Structured Review

Promega pcr master mix
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 <t>RT-PCR.</t> 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. <t>cDNA</t> 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).
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Images

1) 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

2) 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

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 "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

6) 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

7) 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

8) 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

9) 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

10) 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

11) 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

12) 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

13) 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

14) 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

15) 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

16) 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

17) 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

18) 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

19) 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

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 "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

22) 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

23) 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

24) 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

25) 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

26) 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

27) 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

28) 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

29) 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

30) 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

31) 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

32) 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

33) Product Images from "Glial Cell Line-Derived Neurotrophic Factor Mediates the Desirable Actions of the Anti-Addiction Drug Ibogaine against Alcohol Consumption"

Article Title: Glial Cell Line-Derived Neurotrophic Factor Mediates the Desirable Actions of the Anti-Addiction Drug Ibogaine against Alcohol Consumption

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.3959-04.2005

Ibogaine activates the GDNF pathway in SHSY5Y cells. A , Cells were treated with 10 μm ibogaine for the indicated times and lysed for total RNA isolation. Expression of GDNF and control actin was analyzed by RT-PCR ( n =4). B , Cells were treated with 10 μm ibogaine for the indicated times. GDNF in the media was detected by an ELISA assay. Histogram depicts the mean ± SD of GDNF secretion in three experiments. C , Cells were treated with the indicated concentrations of ibogaine for 3 hr. Ret was immunoprecipitated with anti-Ret antibodies, followed by Western blot analysis with anti-GFRα1 antibodies. The levels of GFRα1 in the homogenates were determined by Western blot analysis ( n =3). D , Cells were treated with 10 μm ibogaine for the indicated times. Ret was immunoprecipitated followed by Western blot analysis with anti-phosphotyrosine or anti-Ret antibodies ( n = 3). E , Cells were treated with vehicle (lane 1) or 10 μm ibogaine (lane 2) for 3 hr or with 50 ng/ml BDNF for 10 min (lane 3). Trk phosphorylation was analyzed by Western blot analysis with anti-phospho-Trk antibodies. The levels of TrkB were also determined by Western blot analysis ( n = 3). F , Cells were preincubated with 0.3 U/ml PI-PLC for 1 hr (lanes 3 and 4). Cells were then washed and treated without (lanes 1 and 3) or with (lanes 2 and 4) 10 μm ibogaine for 3 hr. Ret phosphorylation was determined as described above ( n = 4). G , Cells were treated for 3 hr with vehicle (lane 1), 10 μm ibogaine (lane 2), 10 μm ibogaine plus 10 μg/ml of mouse IgG (lane 3), or 10 μm ibogaine plus anti-GDNF neutralizing antibodies (lane 4). Treatment with 50 ng/ml GDNF was used as a positive control (lane 5). The cells were lysed and Ret phosphorylation was analyzed as described above ( n = 3).
Figure Legend Snippet: Ibogaine activates the GDNF pathway in SHSY5Y cells. A , Cells were treated with 10 μm ibogaine for the indicated times and lysed for total RNA isolation. Expression of GDNF and control actin was analyzed by RT-PCR ( n =4). B , Cells were treated with 10 μm ibogaine for the indicated times. GDNF in the media was detected by an ELISA assay. Histogram depicts the mean ± SD of GDNF secretion in three experiments. C , Cells were treated with the indicated concentrations of ibogaine for 3 hr. Ret was immunoprecipitated with anti-Ret antibodies, followed by Western blot analysis with anti-GFRα1 antibodies. The levels of GFRα1 in the homogenates were determined by Western blot analysis ( n =3). D , Cells were treated with 10 μm ibogaine for the indicated times. Ret was immunoprecipitated followed by Western blot analysis with anti-phosphotyrosine or anti-Ret antibodies ( n = 3). E , Cells were treated with vehicle (lane 1) or 10 μm ibogaine (lane 2) for 3 hr or with 50 ng/ml BDNF for 10 min (lane 3). Trk phosphorylation was analyzed by Western blot analysis with anti-phospho-Trk antibodies. The levels of TrkB were also determined by Western blot analysis ( n = 3). F , Cells were preincubated with 0.3 U/ml PI-PLC for 1 hr (lanes 3 and 4). Cells were then washed and treated without (lanes 1 and 3) or with (lanes 2 and 4) 10 μm ibogaine for 3 hr. Ret phosphorylation was determined as described above ( n = 4). G , Cells were treated for 3 hr with vehicle (lane 1), 10 μm ibogaine (lane 2), 10 μm ibogaine plus 10 μg/ml of mouse IgG (lane 3), or 10 μm ibogaine plus anti-GDNF neutralizing antibodies (lane 4). Treatment with 50 ng/ml GDNF was used as a positive control (lane 5). The cells were lysed and Ret phosphorylation was analyzed as described above ( n = 3).

Techniques Used: Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Western Blot, Planar Chromatography, Positive Control

34) Product Images from "Detection of VEGF-Axxxb Isoforms in Human Tissues"

Article Title: Detection of VEGF-Axxxb Isoforms in Human Tissues

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068399

Positive controls are required to interpret lack of amplification of VEGF-A 165 b by competitive RT-PCR. A. Plasmids containing VEGF-A 165 b or VEGF-A 165 a sequence were amplified using primers in exon 8b and exon 7. Two different sized products were generated. B. Densitometric analysis of published RT-PCR gels using plasmids containing VEGF-A 165 b and VEGF-A 165 a and primers in exon 7 and exon 8b. 13/15 show higher intensity for VEGF-A 165 a. p
Figure Legend Snippet: Positive controls are required to interpret lack of amplification of VEGF-A 165 b by competitive RT-PCR. A. Plasmids containing VEGF-A 165 b or VEGF-A 165 a sequence were amplified using primers in exon 8b and exon 7. Two different sized products were generated. B. Densitometric analysis of published RT-PCR gels using plasmids containing VEGF-A 165 b and VEGF-A 165 a and primers in exon 7 and exon 8b. 13/15 show higher intensity for VEGF-A 165 a. p

Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Sequencing, Generated

Isoform specific PCR requires positive controls to ensure specificity. A. Sequence of the VEGF 3′ exon sequence. (i) Exon 7 (red) contains the same last three nucleotides (underlined) as the last three nucleotides of exon 8a (blue, underlined), requiring specific PCR primers that extend into exon 7 (arrow). (ii) mispriming (VEGF-A 165 a -specific primers priming on VEGF-A 165 b, and VEGF-A 165 b -specific primers priming on VEGF-A 165 a) can occur both ways round if the conditions are not tested. B. Published control PCR gels demonstrating specificity of primer conditions. The original description of VEGF-A 165 b describing conditions at which VEGF-A 165 b is not misprimed in the presence of 100ng VEGF-A 165 a (lane highlighted by arrow), but still able to amplify 0.1ng VEGF-A 165 b. C. Annealing temperature dependence of the specificity of the isoform specific primers. Only at > 62°C is specificity resolved. D. qPCR using VEGF-A 165 a specific primers on VEGF-A 165 a and VEGF-A 165 b plasmid E. qPCR using VEGF-A 165 b specific primers on VEGF-A 165 a and VEGF-A 165 b plasmid.
Figure Legend Snippet: Isoform specific PCR requires positive controls to ensure specificity. A. Sequence of the VEGF 3′ exon sequence. (i) Exon 7 (red) contains the same last three nucleotides (underlined) as the last three nucleotides of exon 8a (blue, underlined), requiring specific PCR primers that extend into exon 7 (arrow). (ii) mispriming (VEGF-A 165 a -specific primers priming on VEGF-A 165 b, and VEGF-A 165 b -specific primers priming on VEGF-A 165 a) can occur both ways round if the conditions are not tested. B. Published control PCR gels demonstrating specificity of primer conditions. The original description of VEGF-A 165 b describing conditions at which VEGF-A 165 b is not misprimed in the presence of 100ng VEGF-A 165 a (lane highlighted by arrow), but still able to amplify 0.1ng VEGF-A 165 b. C. Annealing temperature dependence of the specificity of the isoform specific primers. Only at > 62°C is specificity resolved. D. qPCR using VEGF-A 165 a specific primers on VEGF-A 165 a and VEGF-A 165 b plasmid E. qPCR using VEGF-A 165 b specific primers on VEGF-A 165 a and VEGF-A 165 b plasmid.

Techniques Used: Polymerase Chain Reaction, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation

qRT-PCR using protocols shown in figure 2D and E can detect changes in splicing induced by splicing factor knockdown. A. C t max-C t for cDNA extracted from prostate cancer (PC3) cells with lentiviral knockdown of SRPK1 or scrambled. B. Amount of VEGF calculated from standard curves in Figure 2 . C. Amount of VEGF-A 165 b identified by Exon 8b primers (VEGF-A 165 b) or that calculated from mispriming of VEGF-A 165 a. D. Proportion of VEGF that is VEGF-A 165 a or VEGF-A 165 b in control and knockdown cells. Values are Mean±SEM (n = 2). 3E. qPCR for VEGF-A 165 a on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar). 3F qPCR for VEGF-A 165 b on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar).
Figure Legend Snippet: qRT-PCR using protocols shown in figure 2D and E can detect changes in splicing induced by splicing factor knockdown. A. C t max-C t for cDNA extracted from prostate cancer (PC3) cells with lentiviral knockdown of SRPK1 or scrambled. B. Amount of VEGF calculated from standard curves in Figure 2 . C. Amount of VEGF-A 165 b identified by Exon 8b primers (VEGF-A 165 b) or that calculated from mispriming of VEGF-A 165 a. D. Proportion of VEGF that is VEGF-A 165 a or VEGF-A 165 b in control and knockdown cells. Values are Mean±SEM (n = 2). 3E. qPCR for VEGF-A 165 a on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar). 3F qPCR for VEGF-A 165 b on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar).

Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction

35) 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

36) 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

37) Product Images from "Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status"

Article Title: Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054614

Southern blotting analysis of transgene integrants in genomic DNA of transgenic sheep. (A) Genomic DNA extracted from tail tips of transgenic sheep was digested with EcoRI and hybridized with 32 P labeled probe amplified from CMV promoter. (B) Genomic DNA extracted from tail tips of transgenic sheep was double-digested with Sfi I/ Hpa I and hybridized with 32 P labeled probe. NTC, non-transgenic sheep control; # 4–14, transgenic lambs identified by PCR corresponding to Fig. 1A . (C) pLEX-EGFP plasmid was double-digested with Sfi I/ Hpa I and diluted in serial concentrations matched to corresponding copies. Diluted plasmids with copies from 1 to 5 were hybridized with probe double-digested genomic DNA of transgenic lamb in parallel. (D) Standard curve of copy numbers in panel C was generated with diluted plasmid based on the quantification of the blots by densitometric measurement as described in the Materials and Method.
Figure Legend Snippet: Southern blotting analysis of transgene integrants in genomic DNA of transgenic sheep. (A) Genomic DNA extracted from tail tips of transgenic sheep was digested with EcoRI and hybridized with 32 P labeled probe amplified from CMV promoter. (B) Genomic DNA extracted from tail tips of transgenic sheep was double-digested with Sfi I/ Hpa I and hybridized with 32 P labeled probe. NTC, non-transgenic sheep control; # 4–14, transgenic lambs identified by PCR corresponding to Fig. 1A . (C) pLEX-EGFP plasmid was double-digested with Sfi I/ Hpa I and diluted in serial concentrations matched to corresponding copies. Diluted plasmids with copies from 1 to 5 were hybridized with probe double-digested genomic DNA of transgenic lamb in parallel. (D) Standard curve of copy numbers in panel C was generated with diluted plasmid based on the quantification of the blots by densitometric measurement as described in the Materials and Method.

Techniques Used: Southern Blot, Transgenic Assay, Labeling, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Generated

38) Product Images from "Virus detection and identification using random multiplex (RT)-PCR with 3'-locked random primers"

Article Title: Virus detection and identification using random multiplex (RT)-PCR with 3'-locked random primers

Journal: Virology Journal

doi: 10.1186/1743-422X-4-65

Real-Time PCR Quantitation of Adenovirus Type 17 DNA Template . A . PCR-amplified genomic DNA from Adenovirus Type 17 was quantitated by spectrophotometry (OD 260 nm) and the genome equivalents/ml calculated based on Avogadro's number (6.02205 × 10 23 /mol). Real-time PCR was conducted with Adenovirus Type 17 specific primers using 10 9 to 10 1 genome equivalents/ml as template and then using 1 μl of the stock solution of Adenovirus Type 17 as template. A standard curve was produced based on the threshold cycle number of the tested concentrations of Adenovirus Type 17 DNA template and plotted compared to the threshold cycle number (C T ) of the stock solution of Adenovirus Type 17. B . The indicated genome equivalents of Adenovirus Type 17 were added to 1 ml of human plasma, treated with 220 nm filtration and DNAse/RNAse digestion. Total nucleic acids then were purified, amplified by random multiplex PCR (50 cycles) and sequenced as described in the Methods section.
Figure Legend Snippet: Real-Time PCR Quantitation of Adenovirus Type 17 DNA Template . A . PCR-amplified genomic DNA from Adenovirus Type 17 was quantitated by spectrophotometry (OD 260 nm) and the genome equivalents/ml calculated based on Avogadro's number (6.02205 × 10 23 /mol). Real-time PCR was conducted with Adenovirus Type 17 specific primers using 10 9 to 10 1 genome equivalents/ml as template and then using 1 μl of the stock solution of Adenovirus Type 17 as template. A standard curve was produced based on the threshold cycle number of the tested concentrations of Adenovirus Type 17 DNA template and plotted compared to the threshold cycle number (C T ) of the stock solution of Adenovirus Type 17. B . The indicated genome equivalents of Adenovirus Type 17 were added to 1 ml of human plasma, treated with 220 nm filtration and DNAse/RNAse digestion. Total nucleic acids then were purified, amplified by random multiplex PCR (50 cycles) and sequenced as described in the Methods section.

Techniques Used: Real-time Polymerase Chain Reaction, Quantitation Assay, Polymerase Chain Reaction, Amplification, Spectrophotometry, Produced, Filtration, Purification, Multiplex Assay

PCR Screening and Sequencing of Randomly Amplified Coxsackie Virus A7 cDNA . Randomly amplified cDNA from Coxsackie Virus A7-infected plasma was shot-gun ligated into pCR 2.1-TOPO and competent E. coli were transformed. Resultant colonies were screened for the presence of recombinant plasmid DNA (A) and plasmid DNA from positive colonies was then purified and sequenced (B) . Sequence data from recombinant plasmid #7 (see *) was aligned to all sequence data in the Non-Redundant NCBI Database using the NCBI Nucleotide-Nucleotide BLAST (blastn) Search Algorithm (version 2.2.8).
Figure Legend Snippet: PCR Screening and Sequencing of Randomly Amplified Coxsackie Virus A7 cDNA . Randomly amplified cDNA from Coxsackie Virus A7-infected plasma was shot-gun ligated into pCR 2.1-TOPO and competent E. coli were transformed. Resultant colonies were screened for the presence of recombinant plasmid DNA (A) and plasmid DNA from positive colonies was then purified and sequenced (B) . Sequence data from recombinant plasmid #7 (see *) was aligned to all sequence data in the Non-Redundant NCBI Database using the NCBI Nucleotide-Nucleotide BLAST (blastn) Search Algorithm (version 2.2.8).

Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Infection, Transformation Assay, Recombinant, Plasmid Preparation, Purification

Random Multiplex PCR With 5'-VVVVVVVVAA-3' Primers Amplifies Circular Plasmid DNA . A . The indicated plasmids were amplified using random multiplex PCR with either 5'-VVVVVVVVAA-3' or 5'-NNNNNNNNNN-3' primers as indicated in the Methods section. B . The pBabe plasmid was diluted to the indicated amounts and amplified using random multiplex PCR with 5'-VVVVVVVVAA-3' primers.
Figure Legend Snippet: Random Multiplex PCR With 5'-VVVVVVVVAA-3' Primers Amplifies Circular Plasmid DNA . A . The indicated plasmids were amplified using random multiplex PCR with either 5'-VVVVVVVVAA-3' or 5'-NNNNNNNNNN-3' primers as indicated in the Methods section. B . The pBabe plasmid was diluted to the indicated amounts and amplified using random multiplex PCR with 5'-VVVVVVVVAA-3' primers.

Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Plasmid Preparation, Amplification

5'-VVVVVVVVAA-3' Primers Enable Random Multiplex Amplification of DNA from Human Plasma Inoculated with Adenovirus Type 17 . 1 ml of human plasma was inoculated with 0–5 μl of suspended Adenovirus Type 17 (ATCC), filtered and incubated for 2 hours with nucleases. Remaining nucleic acids were purified with the QiaAmp UltrasSens Virus Kit (Qiagen) and subjected to the random multiplex PCR with 5'-VVVVVVVVAA-5' primers as detailed in the Methods section. Amplicons were then visualized on an ethidium bromide impregnated agarose gel.
Figure Legend Snippet: 5'-VVVVVVVVAA-3' Primers Enable Random Multiplex Amplification of DNA from Human Plasma Inoculated with Adenovirus Type 17 . 1 ml of human plasma was inoculated with 0–5 μl of suspended Adenovirus Type 17 (ATCC), filtered and incubated for 2 hours with nucleases. Remaining nucleic acids were purified with the QiaAmp UltrasSens Virus Kit (Qiagen) and subjected to the random multiplex PCR with 5'-VVVVVVVVAA-5' primers as detailed in the Methods section. Amplicons were then visualized on an ethidium bromide impregnated agarose gel.

