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GE Healthcare pcr fragments
Genomic DNA gel-blot analysis of tomato expansin genes. Tomato genomic DNA (10 μg) was digested by Eco RI, Eco RV, and Hin dIII, respectively, and subjected to gel-blot hybridization using gene-specific <t>cDNA</t> probes amplified by <t>PCR</t> using primers corresponding to the 3′-untranslated regions of LeEXP8 (left) and LeEXP10 (right).
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1) Product Images from "Expression of an Expansin Is Associated with Endosperm Weakening during Tomato Seed Germination 1"

Article Title: Expression of an Expansin Is Associated with Endosperm Weakening during Tomato Seed Germination 1

Journal: Plant Physiology

doi:

Genomic DNA gel-blot analysis of tomato expansin genes. Tomato genomic DNA (10 μg) was digested by Eco RI, Eco RV, and Hin dIII, respectively, and subjected to gel-blot hybridization using gene-specific cDNA probes amplified by PCR using primers corresponding to the 3′-untranslated regions of LeEXP8 (left) and LeEXP10 (right).
Figure Legend Snippet: Genomic DNA gel-blot analysis of tomato expansin genes. Tomato genomic DNA (10 μg) was digested by Eco RI, Eco RV, and Hin dIII, respectively, and subjected to gel-blot hybridization using gene-specific cDNA probes amplified by PCR using primers corresponding to the 3′-untranslated regions of LeEXP8 (left) and LeEXP10 (right).

Techniques Used: Western Blot, Hybridization, Amplification, Polymerase Chain Reaction

2) Product Images from "FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)"

Article Title: FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2014.00518

Relative expression profiles of down- (A) or up-regulated (B) monolignol genes in F . × ananassa cv. Elsanta . Total RNA was isolated from a single untreated fruit (wild type; WT), control fruits (pBI-intron), down-regulated fruits (pBI- FaCCRi , pBI- FaCADi , and pBI- FaPODi ) (A) , and up-regulated fruits (pBI- FaCCR , pBI- FaCAD , and pBI- FaPOD ) (B) . Expression levels of all samples were monitored by qRT-PCR with specific primers for target genes ( FaCCR . FaCAD , and FaPOD ) and an interspac er gene. The latter was used as an internal control for normalization. For each box-plot graph, one of the WT group was used as the reference (set to one) and each group contained five biological replicates. The Wilcoxon-Mann-Whitney U -test was used for a non-parametric comparison of two groups from pBI-intron and fruits infiltrated with different constructs. Values indicate statistically decreased and increased levels ( P
Figure Legend Snippet: Relative expression profiles of down- (A) or up-regulated (B) monolignol genes in F . × ananassa cv. Elsanta . Total RNA was isolated from a single untreated fruit (wild type; WT), control fruits (pBI-intron), down-regulated fruits (pBI- FaCCRi , pBI- FaCADi , and pBI- FaPODi ) (A) , and up-regulated fruits (pBI- FaCCR , pBI- FaCAD , and pBI- FaPOD ) (B) . Expression levels of all samples were monitored by qRT-PCR with specific primers for target genes ( FaCCR . FaCAD , and FaPOD ) and an interspac er gene. The latter was used as an internal control for normalization. For each box-plot graph, one of the WT group was used as the reference (set to one) and each group contained five biological replicates. The Wilcoxon-Mann-Whitney U -test was used for a non-parametric comparison of two groups from pBI-intron and fruits infiltrated with different constructs. Values indicate statistically decreased and increased levels ( P

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, MANN-WHITNEY, Construct

Relative expression profiles of monolignol biosynthesis genes in vegetative tissues, flowers, and fruit developmental stages of F . × ananassa cv. Elsanta . Total RNA was isolated from (A) fruit developmental stages at small green (SG), green white (GW), white (W), turning (T), and red (R) after pollination. (B) Expression levels of vegetative tissues (leaf; stem; runner; root), flower (F) and fruit developmental stages (SG, GW, W, T, and R) were monitored by qRT-PCR. FaCCR . FaCAD . FaPOD and FaPOD27 (Ring et al., 2013 ) were target genes. The white fruit was used as the reference with one for each graph. Values are mean ± SE of 5–6 replicates from two sets of cDNAs and are shown as relative changes.
Figure Legend Snippet: Relative expression profiles of monolignol biosynthesis genes in vegetative tissues, flowers, and fruit developmental stages of F . × ananassa cv. Elsanta . Total RNA was isolated from (A) fruit developmental stages at small green (SG), green white (GW), white (W), turning (T), and red (R) after pollination. (B) Expression levels of vegetative tissues (leaf; stem; runner; root), flower (F) and fruit developmental stages (SG, GW, W, T, and R) were monitored by qRT-PCR. FaCCR . FaCAD . FaPOD and FaPOD27 (Ring et al., 2013 ) were target genes. The white fruit was used as the reference with one for each graph. Values are mean ± SE of 5–6 replicates from two sets of cDNAs and are shown as relative changes.

