inG-R in lanes 4 and 5. PCR products from genomic DNA amplified with primer pair for intron-F are eluted in lanes 6, 7, 10, 13 and 14, and with primer pair for intron-G in lanes 8, 9 and 11. Lane 12 is the negative control.

    Article Snippet: PCR was performed individually using PCR Master Mix and the primer pair inF-F and inF-R for intron-F and inG-F and inG-R for intron-G which we newly designed.

    Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Negative Control

    Schematic representation of the insertion deletion. (A) mms16 wild-type gene and different truncated fragments (I to III). Sizes are as indicated. (B) Molecular organization of the mms16 locus before and after insertion-duplication mutagenesis with a truncated fragment. Different fill patterns are used to mark the origins of different parts of the gene after a single crossover. Parts encoding the N and C termini of the corresponding gene products are indicated. (C) Characterization of the Da127 mutant by RT-PCR. The following primer combinations were applied in PCRs: mmpF1 plus mmpB1 (lanes 1 and 2); mmpF1 plus mmpB2 (lanes 3 and 4). A 271-bp truncated fragment was used for mutant construction. Positions of primers are indicated in panel A. cDNA obtained from the wild type (lanes 1 and 3) or the mutant strain Da127 (lanes 2 and 4) was used as a template. Identical reactions with reverse transcriptase omitted were used as negative controls (data not shown).

    Journal: Journal of Bacteriology

    Article Title: The Presumptive Magnetosome Protein Mms16 Is a Poly(3-Hydroxybutyrate) Granule-Bound Protein (Phasin) in Magnetospirillum gryphiswaldense

    doi: 10.1128/JB.187.7.2416-2425.2005

    Figure Lengend Snippet: Schematic representation of the insertion deletion. (A) mms16 wild-type gene and different truncated fragments (I to III). Sizes are as indicated. (B) Molecular organization of the mms16 locus before and after insertion-duplication mutagenesis with a truncated fragment. Different fill patterns are used to mark the origins of different parts of the gene after a single crossover. Parts encoding the N and C termini of the corresponding gene products are indicated. (C) Characterization of the Da127 mutant by RT-PCR. The following primer combinations were applied in PCRs: mmpF1 plus mmpB1 (lanes 1 and 2); mmpF1 plus mmpB2 (lanes 3 and 4). A 271-bp truncated fragment was used for mutant construction. Positions of primers are indicated in panel A. cDNA obtained from the wild type (lanes 1 and 3) or the mutant strain Da127 (lanes 2 and 4) was used as a template. Identical reactions with reverse transcriptase omitted were used as negative controls (data not shown).

    Article Snippet: The obtained cDNA was amplified by using PCR master mix (Promega) and primer pairs mmpF1 plus mmpB1 and mmpF2 plus mmpB2 , which amplify 309- and 367-bp fragments of the mms16 gene, respectively.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction

    The generation of DNA templates from microarrays and parallel analysis. A ssDNA microarray was incubated with a primer (16 h) followed by elongation using Taq polymerase (16 h) producing as dsDNA microarray. The newly synthesized DNA strands were used as templates for solution phase PCR carried out over the microarray leading to amplification of the ssDNA displayed on the microarray. The dsDNA was amplified by PCR to produce fluorescently labeled ssDNA analogous to the ssDNA printed on the microarray. The fluorescently labeled ssDNA was hybridized to a complementary microarray or submitted to Solexa sequencing to allow decoding of the amplified ssDNA. FAM = 5(6)-carboxyfluorescein.

    Journal: PLoS ONE

    Article Title: Microarray Generation of Thousand-Member Oligonucleotide Libraries

    doi: 10.1371/journal.pone.0024906

    Figure Lengend Snippet: The generation of DNA templates from microarrays and parallel analysis. A ssDNA microarray was incubated with a primer (16 h) followed by elongation using Taq polymerase (16 h) producing as dsDNA microarray. The newly synthesized DNA strands were used as templates for solution phase PCR carried out over the microarray leading to amplification of the ssDNA displayed on the microarray. The dsDNA was amplified by PCR to produce fluorescently labeled ssDNA analogous to the ssDNA printed on the microarray. The fluorescently labeled ssDNA was hybridized to a complementary microarray or submitted to Solexa sequencing to allow decoding of the amplified ssDNA. FAM = 5(6)-carboxyfluorescein.

    Article Snippet: PCR in solution The purified products (250 ng) from each of the PCR “read-off” microarrays were used as templates in another round of PCR with primer-1 and 2 (1 µM) or Solexa-primer-1 and 2 (1 µM) in a 1× PCR Master Mix (200 µL; Promega, 25 U/mL Taq Polymerase, 200 µM dNTP, 1.5 mM MgCl2 ) in a Techne TC-312 PCR cycler with the same cycle as on the microarray.

    Techniques: Microarray, Incubation, Synthesized, Polymerase Chain Reaction, Amplification, Labeling, Sequencing

    The kinetics of infection in mice with TgCtwh6 isolates. Kinetics of infection in blood and organs after oral infection with 50 cysts of TgCtwh6 isolate (genotype Chinese 1) in mice. I: the number of DNA copies in blood and tissues of the TgCtwh6 isolate post infection at various intervals by qPCR. Each point represents the mean value of the T. gondii DNA copies for five mice ± SD. II: detection of PCR products of T. gondii 529 bp fragments extracted from brain or ascitic fluid in the recipient mice. Abbreviations: A ; blood, B ; heart, C ; liver, D ; brain, and E ; lymph node. b, n, and p represent blank, negative and positive control. * P

    Journal: PLoS ONE

    Article Title: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

    doi: 10.1371/journal.pone.0053483

    Figure Lengend Snippet: The kinetics of infection in mice with TgCtwh6 isolates. Kinetics of infection in blood and organs after oral infection with 50 cysts of TgCtwh6 isolate (genotype Chinese 1) in mice. I: the number of DNA copies in blood and tissues of the TgCtwh6 isolate post infection at various intervals by qPCR. Each point represents the mean value of the T. gondii DNA copies for five mice ± SD. II: detection of PCR products of T. gondii 529 bp fragments extracted from brain or ascitic fluid in the recipient mice. Abbreviations: A ; blood, B ; heart, C ; liver, D ; brain, and E ; lymph node. b, n, and p represent blank, negative and positive control. * P

    Article Snippet: Briefly, the target DNA sequences were amplified by PCR using PCR Master Mix (Promega, USA) for all markers.

    Techniques: Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control