Techniques Used: Multiplex Assay, Amplification, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

PCR Screening and Sequencing of Randomly Amplified Adenovirus Type 17 DNA . Randomly amplified DNA from Adenovirus 17-infected plasma was shot-gun ligated into pCR 2.1-TOPO and competent E. coli were transformed. Resultant colonies were screened for the presence of recombinant plasmid DNA ( A ) and plasmid DNA from positive colonies was then purified and sequenced ( B ). Sequence data from recombinant plasmid #9 (see *) was aligned to all sequence data in the Non-Redundant NCBI Database using the NCBI Nucleotide-Nucleotide BLAST (blastn) Search Algorithm (version 2.2.8) ( C ).
Figure Legend Snippet: PCR Screening and Sequencing of Randomly Amplified Adenovirus Type 17 DNA . Randomly amplified DNA from Adenovirus 17-infected plasma was shot-gun ligated into pCR 2.1-TOPO and competent E. coli were transformed. Resultant colonies were screened for the presence of recombinant plasmid DNA ( A ) and plasmid DNA from positive colonies was then purified and sequenced ( B ). Sequence data from recombinant plasmid #9 (see *) was aligned to all sequence data in the Non-Redundant NCBI Database using the NCBI Nucleotide-Nucleotide BLAST (blastn) Search Algorithm (version 2.2.8) ( C ).

Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Infection, Transformation Assay, Recombinant, Plasmid Preparation, Purification

5'-VVVVVVVVAA-3' Primers Enable Random Multiplex Amplification of Viral RNA from Human Plasma Inoculated with Coxsackie Virus A7 . A . 1 ml of human plasma was inoculated with 5 μl of suspended Coxsackie Virus A7 (ATCC), filtered and incubated for 2 hours with nucleases. Remaining nucleic acids were purified with the QiaAmp UltrasSens Virus Kit (Qiagen) and subjected to first strand cDNA synthesis using either Omniscript or Superscript reverse transcriptase. Virus-specific primers were used to amplify either a 200 bp or a 1.4 Kb portion of the viral genome. B . The same samples were then subjected to random multiplex PCR with 5'-VVVVVVVVAA-5' primers as detailed in the Methods section. Amplicons were then visualized on an ethidium bromide impregnated agarose gel.
Figure Legend Snippet: 5'-VVVVVVVVAA-3' Primers Enable Random Multiplex Amplification of Viral RNA from Human Plasma Inoculated with Coxsackie Virus A7 . A . 1 ml of human plasma was inoculated with 5 μl of suspended Coxsackie Virus A7 (ATCC), filtered and incubated for 2 hours with nucleases. Remaining nucleic acids were purified with the QiaAmp UltrasSens Virus Kit (Qiagen) and subjected to first strand cDNA synthesis using either Omniscript or Superscript reverse transcriptase. Virus-specific primers were used to amplify either a 200 bp or a 1.4 Kb portion of the viral genome. B . The same samples were then subjected to random multiplex PCR with 5'-VVVVVVVVAA-5' primers as detailed in the Methods section. Amplicons were then visualized on an ethidium bromide impregnated agarose gel.

Techniques Used: Multiplex Assay, Amplification, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Filtration and Nuclease Treatment Improves Viral DNA Amplification by Removing Host DNA and mRNA . Human plasma was inoculated with 1 μl of an Adenovirus Type 17 suspension, filtered and incubated for 2 hrs with either 10 u DNAse or 5 u RNAse as indicated. Remaining nucleic acids were purified with the QiaAmp UltrasSens Virus Kit and subjected to 1 st strand cDNA synthesis and 50 cycles PCR using primers specific for either Adenovirus Type 17 or human β-Actin DNA (upper band; primers cross an intron) or cDNA (lower band). Amplicons were then visualized on an ethidium bromide impregnated agarose gel.
Figure Legend Snippet: Filtration and Nuclease Treatment Improves Viral DNA Amplification by Removing Host DNA and mRNA . Human plasma was inoculated with 1 μl of an Adenovirus Type 17 suspension, filtered and incubated for 2 hrs with either 10 u DNAse or 5 u RNAse as indicated. Remaining nucleic acids were purified with the QiaAmp UltrasSens Virus Kit and subjected to 1 st strand cDNA synthesis and 50 cycles PCR using primers specific for either Adenovirus Type 17 or human β-Actin DNA (upper band; primers cross an intron) or cDNA (lower band). Amplicons were then visualized on an ethidium bromide impregnated agarose gel.

Techniques Used: Filtration, Amplification, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

39) 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

DNA gel electrophoresis. ( a ) PCR products from the 1, 10, 3,875, 10,000 oligonucleotide microarrays. ( b ) Products from 5 repeats of PCR from the 10,000 oligonucleotide array. ( c ) dsDNA-10,000 and dsDNA-3875 (left) and their EcoICRI digestion (right). ( d ) PCR amplification with two primers producing dsDNA-10,000-FAM and dsDNA-3,875-FAM and dsDNA-10-FAM (left) and asymmetric PCR with a single primer producing ssDNA-10,000-FAM and ssDNA-3,875-FAM (right).
Figure Legend Snippet: DNA gel electrophoresis. ( a ) PCR products from the 1, 10, 3,875, 10,000 oligonucleotide microarrays. ( b ) Products from 5 repeats of PCR from the 10,000 oligonucleotide array. ( c ) dsDNA-10,000 and dsDNA-3875 (left) and their EcoICRI digestion (right). ( d ) PCR amplification with two primers producing dsDNA-10,000-FAM and dsDNA-3,875-FAM and dsDNA-10-FAM (left) and asymmetric PCR with a single primer producing ssDNA-10,000-FAM and ssDNA-3,875-FAM (right).

Techniques Used: DNA Gel Electrophoresis, Polymerase Chain Reaction, Amplification

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

40) Product Images from "Screening for Y-chromosome microdeletions in a population of infertile males in the Gaza Strip"

Article Title: Screening for Y-chromosome microdeletions in a population of infertile males in the Gaza Strip

Journal: Journal of Experimental & Clinical Assisted Reproduction

doi:

Representative ethidium bromide-stained agarose gels for detection of classic (full) AZF microdeletions (case #83). The STSs and the PCR product sizes are indicated above each band. L: 100 bp DNA Ladder.
Figure Legend Snippet: Representative ethidium bromide-stained agarose gels for detection of classic (full) AZF microdeletions (case #83). The STSs and the PCR product sizes are indicated above each band. L: 100 bp DNA Ladder.

Techniques Used: Staining, Polymerase Chain Reaction

Ethidium bromide-stained agarose gel demonstrating selected gr/gr deletions. Upper panel “A”: sY1291 STS PCR products show absence of sY1291 (527 bp) in gr/gr deletion cases #17, 47, 49, and 57 (lanes 4–7). Lower panel “B”: sY1191 STS PCR results showing presence of sY1191 (385 bp) in the same gr/gr deleted cases (lanes 4–7). L= 100 bp DNA ladder, Lane 1: negative control (water), Lane 2: negative control (female DNA), Lane 3: positive control (fertile male DNA).
Figure Legend Snippet: Ethidium bromide-stained agarose gel demonstrating selected gr/gr deletions. Upper panel “A”: sY1291 STS PCR products show absence of sY1291 (527 bp) in gr/gr deletion cases #17, 47, 49, and 57 (lanes 4–7). Lower panel “B”: sY1191 STS PCR results showing presence of sY1191 (385 bp) in the same gr/gr deleted cases (lanes 4–7). L= 100 bp DNA ladder, Lane 1: negative control (water), Lane 2: negative control (female DNA), Lane 3: positive control (fertile male DNA).

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

41) Product Images from "Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera"

Article Title: Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020513

Expression of AmelTwdl1 , AmelTwdl2 and Ampxd genes during the pupal-to-adult development coordinated by ecdysteroid titer. (A) Hemolymph ecdysteroid titer redrawn from Pinto et al . (see ref. [59] ), and the successive developmental phases in the interval between the pupal and adult ecdyses: Pw and Pp are early and late pupae; Pdp, Pb, Pbl, Pbm and Pbd are the successive pharate adult phases. (B) Transcriptional profile of AmelTwdl1 in the thoracic, abdominal and wing integument. (C) Developmental profile of thoracic integument proteins stained with Coomassie Brillant Blue (at the left) and the AmeTwdl1 protein (at the right) as detected using Western blot and an antibody against a cuticle protein from B. mori (BmGRP2). (D) Transcriptional profile of AmelTwdl2 in the thoracic, abdominal and wing integument. (E) Transcriptional profile of Ampxd in whole body extracts and wings. (F) AmPXD enzyme activity detected in thoracic integument samples using electrophoresis in polyacrylamide gels stained with a specific substrate. Transcript abundance was investigated by semiquantitative RT-PCR followed by electrophoresis of the amplified cDNAs in ethidium bromide-stained agarose gels. An A. mellifera actin gene, Amactin , was used as endogenous control.
Figure Legend Snippet: Expression of AmelTwdl1 , AmelTwdl2 and Ampxd genes during the pupal-to-adult development coordinated by ecdysteroid titer. (A) Hemolymph ecdysteroid titer redrawn from Pinto et al . (see ref. [59] ), and the successive developmental phases in the interval between the pupal and adult ecdyses: Pw and Pp are early and late pupae; Pdp, Pb, Pbl, Pbm and Pbd are the successive pharate adult phases. (B) Transcriptional profile of AmelTwdl1 in the thoracic, abdominal and wing integument. (C) Developmental profile of thoracic integument proteins stained with Coomassie Brillant Blue (at the left) and the AmeTwdl1 protein (at the right) as detected using Western blot and an antibody against a cuticle protein from B. mori (BmGRP2). (D) Transcriptional profile of AmelTwdl2 in the thoracic, abdominal and wing integument. (E) Transcriptional profile of Ampxd in whole body extracts and wings. (F) AmPXD enzyme activity detected in thoracic integument samples using electrophoresis in polyacrylamide gels stained with a specific substrate. Transcript abundance was investigated by semiquantitative RT-PCR followed by electrophoresis of the amplified cDNAs in ethidium bromide-stained agarose gels. An A. mellifera actin gene, Amactin , was used as endogenous control.