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

Relative expression profiles of phenylpropanoid biosynthesis genes of F . × ananassa cv. Elsanta in response to Agrobacterium . Expression levels in control (gray column) and agroinfiltrated fruits (black column) were monitored by qRT-PCR at different time points (1, 3, 6, 12, 24, 48, and 96 h). FaPAL . FaCHS, FaCCR . FaCAD . FaPOD , and FaPOD27 were target genes. The control fruit (1 h) was used as the reference with one for each graph. Values are mean ± SE of 2–3 replicates from one fruit and are shown as relative changes.
Figure Legend Snippet: Relative expression profiles of phenylpropanoid biosynthesis genes of F . × ananassa cv. Elsanta in response to Agrobacterium . Expression levels in control (gray column) and agroinfiltrated fruits (black column) were monitored by qRT-PCR at different time points (1, 3, 6, 12, 24, 48, and 96 h). FaPAL . FaCHS, FaCCR . FaCAD . FaPOD , and FaPOD27 were target genes. The control fruit (1 h) was used as the reference with one for each graph. Values are mean ± SE of 2–3 replicates from one fruit and are shown as relative changes.

Techniques Used: Expressing, Quantitative RT-PCR

3) Product Images from "Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids"

Article Title: Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055941

Production of transgenic lambs by handmade cloning. ( A ) The recipient #0907 and the transgenic lamb (PP-01). ( B ) Detection of the mfat-1 gene in umbilical cord samples of three cloned lambs by PCR and RT-qPCR. ( C ) Insertion site of mfat-1 vector in the sheep genome. Arrows indicate the mfat-1 transcriptional direction, which is identical with the endogenous putative sheep Cep120 gene. PCR fragment obtained in this study are shown by black bars. ( D ) Southern blot using the 32 P-labled mfat-1 specific sequence as a probe to hybridize the genomic DNA from the transgenic donor cells and the cloned lambs. The genomic DNA was digested with Bam HI before the gel electrophoresis. ( E ) Northern blot analysis. Total RNAs were loaded on each lane (15 µg per sample) and the coding region of mfat-1 was used as a probe. Shown below is the gel electrophoresis of rRNA as control. ( F ) Quantitative PCR analysis of mfat-1 expression in major tissues from the transgenic lambs (PP-02). Compared with the mRNA expression level normalized to the donor cell, the highest level of mfat-1 expression was observed in transgenic muscle sample.
Figure Legend Snippet: Production of transgenic lambs by handmade cloning. ( A ) The recipient #0907 and the transgenic lamb (PP-01). ( B ) Detection of the mfat-1 gene in umbilical cord samples of three cloned lambs by PCR and RT-qPCR. ( C ) Insertion site of mfat-1 vector in the sheep genome. Arrows indicate the mfat-1 transcriptional direction, which is identical with the endogenous putative sheep Cep120 gene. PCR fragment obtained in this study are shown by black bars. ( D ) Southern blot using the 32 P-labled mfat-1 specific sequence as a probe to hybridize the genomic DNA from the transgenic donor cells and the cloned lambs. The genomic DNA was digested with Bam HI before the gel electrophoresis. ( E ) Northern blot analysis. Total RNAs were loaded on each lane (15 µg per sample) and the coding region of mfat-1 was used as a probe. Shown below is the gel electrophoresis of rRNA as control. ( F ) Quantitative PCR analysis of mfat-1 expression in major tissues from the transgenic lambs (PP-02). Compared with the mRNA expression level normalized to the donor cell, the highest level of mfat-1 expression was observed in transgenic muscle sample.

Techniques Used: Transgenic Assay, Clone Assay, Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Southern Blot, Sequencing, Nucleic Acid Electrophoresis, Northern Blot, Real-time Polymerase Chain Reaction, Expressing

Establishment and analysis of transgenic clonal donor cells. ( A ) Schematic representation of n−3 fatty acid desaturase gene with linearized expression vectors. ( B ) Detection of the mfat-1 gene in Geneticin-resistant cell clones by PCR and RT-qPCR. mfat-1 expression vector was used as the template for positive control (PC) and untransfected syngenic cells was used as the negative control (NC). ( C ) Quantitative PCR analysis of mfat-1 expression in positive cell clones. cDNA representing mfat-1 was amplified with sequence specific primers. The beta-actin was used as internal control and the expression level observed in the transgenic donor cell was normalized to the value of A-3-1. ( D ) Partial gas chromatograph traces showing the polyunsaturated fatty acid profiles of total cellular lipids from the H-6-6 cells and the control cells. Note the level of n-6 polyunsaturated acids are lower whereas n−3 fatty acids are abundant in the mfat-1 cells (right) as compared with the control cells (left), in which there is very little n−3 fatty acid.
Figure Legend Snippet: Establishment and analysis of transgenic clonal donor cells. ( A ) Schematic representation of n−3 fatty acid desaturase gene with linearized expression vectors. ( B ) Detection of the mfat-1 gene in Geneticin-resistant cell clones by PCR and RT-qPCR. mfat-1 expression vector was used as the template for positive control (PC) and untransfected syngenic cells was used as the negative control (NC). ( C ) Quantitative PCR analysis of mfat-1 expression in positive cell clones. cDNA representing mfat-1 was amplified with sequence specific primers. The beta-actin was used as internal control and the expression level observed in the transgenic donor cell was normalized to the value of A-3-1. ( D ) Partial gas chromatograph traces showing the polyunsaturated fatty acid profiles of total cellular lipids from the H-6-6 cells and the control cells. Note the level of n-6 polyunsaturated acids are lower whereas n−3 fatty acids are abundant in the mfat-1 cells (right) as compared with the control cells (left), in which there is very little n−3 fatty acid.

Techniques Used: Transgenic Assay, Expressing, Clone Assay, Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Positive Control, Negative Control, Real-time Polymerase Chain Reaction, Amplification, Sequencing

4) Product Images from "Importin ?1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1"

Article Title: Importin ?1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1

Journal: Biochemical Journal

doi: 10.1042/BJ20061295

Importin α 1 acts synergistically with Zac1 on the p21 promoter and protein induction in HeLa cells ( a and b ) HeLa cells were transiently transfected with the p21-LUC reporter gene (0.4 μg) and pSG5HA.Zac1 (0.3 μg) and/or pSG5HA.importin α 1 (0.3 μg) reporter constructs. Luciferase activity in the transfected cell extracts is indicated in RLU. Numbers above the columns indicate the fold induction in RLU relative to that of the control cells in which only p21-LUC was transfected. ( b ) HeLa cell extracts were subjected to Western blot analysis probing with anti-p21, anti-p53, anti-HA and anti-α-tubulin antibodies. ( c ) HeLa cell extracts were used for ChIP assays followed by PCR analysis with two sets of primer for both: Sp1 A (−218/−87) and Sp1 B (−86/+142). Results ( b and c ) are representative of two independent experiments.
Figure Legend Snippet: Importin α 1 acts synergistically with Zac1 on the p21 promoter and protein induction in HeLa cells ( a and b ) HeLa cells were transiently transfected with the p21-LUC reporter gene (0.4 μg) and pSG5HA.Zac1 (0.3 μg) and/or pSG5HA.importin α 1 (0.3 μg) reporter constructs. Luciferase activity in the transfected cell extracts is indicated in RLU. Numbers above the columns indicate the fold induction in RLU relative to that of the control cells in which only p21-LUC was transfected. ( b ) HeLa cell extracts were subjected to Western blot analysis probing with anti-p21, anti-p53, anti-HA and anti-α-tubulin antibodies. ( c ) HeLa cell extracts were used for ChIP assays followed by PCR analysis with two sets of primer for both: Sp1 A (−218/−87) and Sp1 B (−86/+142). Results ( b and c ) are representative of two independent experiments.

Techniques Used: Transfection, Construct, Luciferase, Activity Assay, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction

5) Product Images from "Checkpoint suppressor 1 suppresses transcriptional activity of ERα and breast cancer cell proliferation via deacetylase SIRT1"

Article Title: Checkpoint suppressor 1 suppresses transcriptional activity of ERα and breast cancer cell proliferation via deacetylase SIRT1

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0629-3

CHES1 expression was repressed by E 2 -ERα pathway in breast cancer. a Western blotting tested the protein expression of CHES1 and ERα in MCF7 cells with or without E 2 treatment. b Immunoblotting of CHES1 and ERα in MCF7 cells treated with or without 100 nM E 2 , 1 nM 4-Hydroxytamoxifen, and 0.1 μM ICI 182780. c Schematic representation of conserved ERα-binding motif and ERE site on CHES1 potential promoter region, real-time PCR assay tested the mRNA level of CHES1 in MCF7 cells treated with or without E 2 . d Schematic representation of CHES1 -promoter luc construction. Luciferase reporter assay showed that the activity of CHES1 -promoter luc was repressed in MCF7 cells when treated with E 2 stimulation. * P
Figure Legend Snippet: CHES1 expression was repressed by E 2 -ERα pathway in breast cancer. a Western blotting tested the protein expression of CHES1 and ERα in MCF7 cells with or without E 2 treatment. b Immunoblotting of CHES1 and ERα in MCF7 cells treated with or without 100 nM E 2 , 1 nM 4-Hydroxytamoxifen, and 0.1 μM ICI 182780. c Schematic representation of conserved ERα-binding motif and ERE site on CHES1 potential promoter region, real-time PCR assay tested the mRNA level of CHES1 in MCF7 cells treated with or without E 2 . d Schematic representation of CHES1 -promoter luc construction. Luciferase reporter assay showed that the activity of CHES1 -promoter luc was repressed in MCF7 cells when treated with E 2 stimulation. * P

Techniques Used: Expressing, Western Blot, Binding Assay, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Activity Assay

CHES1 has little effect on the stability, dimerization, subcellular location of ERα, and it also does not influence the interaction between ERα and HDAC1/2. a Western blotting and real-time PCR assays tested the mRNA and protein level of endogenous ERα with overexpression of Flag-CHES1 in MCF7 cells. b Western blotting assay showed that the protein level of exogenous ERα were not affected when co-transfected with CHES1 in HeLa cells. c CoIP assay indicated that the dimerization of ERα were not interfered with knockdown of CHES1 in HEK293T cells. d Cytoplasmic and nucleus fractions separation assay were conducted in MCF7 cells with or without knockdown of CHES1 to test the subcellular distribution of endogenous ERα. The β-tubulin used as cytoplasmic marker and Fibrillarin as nuclear marker. e CoIP assay showed the endogenous and exogenous interaction between CHES1 and HDAC1/2 in MCF7 and HEK293T cells. f CoIP assay assessed the effect of CHES1 on the interaction between ERα and HDAC1/2
Figure Legend Snippet: CHES1 has little effect on the stability, dimerization, subcellular location of ERα, and it also does not influence the interaction between ERα and HDAC1/2. a Western blotting and real-time PCR assays tested the mRNA and protein level of endogenous ERα with overexpression of Flag-CHES1 in MCF7 cells. b Western blotting assay showed that the protein level of exogenous ERα were not affected when co-transfected with CHES1 in HeLa cells. c CoIP assay indicated that the dimerization of ERα were not interfered with knockdown of CHES1 in HEK293T cells. d Cytoplasmic and nucleus fractions separation assay were conducted in MCF7 cells with or without knockdown of CHES1 to test the subcellular distribution of endogenous ERα. The β-tubulin used as cytoplasmic marker and Fibrillarin as nuclear marker. e CoIP assay showed the endogenous and exogenous interaction between CHES1 and HDAC1/2 in MCF7 and HEK293T cells. f CoIP assay assessed the effect of CHES1 on the interaction between ERα and HDAC1/2