Techniques Used: Expressing, Staining, Western Blot, Activity Assay, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification

Abundance of AmelTwdl1 , AmelTwdl2 and Ampxd transcripts in the integument of ligated (L) abdomens compared to non-ligated controls (NL). Transcript levels were investigated at days (d) 1, 2, 3, 4, 5 and 6 after the abdominal ligature of newly ecdysed pupae. Transcript levels were assessed by semiquantitative RT-PCR assays followed by electrophoresis of the amplified cDNAs in ethidium bromide stained agarose gels. The Amactin gene was used as endogenous control.
Figure Legend Snippet: Abundance of AmelTwdl1 , AmelTwdl2 and Ampxd transcripts in the integument of ligated (L) abdomens compared to non-ligated controls (NL). Transcript levels were investigated at days (d) 1, 2, 3, 4, 5 and 6 after the abdominal ligature of newly ecdysed pupae. Transcript levels were assessed by semiquantitative RT-PCR assays followed by electrophoresis of the amplified cDNAs in ethidium bromide stained agarose gels. The Amactin gene was used as endogenous control.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Amplification, Staining

42) Product Images from "Evaluation of Reverse Transcription-PCR and a Bacteriophage-Based Assay for Rapid Phenotypic Detection of Rifampin Resistance in Clinical Isolates of Mycobacterium tuberculosis"

Article Title: Evaluation of Reverse Transcription-PCR and a Bacteriophage-Based Assay for Rapid Phenotypic Detection of Rifampin Resistance in Clinical Isolates of Mycobacterium tuberculosis

Journal: Journal of Clinical Microbiology

doi:

Agarose (1.5%) gel demonstrating loss of the 274-bp dnaK product with increasing concentrations of rifampin. RT-PCR was performed on five rifampin-susceptible clinical isolates of M. tuberculosis after overnight incubation in Middlebrook 7H9 broth with no drug (lanes 3 to 7) and with rifampin at 1 μg/ml (lanes 8 to 12), 2 μg/ml (lanes 13 to 17), and 4 μg/ml (lanes 18 to 22) following incubation at 45°C for 45 min. Lane 1, positive DNA control; lane 2, negative PCR control; lanes M, molecular weight markers.
Figure Legend Snippet: Agarose (1.5%) gel demonstrating loss of the 274-bp dnaK product with increasing concentrations of rifampin. RT-PCR was performed on five rifampin-susceptible clinical isolates of M. tuberculosis after overnight incubation in Middlebrook 7H9 broth with no drug (lanes 3 to 7) and with rifampin at 1 μg/ml (lanes 8 to 12), 2 μg/ml (lanes 13 to 17), and 4 μg/ml (lanes 18 to 22) following incubation at 45°C for 45 min. Lane 1, positive DNA control; lane 2, negative PCR control; lanes M, molecular weight markers.

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

43) Product Images from "The Cellular Processing Capacity Limits the Amounts of Chimeric U7 snRNA Available for Antisense Delivery"

Article Title: The Cellular Processing Capacity Limits the Amounts of Chimeric U7 snRNA Available for Antisense Delivery

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1038/mtna.2012.24

U7-mediated exon 23 skipping on the Duchenne muscular dystrophy ( Dmd ) pre-mRNA . ( a ) Reverse transcription and nested PCR detection of Dmd exon 23 skipping after transduction of C2C12 cells with AAV2/5-U7DTex23 (–MHCK) or AAV2/5-MHCK-U7DTex23 (+MHCK) with different multiplicities of infection (MOI) (10 5 or 10 6 viral genome per cell). Controls include nontransduced cells (NT) and no reverse transcriptase (RT–). The native Dmd mRNA containing exon 23 is detected as a 901-bp fragment and the skipped mRNA yields a 688-bp product. Results are representative of at least three independent transductions. ( b ) Analysis of mouse exon 23 skipping by quantitative PCR. The graph represents the ratio between the exon 22–24 junction (skipped mRNA species) and the exon 22–23 junction (full-length mRNA species). Quantification was performed on mRNA extract from C2C12 cells transduced with AAV2/5-U7DTex23 or AAV2/5-MHCK-U7DTex23 at the indicated MOI. A significant difference is observed at MOI 10 5 as shown by the P value ( P = 0.023), according to Student's t-test. Error bars are shown as mean ± SEM ( n = 6). MHCK, muscle- and heart-specific enhancer.
Figure Legend Snippet: U7-mediated exon 23 skipping on the Duchenne muscular dystrophy ( Dmd ) pre-mRNA . ( a ) Reverse transcription and nested PCR detection of Dmd exon 23 skipping after transduction of C2C12 cells with AAV2/5-U7DTex23 (–MHCK) or AAV2/5-MHCK-U7DTex23 (+MHCK) with different multiplicities of infection (MOI) (10 5 or 10 6 viral genome per cell). Controls include nontransduced cells (NT) and no reverse transcriptase (RT–). The native Dmd mRNA containing exon 23 is detected as a 901-bp fragment and the skipped mRNA yields a 688-bp product. Results are representative of at least three independent transductions. ( b ) Analysis of mouse exon 23 skipping by quantitative PCR. The graph represents the ratio between the exon 22–24 junction (skipped mRNA species) and the exon 22–23 junction (full-length mRNA species). Quantification was performed on mRNA extract from C2C12 cells transduced with AAV2/5-U7DTex23 or AAV2/5-MHCK-U7DTex23 at the indicated MOI. A significant difference is observed at MOI 10 5 as shown by the P value ( P = 0.023), according to Student's t-test. Error bars are shown as mean ± SEM ( n = 6). MHCK, muscle- and heart-specific enhancer.

Techniques Used: Nested PCR, Transduction, Infection, Real-time Polymerase Chain Reaction

44) Product Images from "CTNND1 755 T > G Promoter Polymorphism and Risk of Pancreatic Carcinoma in Chinese"

Article Title: CTNND1 755 T > G Promoter Polymorphism and Risk of Pancreatic Carcinoma in Chinese

Journal: Journal of Clinical Laboratory Analysis

doi: 10.1002/jcla.22055

PCR ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: DNA ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.
Figure Legend Snippet: PCR ‐ RFLP analysis of ‐755 T > G CTNND 1 polymorphism. lane M: DNA ladder; lane 1: TT genotype; lane 2: TG genotype; lane 3: GG genotype.

Techniques Used: Polymerase Chain Reaction

45) Product Images from "Inactivation of the Flagellin Gene flaA in Magnetospirillum gryphiswaldense Results in Nonmagnetotactic Mutants Lacking Flagellar Filaments"

Article Title: Inactivation of the Flagellin Gene flaA in Magnetospirillum gryphiswaldense Results in Nonmagnetotactic Mutants Lacking Flagellar Filaments

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.70.6.3624-3631.2004

(A) Analysis of the expression of the flaA gene in M. gryphiswaldense wild type and mutant strain Da136 by RT-PCR using various DNA templates. Lane 1, genomic DNA used as a template (positive control); lane 2, cDNA of the wild type; lane 3, cDNA of strain Da136; lane 4, wild-type RNA, reverse transcriptase omitted (negative control); lane 5, Da136 RNA, reverse transcriptase omitted (negative control). DNA was amplified by using primers flaRTfo and flaRTrw. (B) Analysis of the flagellin synthesis by SDS-12% PAGE. Whole-cell extracts of the wild type (lane 1) and the mutant Da136 (lane 2) were stained with Coomassie brilliant blue. A putative flagellin polypeptide was present in the wild-type strain (arrow) but absent from the mutant strain. M, molecular weight markers.
Figure Legend Snippet: (A) Analysis of the expression of the flaA gene in M. gryphiswaldense wild type and mutant strain Da136 by RT-PCR using various DNA templates. Lane 1, genomic DNA used as a template (positive control); lane 2, cDNA of the wild type; lane 3, cDNA of strain Da136; lane 4, wild-type RNA, reverse transcriptase omitted (negative control); lane 5, Da136 RNA, reverse transcriptase omitted (negative control). DNA was amplified by using primers flaRTfo and flaRTrw. (B) Analysis of the flagellin synthesis by SDS-12% PAGE. Whole-cell extracts of the wild type (lane 1) and the mutant Da136 (lane 2) were stained with Coomassie brilliant blue. A putative flagellin polypeptide was present in the wild-type strain (arrow) but absent from the mutant strain. M, molecular weight markers.