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Transfection, Co-Immunoprecipitation Assay, Marker

6) Product Images from "The Caenorhabditis elegans unc-49 Locus Encodes Multiple Subunits of a Heteromultimeric GABA Receptor"

Article Title: The Caenorhabditis elegans unc-49 Locus Encodes Multiple Subunits of a Heteromultimeric GABA Receptor

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.19-13-05348.1999

unc-49 produces three distinct GABA receptor subunits. A , Structure of the unc-49 locus showing the positions of conserved GABA receptor structural motifs. Domain structure of the locus is indicated by bars at the top (see Results). B , unc-49 mRNA structure. Transcripts were isolated both from cDNA libraries and from RT-PCR experiments. Multiple UNC-49A cDNA clones were identified with different splicing patterns, all resulting in premature stops. One representative example is shown here. Properly spliced RNAs were identified by RT-PCR; the short arrows and circled numbers represent PCR primers. Two superimposed primers (for example, 68 and 73) represent a set of nested PCR primers. The shaded boxes represent coding exons, and the open boxes represent untranslated regions. The SL1 splice leader was found at the 5′ ends of the mRNA species where indicated. C , Northern analysis of poly(A + ) RNA. The probes, indicated below each lane, correspond to the C-terminal repeats. Labels to the right of each lane indicate the probable identity of each band. In the UNC-49C lane the UNC-49B mRNA is visible because it contains the UNC-49C open reading frame in its 3′ UTR. Asterisks indicate higher molecular weight bands that may correspond to partially spliced unc-49 pre-mRNA. All lanes were exposed for the same length of time.
Figure Legend Snippet: unc-49 produces three distinct GABA receptor subunits. A , Structure of the unc-49 locus showing the positions of conserved GABA receptor structural motifs. Domain structure of the locus is indicated by bars at the top (see Results). B , unc-49 mRNA structure. Transcripts were isolated both from cDNA libraries and from RT-PCR experiments. Multiple UNC-49A cDNA clones were identified with different splicing patterns, all resulting in premature stops. One representative example is shown here. Properly spliced RNAs were identified by RT-PCR; the short arrows and circled numbers represent PCR primers. Two superimposed primers (for example, 68 and 73) represent a set of nested PCR primers. The shaded boxes represent coding exons, and the open boxes represent untranslated regions. The SL1 splice leader was found at the 5′ ends of the mRNA species where indicated. C , Northern analysis of poly(A + ) RNA. The probes, indicated below each lane, correspond to the C-terminal repeats. Labels to the right of each lane indicate the probable identity of each band. In the UNC-49C lane the UNC-49B mRNA is visible because it contains the UNC-49C open reading frame in its 3′ UTR. Asterisks indicate higher molecular weight bands that may correspond to partially spliced unc-49 pre-mRNA. All lanes were exposed for the same length of time.

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Polymerase Chain Reaction, Nested PCR, Northern Blot, Molecular Weight

7) Product Images from "Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis"

Article Title: Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.69.1.475-482.2003

DGGE profiles of PCR-amplified 16S rDNA fragments obtained with primer pair L1GC-HDA2 and DNA isolated during sourdough fermentations A, B, C, and D at different fermentation days. (A) After 0 (0 d), 1 (1 d), and 9 (9 d) days. (B) After 0 (0 d), 1 (1 d), 3 (3 d), and 5 (5 d) days. (C) After 0 (0 d), 1 (1 d), 2 (2 d), and 7 (7 d) days. (D) After 0 (0 d), 2 (2 d), 6 (6 d), and 14 (14 d) days. The fragments were excised and sequenced at the end of the fermentation times. Based on sequence comparisons, the fragments were allotted to the following species: b 1 and b 2 , L . mindensis ; c, L . sanfranciscensis ; i, L . crispatus ; j 1 and j 2 , L . pontis ; k, L . panis ; l, L . frumenti ; m, P . acidilactici ; n, L . johnsonii ; and o, L . reuteri . Lanes RI, reference strains.
Figure Legend Snippet: DGGE profiles of PCR-amplified 16S rDNA fragments obtained with primer pair L1GC-HDA2 and DNA isolated during sourdough fermentations A, B, C, and D at different fermentation days. (A) After 0 (0 d), 1 (1 d), and 9 (9 d) days. (B) After 0 (0 d), 1 (1 d), 3 (3 d), and 5 (5 d) days. (C) After 0 (0 d), 1 (1 d), 2 (2 d), and 7 (7 d) days. (D) After 0 (0 d), 2 (2 d), 6 (6 d), and 14 (14 d) days. The fragments were excised and sequenced at the end of the fermentation times. Based on sequence comparisons, the fragments were allotted to the following species: b 1 and b 2 , L . mindensis ; c, L . sanfranciscensis ; i, L . crispatus ; j 1 and j 2 , L . pontis ; k, L . panis ; l, L . frumenti ; m, P . acidilactici ; n, L . johnsonii ; and o, L . reuteri . Lanes RI, reference strains.