Techniques Used: Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Amplification, Polyacrylamide Gel Electrophoresis, Staining, Molecular Weight

46) Product Images from "Identification of Piwil2-Like (PL2L) Proteins that Promote Tumorigenesis"

Article Title: Identification of Piwil2-Like (PL2L) Proteins that Promote Tumorigenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0013406

Identification and characterization of PL2L proteins of humans and mice. A B , Western blot analysis of Piwil2 and PL2L proteins expressed in mouse testis (A) and HeLa cells (B), using rabbit polyclonal antibody to Piwil2 peptide. A, Testicular cell lysates of mili −/− and mili +/+ mice. B, HeLa cell lysate: Lane 1 5: 4 µg/mL antibody without peptide; Lane 2 6: 4 µg/mL antibody with 16 µg/mL Peptide; Lane 3: 2 µg/mL antibody alone; Lane 4: 2 µg/mL antibody with 8 µg/mL Peptide. P: Piwil2 peptide; Ab: antibody to Piwil2 peptide. C , GEM RT-PCR analysis of testicular tissues from wild-type (Lane 1) and mili −/− mice (Lane 2): the primer pairs specific for E1-7, E6-14, E13-21 and E21-23 of Piwil2 were used for GEM RT-PCR analysis, and the primer pair specific for E18-21 was used as a positive control. Lane 3: no cDNA control.
Figure Legend Snippet: Identification and characterization of PL2L proteins of humans and mice. A B , Western blot analysis of Piwil2 and PL2L proteins expressed in mouse testis (A) and HeLa cells (B), using rabbit polyclonal antibody to Piwil2 peptide. A, Testicular cell lysates of mili −/− and mili +/+ mice. B, HeLa cell lysate: Lane 1 5: 4 µg/mL antibody without peptide; Lane 2 6: 4 µg/mL antibody with 16 µg/mL Peptide; Lane 3: 2 µg/mL antibody alone; Lane 4: 2 µg/mL antibody with 8 µg/mL Peptide. P: Piwil2 peptide; Ab: antibody to Piwil2 peptide. C , GEM RT-PCR analysis of testicular tissues from wild-type (Lane 1) and mili −/− mice (Lane 2): the primer pairs specific for E1-7, E6-14, E13-21 and E21-23 of Piwil2 were used for GEM RT-PCR analysis, and the primer pair specific for E18-21 was used as a positive control. Lane 3: no cDNA control.

Techniques Used: Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Positive Control

47) Product Images from "Comparison of PCR- and HinfI Restriction Endonuclease-Based Methods for Typing of Candida krusei Isolates"

Article Title: Comparison of PCR- and HinfI Restriction Endonuclease-Based Methods for Typing of Candida krusei Isolates

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.42.12.5889-5891.2004

(A) Agarose gel electrophoresis of REAG patterns of HinfI-digested DNA. Patterns for different isolates C. krusei from two different patients in lanes 1 to 5 (patient A) and 6 and 7 (patient B) are shown. (B) Agarose gel electrophoresis of PCR-amplified DNA products generated with C. krusei- specific primers Arno1 and Arno2. Minor banding variations among isolates of C. krusei are seen for the four individual patients (lanes 1 and 2, 3 to 7, 8 to 10, and 11 to 13). Lane M is a molecular weight marker (1-kb DNA ladder).
Figure Legend Snippet: (A) Agarose gel electrophoresis of REAG patterns of HinfI-digested DNA. Patterns for different isolates C. krusei from two different patients in lanes 1 to 5 (patient A) and 6 and 7 (patient B) are shown. (B) Agarose gel electrophoresis of PCR-amplified DNA products generated with C. krusei- specific primers Arno1 and Arno2. Minor banding variations among isolates of C. krusei are seen for the four individual patients (lanes 1 and 2, 3 to 7, 8 to 10, and 11 to 13). Lane M is a molecular weight marker (1-kb DNA ladder).

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

48) Product Images from "A Combined Approach to Assess the Microbial Contamination of the Archimedes Palimpsest"

Article Title: A Combined Approach to Assess the Microbial Contamination of the Archimedes Palimpsest

Journal: Microbial Ecology

doi: 10.1007/s00248-014-0481-7

PCR-DGGE-fingerprints derived from a the bacterial community colonizing all three pooled swab samples and b the fungal communities colonizing the swab sample AP1, c the swab sample AP2, and d the swab sample AP3 of the Archimedes Palimpsest, as well as the PCR-DGGE profiles of sequenced clones containing 16S rDNA bacterial fragments ( a ) and ITS regions ( b – d ) producing PCR products with different motility behavior. O Original fingerprint derived from swab samples. P clones matching plant DNA. The numbers of the lanes indicate the number of the corresponding sequenced clones quoted in Table 1 and 2 . The linear chemical gradient of denaturants used was 25–60 % for bacteria and 20–50 % for fungi
Figure Legend Snippet: PCR-DGGE-fingerprints derived from a the bacterial community colonizing all three pooled swab samples and b the fungal communities colonizing the swab sample AP1, c the swab sample AP2, and d the swab sample AP3 of the Archimedes Palimpsest, as well as the PCR-DGGE profiles of sequenced clones containing 16S rDNA bacterial fragments ( a ) and ITS regions ( b – d ) producing PCR products with different motility behavior. O Original fingerprint derived from swab samples. P clones matching plant DNA. The numbers of the lanes indicate the number of the corresponding sequenced clones quoted in Table 1 and 2 . The linear chemical gradient of denaturants used was 25–60 % for bacteria and 20–50 % for fungi

Techniques Used: Polymerase Chain Reaction, Denaturing Gradient Gel Electrophoresis, Derivative Assay, Clone Assay

49) Product Images from "Overexpression of DDB2 enhances the sensitivity of human ovarian cancer cells to cisplatin by augmenting cellular apoptosis"

Article Title: Overexpression of DDB2 enhances the sensitivity of human ovarian cancer cells to cisplatin by augmenting cellular apoptosis

Journal: International journal of cancer. Journal international du cancer

doi: 10.1002/ijc.25112

DDB2 enhances cisplatin-induced apoptosis through downregulating Bcl-2 expression. ( a ) Cell lysates were prepared from A2780, CP70, CP70-vector and CP70-DDB2-1 cell lines, and analyzed by immunoblotting for the expression of DDB2 and Bcl-2. ( b, c ) Total RNA was isolated from cell lines described in ( a ) and subjected to RT-PCR and real-time RT-PCR analysis to detect the mRNA levels of DDB2 and Bcl-2 . The PCR products were resolved in agarose gel ( b ), and the relative transcript levels obtained from quantitative RT-PCR were plotted in ( c ) (Bar: SD, N = 3). ( d ) CP70 cells were transiently transfected with 2 µg DDB2 cDNA for 24, 48 and 72 hr, whole cell lysates were prepared and analyzed by Western blotting. ( e ) CP70 cells were transiently transfected with 2 µg DDB2 cDNA for 24 hr, treated with 20 µM cisplatin for 1 hr and left in culture for 24 and 48 hr. Cell lysates were then prepared and analyzed by Western blotting. ( f ) CP70 cells were transiently transfected with 2 µg DDB2 cDNA for 24 hr, then treated with MG132 (5, 10 µM) or Bortezomib (5, 10 nM) for 24 hr. The whole cell lysates were prepared and subjected to Western blotting.
Figure Legend Snippet: DDB2 enhances cisplatin-induced apoptosis through downregulating Bcl-2 expression. ( a ) Cell lysates were prepared from A2780, CP70, CP70-vector and CP70-DDB2-1 cell lines, and analyzed by immunoblotting for the expression of DDB2 and Bcl-2. ( b, c ) Total RNA was isolated from cell lines described in ( a ) and subjected to RT-PCR and real-time RT-PCR analysis to detect the mRNA levels of DDB2 and Bcl-2 . The PCR products were resolved in agarose gel ( b ), and the relative transcript levels obtained from quantitative RT-PCR were plotted in ( c ) (Bar: SD, N = 3). ( d ) CP70 cells were transiently transfected with 2 µg DDB2 cDNA for 24, 48 and 72 hr, whole cell lysates were prepared and analyzed by Western blotting. ( e ) CP70 cells were transiently transfected with 2 µg DDB2 cDNA for 24 hr, treated with 20 µM cisplatin for 1 hr and left in culture for 24 and 48 hr. Cell lysates were then prepared and analyzed by Western blotting. ( f ) CP70 cells were transiently transfected with 2 µg DDB2 cDNA for 24 hr, then treated with MG132 (5, 10 µM) or Bortezomib (5, 10 nM) for 24 hr. The whole cell lysates were prepared and subjected to Western blotting.

Techniques Used: Expressing, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Western Blot

50) Product Images from "The genetic basis of anoxygenic photosynthetic arsenite oxidation"

Article Title: The genetic basis of anoxygenic photosynthetic arsenite oxidation

Journal: Environmental microbiology

doi: 10.1111/1462-2920.13509

Evidence for environmental photoarsenotrophy activity determined by arxA gene expression in situ . Red biofilm-like microbial mats within hot springs of Paoha Island Mono Lake, CA were collected and the RNA and DNA extracts analysed for arxA ( arxA _1824_deg_F and arxA _2380_deg_R primers) (A) and 16S rRNA (B) by PCR and RT-PCR respectively. Agarose gel electrophoresis of the PCRs and RT-PCRs are shown in A and B. For both panels lanes correspond to: DNA ladder (1, 10), BSL-9 genomic DNA (2), water negative control (3), environmental DNA (4–5), and cDNA of environmental RNA samples with (6–7) and without (8–9) reverse transcriptase.
Figure Legend Snippet: Evidence for environmental photoarsenotrophy activity determined by arxA gene expression in situ . Red biofilm-like microbial mats within hot springs of Paoha Island Mono Lake, CA were collected and the RNA and DNA extracts analysed for arxA ( arxA _1824_deg_F and arxA _2380_deg_R primers) (A) and 16S rRNA (B) by PCR and RT-PCR respectively. Agarose gel electrophoresis of the PCRs and RT-PCRs are shown in A and B. For both panels lanes correspond to: DNA ladder (1, 10), BSL-9 genomic DNA (2), water negative control (3), environmental DNA (4–5), and cDNA of environmental RNA samples with (6–7) and without (8–9) reverse transcriptase.