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

DGGE profiles of PCR products obtained with primer pair L1GC-HDA2 and DNA isolated from commercially available sourdough starters and baker's yeast. Lane RI, reference strains: 1, W . confusa ; 2, L . johnsonii ; 3, L . fermentum ; 4, L . brevis ; 5, L . crispatus / L . acidophilus ; 6, P . pentosaceus ; 7, L . farciminis ; 8, L . panis ; 9, P . acidilactici ; 10, L . sanfranciscensis / L . pontis ; 11, L . frumenti ; 12, L . reuteri ; and 13, L . paracasei . Lane M, starter mixture (S1, S2, S3, and Y). Lanes S1 to S3, sourdough starters. Lane Y, baker's yeast. Sequence characterization of the excised fragments indicated the presence of the following: a 1 and a 2 , W . confusa ; b 1 to b 6 , L . mindensis ; c 1 to c 3 , L . sanfranciscensis ; d 1 , W . viridescens ; e 1 and e 2 , L . brevis ; f 1 , L . curvatus ; g 1 , L . plantarum ; h 1 , L . fermentum ; and i 1 , L . crispatus .
Figure Legend Snippet: DGGE profiles of PCR products obtained with primer pair L1GC-HDA2 and DNA isolated from commercially available sourdough starters and baker's yeast. Lane RI, reference strains: 1, W . confusa ; 2, L . johnsonii ; 3, L . fermentum ; 4, L . brevis ; 5, L . crispatus / L . acidophilus ; 6, P . pentosaceus ; 7, L . farciminis ; 8, L . panis ; 9, P . acidilactici ; 10, L . sanfranciscensis / L . pontis ; 11, L . frumenti ; 12, L . reuteri ; and 13, L . paracasei . Lane M, starter mixture (S1, S2, S3, and Y). Lanes S1 to S3, sourdough starters. Lane Y, baker's yeast. Sequence characterization of the excised fragments indicated the presence of the following: a 1 and a 2 , W . confusa ; b 1 to b 6 , L . mindensis ; c 1 to c 3 , L . sanfranciscensis ; d 1 , W . viridescens ; e 1 and e 2 , L . brevis ; f 1 , L . curvatus ; g 1 , L . plantarum ; h 1 , L . fermentum ; and i 1 , L . crispatus .

Techniques Used: Denaturing Gradient Gel Electrophoresis, Polymerase Chain Reaction, Isolation, Sequencing

8) Product Images from "Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis"

Article Title: Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2013.1

Grail reduces p53 protein levels. ( a and b ) Saos-2 cells were infected with adenoviral p53 and/or Grail. After 48 h, protein ( a ) and mRNA ( b ) were extracted and analyzed by western blotting or RT-PCR, respectively. ( c and d ) Saos-2 cells were
Figure Legend Snippet: Grail reduces p53 protein levels. ( a and b ) Saos-2 cells were infected with adenoviral p53 and/or Grail. After 48 h, protein ( a ) and mRNA ( b ) were extracted and analyzed by western blotting or RT-PCR, respectively. ( c and d ) Saos-2 cells were

Techniques Used: Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction

The p53/Grail negative feedback loop. ( a and b ) H1299 cells were infected with Ad-p53 expression vector. At indicated time, cells were harvested to analyze the cell cycle profiles by FACS ( a ), and analyze the mRNA amount by quantitative real-time PCR
Figure Legend Snippet: The p53/Grail negative feedback loop. ( a and b ) H1299 cells were infected with Ad-p53 expression vector. At indicated time, cells were harvested to analyze the cell cycle profiles by FACS ( a ), and analyze the mRNA amount by quantitative real-time PCR

Techniques Used: Infection, Expressing, Plasmid Preparation, FACS, Real-time Polymerase Chain Reaction

9) Product Images from "Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis"

Article Title: Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.67.6.2578-2585.2001

DGGE analysis of 16S rDNA fragments obtained from the fecal samples of subjects 2 and 4 before, during, and after consumption of a probiotic product. Subject 2: R1, L. ruminis DSM 20403 T ; R2, L. rhamnosus DSM 20021 T ). Subject 4: R2, L. rhamnosus DSM 20021 T ; R3, L. acidophilus DSM 20079 T ) and sample 7 of the posttest period of subject 4 did not give PCR products. DNA fragments which did not match fragments of the reference strains were allotted upon sequencing to the following species: 1, L. sakei ; 2, L. delbrueckii ; 3, L. curvatus ; and 4, Leuconostoc mesenteroides .
Figure Legend Snippet: DGGE analysis of 16S rDNA fragments obtained from the fecal samples of subjects 2 and 4 before, during, and after consumption of a probiotic product. Subject 2: R1, L. ruminis DSM 20403 T ; R2, L. rhamnosus DSM 20021 T ). Subject 4: R2, L. rhamnosus DSM 20021 T ; R3, L. acidophilus DSM 20079 T ) and sample 7 of the posttest period of subject 4 did not give PCR products. DNA fragments which did not match fragments of the reference strains were allotted upon sequencing to the following species: 1, L. sakei ; 2, L. delbrueckii ; 3, L. curvatus ; and 4, Leuconostoc mesenteroides .