Techniques Used: Activity Assay, Expressing, In Situ, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control

Development and verification of arxA disruption mutation in Ectothiorhodospira sp. str. BSL-9. (A) The pSMV20 vector was inserted within arxA via homologous recombination. (B) PCR analysis for DNA from wild-type BLS-9 (2, 7, 12), ARXA1 (3, 8, 13), empty pSMV20 vector (4, 9, 14), and water (5, 10, 15). The following PCR primers were used: (i) arxA (Ext_arxA_F, arrow 1) gene and vector-specific (M13_R, arrow 2) (lanes 2-5), (ii) 16S rRNA (0341_16SV3V4-F and 0785_16SV3V4-R) gene (lanes 7-10) and (iii) sacB ( sacB _F and sacB _R, arrows 3 and 4) (lanes 12-15). PCRs were analysed by agarose (1%) gel electrophoresis.
Figure Legend Snippet: Development and verification of arxA disruption mutation in Ectothiorhodospira sp. str. BSL-9. (A) The pSMV20 vector was inserted within arxA via homologous recombination. (B) PCR analysis for DNA from wild-type BLS-9 (2, 7, 12), ARXA1 (3, 8, 13), empty pSMV20 vector (4, 9, 14), and water (5, 10, 15). The following PCR primers were used: (i) arxA (Ext_arxA_F, arrow 1) gene and vector-specific (M13_R, arrow 2) (lanes 2-5), (ii) 16S rRNA (0341_16SV3V4-F and 0785_16SV3V4-R) gene (lanes 7-10) and (iii) sacB ( sacB _F and sacB _R, arrows 3 and 4) (lanes 12-15). PCRs were analysed by agarose (1%) gel electrophoresis.

Techniques Used: Mutagenesis, Plasmid Preparation, Homologous Recombination, Polymerase Chain Reaction, Nucleic Acid Electrophoresis

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TA Cloning:

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Article Title: Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity
Article Snippet: Paragraph title: PCR and quantitative PCR conditions ... Reactions were performed in a 50 µl volume containing 1 µl of DNA, 25 µl of Promega PCR master mix (Promega Ltd., Southampton, UK), 1 µl (10 µmol) of each primer, 2.5 µl DMSO, 2 µl (10 mg/ml) Bovine Serum Albumine (BSA)(Sigma-Aldrich, Dorset, UK), and 17.5 µl of filter sterilized MonoQ water.

Article Title: Bacterial Community Dynamics During the Application of a Myxococcus xanthus-Inoculated Culture Medium Used for Consolidation of Ornamental Limestone
Article Snippet: .. Quantification of M. xanthus by Real-Time PCR For qPCR reactions, 2× PCR Master Mix (SensiMix dT, Quantace) was diluted as recommended by the manufacturers. ..

Article Title: Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection *
Article Snippet: For PCR analysis, the cDNA was analyzed using the PCR Master Mix (Promega), and the PCR cycle conditions used were 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. .. For qPCR analysis, the cDNA was analyzed with the MESA BLUE qPCR MasterMix Plus for SYBR (Eurogentec, Southampton, UK) using a Stratagene MX3005 (Stratagene, La Jolla, CA).

Microarray:

Article Title: Microarray Generation of Thousand-Member Oligonucleotide Libraries
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. .. After PCR the samples were concentrated in a speed-vac followed by purification by preparative DNA gel electrophoresis as described above (dsDNA-3875-2: 1.11 µg, 27% isolated yield, dsDNA-10,000-2: 16.8 µg, 30% isolated yield).

Incubation:

Article Title: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China
Article Snippet: Briefly, the target DNA sequences were amplified by PCR using PCR Master Mix (Promega, USA) for all markers. .. The reactions were performed in a thermal cycler (Biometra, Germany) with an initial denaturation step of 94°C for 5 min, followed by 38 cycles of denaturation at 94°C, annealing temperatures appropriate for the primer, and extension at 72°C for 1 min each and a final 10 min incubation at 72°C to complete partial polymerisation.

Derivative Assay:

Article Title: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China
Article Snippet: These had been derived from cats from different areas in China: Anhui (two samples), Guangdong (two samples), Shanxi (two samples), and Hubei (eight samples) provinces. .. Briefly, the target DNA sequences were amplified by PCR using PCR Master Mix (Promega, USA) for all markers.

Inverse PCR:

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: .. To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 O to 50 μl. .. The initial denaturation step was 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 63°C for 30 s, 73°C for 3 min and final elongation of 73°C for 5 min. Fifteen microliters of the PCR reaction was analyzed on a 1% (w/v) agarose gel.

Infection:

Article Title: Importation of exotic ticks and tick-borne spotted fever group rickettsiae into the United States by migrating songbirds
Article Snippet: Spotted fever group rickettsial infection was detected by using rickettsial outer membrane protein A ( rompA ) gene-specific primers in a nested PCR assay ( ). .. In the primary reaction, 2.5 μL of DNA template (~62.5 ng) was added to 12.5 μL of 2× PCR Master Mix (Promega, Madison, WI), 8 μL of nuclease-free water, and 1 μL of each primer (10 μM).

Reverse Transcription Polymerase Chain Reaction:

Article Title: The Presumptive Magnetosome Protein Mms16 Is a Poly(3-Hydroxybutyrate) Granule-Bound Protein (Phasin) in Magnetospirillum gryphiswaldense
Article Snippet: Paragraph title: Reverse transcription (RT)-PCR. ... 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.

Imaging:

Article Title: LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling
Article Snippet: ChIP-purified DNA was amplified by standard PCR using primers specific for the MyD88 promoter and the 2 × PCR Master Mix (Promega). .. After PCR reaction, PCR products were separated on 1.2% agarose gels and visualized by Gel imaging system software (Tanon, Shanghai).

Polymerase Chain Reaction:

Article Title: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China
Article Snippet: .. Briefly, the target DNA sequences were amplified by PCR using PCR Master Mix (Promega, USA) for all markers. .. Each PCR reaction was conducted on 1.5 µl (100 ng) of each DNA extraction sample with 25 µl of PCR Master Mix, 50 pmol of each primer with the total reaction volume reaching 50 µl.

Article Title: Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity
Article Snippet: .. Reactions were performed in a 50 µl volume containing 1 µl of DNA, 25 µl of Promega PCR master mix (Promega Ltd., Southampton, UK), 1 µl (10 µmol) of each primer, 2.5 µl DMSO, 2 µl (10 mg/ml) Bovine Serum Albumine (BSA)(Sigma-Aldrich, Dorset, UK), and 17.5 µl of filter sterilized MonoQ water. .. The PCR cycle was 94°C for 1 min, followed by 40 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 1.30 min and a single extension step of 72°C for 7 mins.

Article Title: Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity
Article Snippet: .. Subsequently, specific 16S rRNA gene regions were amplified in the following manner: each 50 µl reaction contained 1 µl of DNA, 25 µl of Promega PCR master mix (Promega Ltd., Southampton, UK), 1 µl (1µmol) of each primer, 2.5 µl DMSO, 2 µl (10 mg/ml) Bovine Serum Albumine (BSA)(Sigma-Aldrich, Dorset, UK), and 17.5 µl of filter sterilized MonoQ water. ..

Article Title: LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling
Article Snippet: .. ChIP-purified DNA was amplified by standard PCR using primers specific for the MyD88 promoter and the 2 × PCR Master Mix (Promega). .. After PCR reaction, PCR products were separated on 1.2% agarose gels and visualized by Gel imaging system software (Tanon, Shanghai).

Article Title: Bacterial Community Dynamics During the Application of a Myxococcus xanthus-Inoculated Culture Medium Used for Consolidation of Ornamental Limestone
Article Snippet: .. Quantification of M. xanthus by Real-Time PCR For qPCR reactions, 2× PCR Master Mix (SensiMix dT, Quantace) was diluted as recommended by the manufacturers. ..

Article Title: Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods
Article Snippet: .. After comparison of all protocols/polymerase kits the protocol of Verhaeghen et al [ ] using the 2 × PCR master mix (Promega) was selected to genotype the 96 sample reference plate. .. TaqMan Previous work characterizing the para gene region encoding domain II S4–S6 of the sodium channel from a range of insect species has shown that this region contains an intron very close to the kdr mutation site.

Article Title: An Insight Into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae)
Article Snippet: .. In the primary reaction, ≈150 ng of DNA template was added to 2× PCR Master Mix (Promega, Madison, WI) and the appropriate primers (400 nM). .. SFGR PCR was performed in a MyCycler Thermal Cycler (Bio-Rad Laboratories, Richmond, CA) as follows: one cycle at 95°C for 3 min, 35 cycles of 95° for 20 s, 46°C for 30 s, and 63°C for 60 s, and one cycle at 72°C for 7 min. For each reaction, two negative controls (no-template and no-primer) and one positive control (50 ng of a known SFGR) were included.

Article Title: Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection *
Article Snippet: .. For PCR analysis, the cDNA was analyzed using the PCR Master Mix (Promega), and the PCR cycle conditions used were 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. ..

Article Title: Expression and ecdysteroid responsiveness of the nuclear receptors HR3 and E75 in the crustacean Daphnia magna
Article Snippet: .. The PCR product was amplified from 200 ng cDNA using 10 µL PCR Master Mix (Promega) and 0.4 µM of each primer. .. The first round of PCR cycling followed standard 3-step PCR (denaturation at 95°C for 1 min., annealing at 52°C for 45 sec, and extension at 72°C for 1 min) for 40 cycles.

Article Title: The Presumptive Magnetosome Protein Mms16 Is a Poly(3-Hydroxybutyrate) Granule-Bound Protein (Phasin) in Magnetospirillum gryphiswaldense
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. .. For complementation of the mms16 insertion mutant Da127, the apdA - eyfp gene fusion ( ) was excised from plasmid pSN2389 by XbaI and SalI digestion and ligated under control of the lac promoter into the broad-host-range vector pBBR1MCS5.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: .. To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 O to 50 μl. .. The initial denaturation step was 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 63°C for 30 s, 73°C for 3 min and final elongation of 73°C for 5 min. Fifteen microliters of the PCR reaction was analyzed on a 1% (w/v) agarose gel.

Article Title: Microarray Generation of Thousand-Member Oligonucleotide Libraries
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. .. After PCR the samples were concentrated in a speed-vac followed by purification by preparative DNA gel electrophoresis as described above (dsDNA-3875-2: 1.11 µg, 27% isolated yield, dsDNA-10,000-2: 16.8 µg, 30% isolated yield).

Article Title: Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan
Article Snippet: .. 16S rRNA Gene Sequencing The 16S rRNA gene was amplified by PCR mixtures (25 μL) contained approximately 30–50 ng template DNA, 1 μM forward primer 27F and 1 μM reverse primer 1492R ( ) using a PCR Master Mix (Promega, Canada) performed under the standard PCR amplification procedure in a SimpliAmpTM Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). .. The PCR amplicons were checked for their purity on 1% agarose gel electrophoresis in the presence of ethidium bromide (10 mg/mL), which was later analyzed by the Gel Doc System (Ultra-Violet Products Ltd, UK).

Article Title: Importation of exotic ticks and tick-borne spotted fever group rickettsiae into the United States by migrating songbirds
Article Snippet: .. In the primary reaction, 2.5 μL of DNA template (~62.5 ng) was added to 12.5 μL of 2× PCR Master Mix (Promega, Madison, WI), 8 μL of nuclease-free water, and 1 μL of each primer (10 μM). .. In the nested reaction, 12.5 μL of 2× PCR Master Mix was used, 8 μL of nuclease-free water, 1 μL of each nested primer (10 μM), and 2.5 μL of the primary PCR reaction.

Article Title: Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus
Article Snippet: .. An 8-μl reaction mixture consisted of 0.5 μl of the diluted fungal DNA extract, 4 μl of 2× PCR Master Mix (Promega), 2.5 μl of nuclease-free water, and 0.5 μl of each primer (each 12.5 pmol/μl). .. PCR cycling depended on the size of the target fragment.

Denaturing Gradient Gel Electrophoresis:

Article Title: Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity
Article Snippet: PCR and quantitative PCR conditions In order to reliably generate PCR amplicons for DGGE, nesting was applied to all soil extracted DNA using the primer set pA and pH ( ) to first amplify the whole 16S rRNA gene . .. Reactions were performed in a 50 µl volume containing 1 µl of DNA, 25 µl of Promega PCR master mix (Promega Ltd., Southampton, UK), 1 µl (10 µmol) of each primer, 2.5 µl DMSO, 2 µl (10 mg/ml) Bovine Serum Albumine (BSA)(Sigma-Aldrich, Dorset, UK), and 17.5 µl of filter sterilized MonoQ water.

DNA Gel Electrophoresis:

Article Title: Microarray Generation of Thousand-Member Oligonucleotide Libraries
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. .. After PCR the samples were concentrated in a speed-vac followed by purification by preparative DNA gel electrophoresis as described above (dsDNA-3875-2: 1.11 µg, 27% isolated yield, dsDNA-10,000-2: 16.8 µg, 30% isolated yield).

DNA Extraction:

Article Title: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China
Article Snippet: Paragraph title: DNA Extraction and Genotyping of T. gondii Isolates ... Briefly, the target DNA sequences were amplified by PCR using PCR Master Mix (Promega, USA) for all markers.

Article Title: Importation of exotic ticks and tick-borne spotted fever group rickettsiae into the United States by migrating songbirds
Article Snippet: Paragraph title: DNA extraction and nested PCR amplification of spotted fever group rickettsiae ... In the primary reaction, 2.5 μL of DNA template (~62.5 ng) was added to 12.5 μL of 2× PCR Master Mix (Promega, Madison, WI), 8 μL of nuclease-free water, and 1 μL of each primer (10 μM).

Isolation:

Article Title: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China
Article Snippet: All T. gondii strains isolated from animals and humans were submitted for genotyping. .. Briefly, the target DNA sequences were amplified by PCR using PCR Master Mix (Promega, USA) for all markers.

Article Title: Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection *
Article Snippet: Paragraph title: RNA Isolation, PCR, and Quantitative Real-time RT-PCR ... For PCR analysis, the cDNA was analyzed using the PCR Master Mix (Promega), and the PCR cycle conditions used were 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.

Article Title: The Presumptive Magnetosome Protein Mms16 Is a Poly(3-Hydroxybutyrate) Granule-Bound Protein (Phasin) in Magnetospirillum gryphiswaldense
Article Snippet: Isolated RNA was treated with DNase (MBI Fermentas) and then used in a reverse transcriptase reaction (Moloney murine leukemia virus reverse transcriptase; MBI Fermentas) with primer mmpF1. .. 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.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Genomic DNA was isolated with a Wizard® Genomic DNA purification Kit (Promega). .. To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 O to 50 μl.

Article Title: Microarray Generation of Thousand-Member Oligonucleotide Libraries
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. .. After PCR the samples were concentrated in a speed-vac followed by purification by preparative DNA gel electrophoresis as described above (dsDNA-3875-2: 1.11 µg, 27% isolated yield, dsDNA-10,000-2: 16.8 µg, 30% isolated yield).

Size-exclusion Chromatography:

Article Title: Expression and ecdysteroid responsiveness of the nuclear receptors HR3 and E75 in the crustacean Daphnia magna
Article Snippet: The PCR product was amplified from 200 ng cDNA using 10 µL PCR Master Mix (Promega) and 0.4 µM of each primer. .. The first round of PCR cycling followed standard 3-step PCR (denaturation at 95°C for 1 min., annealing at 52°C for 45 sec, and extension at 72°C for 1 min) for 40 cycles.

Purification:

Article Title: An Insight Into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae)
Article Snippet: In the primary reaction, ≈150 ng of DNA template was added to 2× PCR Master Mix (Promega, Madison, WI) and the appropriate primers (400 nM). .. In the primary reaction, ≈150 ng of DNA template was added to 2× PCR Master Mix (Promega, Madison, WI) and the appropriate primers (400 nM).

Article Title: Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection *
Article Snippet: Using RNA purified from the total RNA or polysome fractions, first strand cDNA synthesis was performed using equivalent amounts of starting RNA from all samples (first strand cDNA synthesis kit, Roche Applied Science). .. For PCR analysis, the cDNA was analyzed using the PCR Master Mix (Promega), and the PCR cycle conditions used were 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 O to 50 μl. .. The brightest bands were cut out, gel purified (Qiagen) and cloned into pJET plasmid using the CloneJET PCR Cloning Kit (ThermoFisher Scientific) according to the manufacturer's protocol.

Article Title: Microarray Generation of Thousand-Member Oligonucleotide Libraries
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. .. After PCR the samples were concentrated in a speed-vac followed by purification by preparative DNA gel electrophoresis as described above (dsDNA-3875-2: 1.11 µg, 27% isolated yield, dsDNA-10,000-2: 16.8 µg, 30% isolated yield).

Sequencing:

Article Title: Expression and ecdysteroid responsiveness of the nuclear receptors HR3 and E75 in the crustacean Daphnia magna
Article Snippet: Primers were designed using the CO nsensus- DE generate H ybrid O ligonucleotide Primers (CODEHOP) program with a blocks format sequence alignment (Rose et al, 1998) of E75 from other species (GenBank accession numbers: , , , ). .. The PCR product was amplified from 200 ng cDNA using 10 µL PCR Master Mix (Promega) and 0.4 µM of each primer.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Paragraph title: Sequencing of eukaryotic genome integration sites ... To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 O to 50 μl.