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

PCR-DGGE analysis of 16S rDNA fragments generated by PCR with the specific primers Lac1 and Lac2GC and DNA extracted from Lactobacillus species or mixed populations. Lanes: 1, L. sharpeae DSM 20505 T ; 2, L. acidophilus DSM 20079 T ; 3, L. salivarius subsp. salicinius DSM 20554 T ; 4, L. paracasei subsp. paracasei DSM 5622 T ; 5, L. reuteri DSM 20016 T ; M1 and M2, mixture of the Lactobacillus species containing 5 × 10 7 and 5 × 10 8 cells of each species per ml, respectively.
Figure Legend Snippet: PCR-DGGE analysis of 16S rDNA fragments generated by PCR with the specific primers Lac1 and Lac2GC and DNA extracted from Lactobacillus species or mixed populations. Lanes: 1, L. sharpeae DSM 20505 T ; 2, L. acidophilus DSM 20079 T ; 3, L. salivarius subsp. salicinius DSM 20554 T ; 4, L. paracasei subsp. paracasei DSM 5622 T ; 5, L. reuteri DSM 20016 T ; M1 and M2, mixture of the Lactobacillus species containing 5 × 10 7 and 5 × 10 8 cells of each species per ml, respectively.

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

DGGE analysis of PCR-amplified 16S rDNA fragments obtained with primer pair Lac1 and Lac2GC and DNA isolated from human feces. The fragments were excised and sequenced. Based on sequence comparisons, the fragments were allotted to the following species: 1A to 1C, Lactobacillus sakei ; 2A to 2C, L. curvatus ; 3, L. acidophilus ; 4, L. crispatus ; 5, W. confusa ; 6, P. pentosaceus ; 7A and 7B, Leuconostoc mesenteroides ; 8, L. fructivorans ; 9A and 9B, L. casei group. Bands not resulting in sequences upon purification and sequencing are indicated by arrows.
Figure Legend Snippet: DGGE analysis of PCR-amplified 16S rDNA fragments obtained with primer pair Lac1 and Lac2GC and DNA isolated from human feces. The fragments were excised and sequenced. Based on sequence comparisons, the fragments were allotted to the following species: 1A to 1C, Lactobacillus sakei ; 2A to 2C, L. curvatus ; 3, L. acidophilus ; 4, L. crispatus ; 5, W. confusa ; 6, P. pentosaceus ; 7A and 7B, Leuconostoc mesenteroides ; 8, L. fructivorans ; 9A and 9B, L. casei group. Bands not resulting in sequences upon purification and sequencing are indicated by arrows.

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

10) Product Images from "RimM and RbfA Are Essential for Efficient Processing of 16S rRNA in Escherichia coli"

Article Title: RimM and RbfA Are Essential for Efficient Processing of 16S rRNA in Escherichia coli

Journal: Journal of Bacteriology

doi:

Northern blot analysis of nusA operon mRNA. (A) Genetic organization of the nusA ), respectively; (B through D) P, promoter; T, terminator. Five micrograms of total RNA was subjected to electrophoresis in an agarose gel containing formaldehyde, transferred to a Hybond N filter, and probed with a radiolabeled PCR fragment (probe D). The probe was removed by washing, and the filter was reprobed twice (probes C and B). The exposure times in the experiments for which results are shown in panels B and C were shorter than those in the experiment for which results are shown in panel D in order to avoid overexposure of the bands in strains PW109 and GOB083. The sizes of the γ- 32 P-labeled ATP kinase-treated fragments of the 1-kb DNA ladder from GIBCO BRL Life Technologies Inc. (Gaithersburg, Md.) are indicated. The strains used (with the relevant genetic markers in parentheses) were MW38 ( rimM + ), MW37 (Δ rimM-2 ), PW109 (Δ rimM-2 sdr-43 ), PW100 (Δ rimM-2 sdr-34 ), GOB113 ( rimM + sdr + ), and GOB083 ( rimM + sdr-43 ).
Figure Legend Snippet: Northern blot analysis of nusA operon mRNA. (A) Genetic organization of the nusA ), respectively; (B through D) P, promoter; T, terminator. Five micrograms of total RNA was subjected to electrophoresis in an agarose gel containing formaldehyde, transferred to a Hybond N filter, and probed with a radiolabeled PCR fragment (probe D). The probe was removed by washing, and the filter was reprobed twice (probes C and B). The exposure times in the experiments for which results are shown in panels B and C were shorter than those in the experiment for which results are shown in panel D in order to avoid overexposure of the bands in strains PW109 and GOB083. The sizes of the γ- 32 P-labeled ATP kinase-treated fragments of the 1-kb DNA ladder from GIBCO BRL Life Technologies Inc. (Gaithersburg, Md.) are indicated. The strains used (with the relevant genetic markers in parentheses) were MW38 ( rimM + ), MW37 (Δ rimM-2 ), PW109 (Δ rimM-2 sdr-43 ), PW100 (Δ rimM-2 sdr-34 ), GOB113 ( rimM + sdr + ), and GOB083 ( rimM + sdr-43 ).

Techniques Used: Northern Blot, Electrophoresis, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Labeling

11) Product Images from "Coactivator-Dependent Acetylation Stabilizes Members of the SREBP Family of Transcription Factors"

Article Title: Coactivator-Dependent Acetylation Stabilizes Members of the SREBP Family of Transcription Factors

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.7.2587-2599.2003

Stabilization of SREBP1a affects cholesterol metabolism. (A) 293T cells were transfected with the vector alone (−) or Flag-tagged wild-type SREBP1a (WT) or K333Q (MUT). Thirty-six hours after transfection, total RNA was extracted and used to determine the expression of the LDLR and HMG-CoA reductase genes by quantitative real-time PCR. The results are presented as the factors by which mRNA levels increased relative to the mRNA levels found in cells transfected with the vector alone. The results represent the averages ± standard deviations of one representative experiment performed in triplicate. (B) 293T cells were transfected with either the vector alone (−) or Flag-tagged wild-type SREBP1a or K333Q (MUT). Thirty-six hours after transfection, cells were placed in fresh media supplemented with [ 14 C]acetate. Nonpolar lipids were extracted and resolved by thin-layer chromatography (TLC). Radioactive products were visualized after exposure of the TLC plates to X-ray film.
Figure Legend Snippet: Stabilization of SREBP1a affects cholesterol metabolism. (A) 293T cells were transfected with the vector alone (−) or Flag-tagged wild-type SREBP1a (WT) or K333Q (MUT). Thirty-six hours after transfection, total RNA was extracted and used to determine the expression of the LDLR and HMG-CoA reductase genes by quantitative real-time PCR. The results are presented as the factors by which mRNA levels increased relative to the mRNA levels found in cells transfected with the vector alone. The results represent the averages ± standard deviations of one representative experiment performed in triplicate. (B) 293T cells were transfected with either the vector alone (−) or Flag-tagged wild-type SREBP1a or K333Q (MUT). Thirty-six hours after transfection, cells were placed in fresh media supplemented with [ 14 C]acetate. Nonpolar lipids were extracted and resolved by thin-layer chromatography (TLC). Radioactive products were visualized after exposure of the TLC plates to X-ray film.

Techniques Used: Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Thin Layer Chromatography

12) Product Images from "ARIA, an Arabidopsis Arm Repeat Protein Interacting with a Transcriptional Regulator of Abscisic Acid-Responsive Gene Expression, Is a Novel Abscisic Acid Signaling Component 1"

Article Title: ARIA, an Arabidopsis Arm Repeat Protein Interacting with a Transcriptional Regulator of Abscisic Acid-Responsive Gene Expression, Is a Novel Abscisic Acid Signaling Component 1

Journal: Plant Physiology

doi: 10.1104/pp.104.049189

Expression of ABA-responsive genes in 35S-ARIA and aria plants. RNA levels were determined by RT-PCR using total RNA isolated from 2-week-old seedlings. The number of amplification cycles is different for different genes and for overexpression and knockout lines.
Figure Legend Snippet: Expression of ABA-responsive genes in 35S-ARIA and aria plants. RNA levels were determined by RT-PCR using total RNA isolated from 2-week-old seedlings. The number of amplification cycles is different for different genes and for overexpression and knockout lines.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Over Expression, Knock-Out

13) Product Images from "Checkpoint suppressor 1 suppresses transcriptional activity of ERα and breast cancer cell proliferation via deacetylase SIRT1"

Article Title: Checkpoint suppressor 1 suppresses transcriptional activity of ERα and breast cancer cell proliferation via deacetylase SIRT1

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0629-3

CHES1 expression was repressed by E 2 -ERα pathway in breast cancer. a Western blotting tested the protein expression of CHES1 and ERα in MCF7 cells with or without E 2 treatment. b Immunoblotting of CHES1 and ERα in MCF7 cells treated with or without 100 nM E 2 , 1 nM 4-Hydroxytamoxifen, and 0.1 μM ICI 182780. c Schematic representation of conserved ERα-binding motif and ERE site on CHES1 potential promoter region, real-time PCR assay tested the mRNA level of CHES1 in MCF7 cells treated with or without E 2 . d Schematic representation of CHES1 -promoter luc construction. Luciferase reporter assay showed that the activity of CHES1 -promoter luc was repressed in MCF7 cells when treated with E 2 stimulation. * P
Figure Legend Snippet: CHES1 expression was repressed by E 2 -ERα pathway in breast cancer. a Western blotting tested the protein expression of CHES1 and ERα in MCF7 cells with or without E 2 treatment. b Immunoblotting of CHES1 and ERα in MCF7 cells treated with or without 100 nM E 2 , 1 nM 4-Hydroxytamoxifen, and 0.1 μM ICI 182780. c Schematic representation of conserved ERα-binding motif and ERE site on CHES1 potential promoter region, real-time PCR assay tested the mRNA level of CHES1 in MCF7 cells treated with or without E 2 . d Schematic representation of CHES1 -promoter luc construction. Luciferase reporter assay showed that the activity of CHES1 -promoter luc was repressed in MCF7 cells when treated with E 2 stimulation. * P

Techniques Used: Expressing, Western Blot, Binding Assay, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Activity Assay