Article Title: Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan
Article Snippet: .. 16S rRNA Gene Sequencing The 16S rRNA gene was amplified by PCR mixtures (25 μL) contained approximately 30–50 ng template DNA, 1 μM forward primer 27F and 1 μM reverse primer 1492R ( ) using a PCR Master Mix (Promega, Canada) performed under the standard PCR amplification procedure in a SimpliAmpTM Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). .. The PCR amplicons were checked for their purity on 1% agarose gel electrophoresis in the presence of ethidium bromide (10 mg/mL), which was later analyzed by the Gel Doc System (Ultra-Violet Products Ltd, UK).

Construct:

Article Title: Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus
Article Snippet: Gene disruption events were confirmed by PCR analyses using different combinations of primers, depending on the different gene constructs in the genome between the mutants and the recipient strain SYS-4. .. An 8-μl reaction mixture consisted of 0.5 μl of the diluted fungal DNA extract, 4 μl of 2× PCR Master Mix (Promega), 2.5 μl of nuclease-free water, and 0.5 μl of each primer (each 12.5 pmol/μl).

Nested PCR:

Article Title: An Insight Into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae)
Article Snippet: The primers RR 190–70 and RR 190–701 ( ) were used for the primary reaction, and 190-FN1 and 190-RN1 ( ) for the nested PCR reaction. .. In the primary reaction, ≈150 ng of DNA template was added to 2× PCR Master Mix (Promega, Madison, WI) and the appropriate primers (400 nM).

Article Title: Importation of exotic ticks and tick-borne spotted fever group rickettsiae into the United States by migrating songbirds
Article Snippet: Paragraph title: DNA extraction and nested PCR amplification of spotted fever group rickettsiae ... In the primary reaction, 2.5 μL of DNA template (~62.5 ng) was added to 12.5 μL of 2× PCR Master Mix (Promega, Madison, WI), 8 μL of nuclease-free water, and 1 μL of each primer (10 μM).

Chromatin Immunoprecipitation:

Article Title: LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling
Article Snippet: .. ChIP-purified DNA was amplified by standard PCR using primers specific for the MyD88 promoter and the 2 × PCR Master Mix (Promega). .. After PCR reaction, PCR products were separated on 1.2% agarose gels and visualized by Gel imaging system software (Tanon, Shanghai).

Plasmid Preparation:

Article Title: Expression and ecdysteroid responsiveness of the nuclear receptors HR3 and E75 in the crustacean Daphnia magna
Article Snippet: The PCR product was amplified from 200 ng cDNA using 10 µL PCR Master Mix (Promega) and 0.4 µM of each primer. .. The fragment was cloned into the vector pCR®4-TOPO using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA).

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 O to 50 μl. .. The brightest bands were cut out, gel purified (Qiagen) and cloned into pJET plasmid using the CloneJET PCR Cloning Kit (ThermoFisher Scientific) according to the manufacturer's protocol.

Software:

Article Title: LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling
Article Snippet: ChIP-purified DNA was amplified by standard PCR using primers specific for the MyD88 promoter and the 2 × PCR Master Mix (Promega). .. After PCR reaction, PCR products were separated on 1.2% agarose gels and visualized by Gel imaging system software (Tanon, Shanghai).

Article Title: Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection *
Article Snippet: For PCR analysis, the cDNA was analyzed using the PCR Master Mix (Promega), and the PCR cycle conditions used were 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. .. The PCR cycle conditions used were 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, and the Ct values were determined using the MxPro software (Stratagene).

Electrophoresis:

Article Title: Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus
Article Snippet: An 8-μl reaction mixture consisted of 0.5 μl of the diluted fungal DNA extract, 4 μl of 2× PCR Master Mix (Promega), 2.5 μl of nuclease-free water, and 0.5 μl of each primer (each 12.5 pmol/μl). .. PCR conditions for amplifying fragments shorter than 2.5 kb were (i) 94°C for 5 min; (ii) 35 cycles of 94°C for 40 s, 56°C for 40 s, 72°C for n min (determined by the size of the target fragment); and (iii) 72°C for 10 min. For fragments larger than 2.5 kb, PCR conditions were (i) 94°C for 5 min; (ii) 40 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for n min; and (iii) 72°C for 10 min. PCR products were visualized by performing electrophoresis on a 1% agarose gel.

RNA Extraction:

Article Title: Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection *
Article Snippet: Total RNA was extracted using ZR Miniprep RNA extraction columns following the manufacturer's instructions and including an on-column TURBO DNase I digestion. .. For PCR analysis, the cDNA was analyzed using the PCR Master Mix (Promega), and the PCR cycle conditions used were 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.

Agarose Gel Electrophoresis:

Article Title: An Insight Into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae)
Article Snippet: In the primary reaction, ≈150 ng of DNA template was added to 2× PCR Master Mix (Promega, Madison, WI) and the appropriate primers (400 nM). .. The amplicons were analyzed on a 2% agarose gel containing ethidium bromide and observed using a ultraviolet transilluminator.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 O to 50 μl. .. The initial denaturation step was 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 63°C for 30 s, 73°C for 3 min and final elongation of 73°C for 5 min. Fifteen microliters of the PCR reaction was analyzed on a 1% (w/v) agarose gel.

Article Title: Some Technological Properties of Lactic Acid Bacteria Isolated from Dahi and Datshi, Naturally Fermented Milk Products of Bhutan
Article Snippet: 16S rRNA Gene Sequencing The 16S rRNA gene was amplified by PCR mixtures (25 μL) contained approximately 30–50 ng template DNA, 1 μM forward primer 27F and 1 μM reverse primer 1492R ( ) using a PCR Master Mix (Promega, Canada) performed under the standard PCR amplification procedure in a SimpliAmpTM Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). .. The PCR amplicons were checked for their purity on 1% agarose gel electrophoresis in the presence of ethidium bromide (10 mg/mL), which was later analyzed by the Gel Doc System (Ultra-Violet Products Ltd, UK).

Article Title: Importation of exotic ticks and tick-borne spotted fever group rickettsiae into the United States by migrating songbirds
Article Snippet: In the primary reaction, 2.5 μL of DNA template (~62.5 ng) was added to 12.5 μL of 2× PCR Master Mix (Promega, Madison, WI), 8 μL of nuclease-free water, and 1 μL of each primer (10 μM). .. The amplicons were analyzed with a 2% agarose gel containing ethidium bromide and observed using a UV transilluminator.

Article Title: Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus
Article Snippet: An 8-μl reaction mixture consisted of 0.5 μl of the diluted fungal DNA extract, 4 μl of 2× PCR Master Mix (Promega), 2.5 μl of nuclease-free water, and 0.5 μl of each primer (each 12.5 pmol/μl). .. PCR conditions for amplifying fragments shorter than 2.5 kb were (i) 94°C for 5 min; (ii) 35 cycles of 94°C for 40 s, 56°C for 40 s, 72°C for n min (determined by the size of the target fragment); and (iii) 72°C for 10 min. For fragments larger than 2.5 kb, PCR conditions were (i) 94°C for 5 min; (ii) 40 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for n min; and (iii) 72°C for 10 min. PCR products were visualized by performing electrophoresis on a 1% agarose gel.

Concentration Assay:

Article Title: LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling
Article Snippet: Chromatin immunoprecipitation (ChIP) assay Approximately 1 × 106 HAPI cells were cross-linked with a 1% final concentration of formaldehyde (Sigma-Aldrich) at 37 °C for 10 mins. .. ChIP-purified DNA was amplified by standard PCR using primers specific for the MyD88 promoter and the 2 × PCR Master Mix (Promega).

DNA Purification:

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Genomic DNA was isolated with a Wizard® Genomic DNA purification Kit (Promega). .. To amplify both junctions flanking the neomycin resistance cassette, inverse PCR was performed using PCR Master Mix (Promega) with the addition of Pfu DNA Polymerase (Agilent Technologies) in a final volume of 50 μl: 25 μl of Master Mix, 160 μM dNTPs, 500 nM of primers (5΄-gaggctaactgaaacacggaaggag-3΄ and 5΄-cgggactatggttgctgactaattg-3΄), 10 μl of ligated DNA and dH2 O to 50 μl.

CTG Assay:

Article Title: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China
Article Snippet: Reference strains of T. gondii were used as controls for genotyping, including type I (GT1), type II (PTG), type III (CTG) and other strains (TgCgCa1 (a.k.a. .. Briefly, the target DNA sequences were amplified by PCR using PCR Master Mix (Promega, USA) for all markers.

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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.
    Pcr Master Mix, supplied by Promega, used in various techniques. Bioz Stars score: 98/100, based on 750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    (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.

    Journal: Ticks and tick-borne diseases

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

    doi: 10.1016/j.ttbdis.2013.09.009

    Figure Lengend 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.

    Article Snippet: In the primary reaction, 2.5 μL of DNA template (~62.5 ng) was added to 12.5 μL of 2× PCR Master Mix (Promega, Madison, WI), 8 μL of nuclease-free water, and 1 μL of each primer (10 μM).

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

    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