CHES1 has little effect on the stability, dimerization, subcellular location of ERα, and it also does not influence the interaction between ERα and HDAC1/2. a Western blotting and real-time PCR assays tested the mRNA and protein level of endogenous ERα with overexpression of Flag-CHES1 in MCF7 cells. b Western blotting assay showed that the protein level of exogenous ERα were not affected when co-transfected with CHES1 in HeLa cells. c CoIP assay indicated that the dimerization of ERα were not interfered with knockdown of CHES1 in HEK293T cells. d Cytoplasmic and nucleus fractions separation assay were conducted in MCF7 cells with or without knockdown of CHES1 to test the subcellular distribution of endogenous ERα. The β-tubulin used as cytoplasmic marker and Fibrillarin as nuclear marker. e CoIP assay showed the endogenous and exogenous interaction between CHES1 and HDAC1/2 in MCF7 and HEK293T cells. f CoIP assay assessed the effect of CHES1 on the interaction between ERα and HDAC1/2
Figure Legend Snippet: CHES1 has little effect on the stability, dimerization, subcellular location of ERα, and it also does not influence the interaction between ERα and HDAC1/2. a Western blotting and real-time PCR assays tested the mRNA and protein level of endogenous ERα with overexpression of Flag-CHES1 in MCF7 cells. b Western blotting assay showed that the protein level of exogenous ERα were not affected when co-transfected with CHES1 in HeLa cells. c CoIP assay indicated that the dimerization of ERα were not interfered with knockdown of CHES1 in HEK293T cells. d Cytoplasmic and nucleus fractions separation assay were conducted in MCF7 cells with or without knockdown of CHES1 to test the subcellular distribution of endogenous ERα. The β-tubulin used as cytoplasmic marker and Fibrillarin as nuclear marker. e CoIP assay showed the endogenous and exogenous interaction between CHES1 and HDAC1/2 in MCF7 and HEK293T cells. f CoIP assay assessed the effect of CHES1 on the interaction between ERα and HDAC1/2

Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Transfection, Co-Immunoprecipitation Assay, Marker

Related Articles

Clone Assay:

Article Title: Checkpoint suppressor 1 suppresses transcriptional activity of ERα and breast cancer cell proliferation via deacetylase SIRT1
Article Snippet: .. GST-tagged full-length and truncated CHES1 were constructed using similar methods by cloning PCR fragments into pGEX-4T-3 (Amersham Pharmacia, UK). .. Myc-CHES1 was obtained by using pcDNA3.1-Myc expression vector.

Polymerase Chain Reaction:

Article Title: Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis
Article Snippet: .. Bacterial expression vectors for various GST-p53 or -Grail fusions were constructed by inserting the appropriate PCR fragments into the Eco RI– Xho I sites of pGEX-4T1 (GE HealthCare, Waukesha, WI, USA). .. Reporters for pG13-, p21 -, Mdm2 -, PUMA -, and Bax -LUC have been described previously., Cell transfections were performed using Fugene 6 (Roche, Basel, Switzerland) according to the manufacturer's instructions.

Article Title: Expression of an Expansin Is Associated with Endosperm Weakening during Tomato Seed Germination 1
Article Snippet: .. The PCR fragments were used to screen a cDNA library prepared from whole gib-1 seeds imbibed in 100 μ m GA4+7 for 24 h. The cDNAs were labeled with enhanced chemiluminescence (ECL) nucleic acid labeling reagents (ECL kit, Amersham Life Science, Arlington Heights, IL) and were added to prehybridization solution at a final concentration of 10 ng/mL. .. Prehybridization was for 30 min at 42°C and hybridization was for 3 h at 42°C.

Article Title: FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)
Article Snippet: .. PCR fragments of the FaCCR and FaPOD cut with Bam HI and Sma I were subcloned into a Bam HI-Sma I cut pGEX-4T-1 vector (GE Healthcare, Munich, Germany), in frame with a coding region for an N-terminal GST (glutathione S-transferase) tag. .. In addition, the full-length coding region of FaCAD was constructed in pET-29a(+) (Novagen, Darmstadt, Germany) as PCR template DNA.

Article Title: The Caenorhabditis elegans unc-49 Locus Encodes Multiple Subunits of a Heteromultimeric GABA Receptor
Article Snippet: .. Genomic sequencing was performed on genomic PCR fragments corresponding to UNC-49B by using the ThermoSequenase cycle sequencing kit (Amersham Pharmacia, Piscataway, NJ). .. The S65C variant of GFP containing three introns (1997 Fire vector kit) was cloned into the T21C12ΔMlu construct such that GFP was inserted, in frame, into the large intracellular loop of one subunit, whereas the other subunits were wild type.

Article Title: Checkpoint suppressor 1 suppresses transcriptional activity of ERα and breast cancer cell proliferation via deacetylase SIRT1
Article Snippet: .. GST-tagged full-length and truncated CHES1 were constructed using similar methods by cloning PCR fragments into pGEX-4T-3 (Amersham Pharmacia, UK). .. Myc-CHES1 was obtained by using pcDNA3.1-Myc expression vector.

Article Title: Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids
Article Snippet: .. The probes were 32 P -labeled PCR fragments of mfat-1 sequence, generated using the Random Prime Labeling System Redi PrimeTMII (GE Healthcare, Piscataway, USA). .. Northern blot analysis was performed as described previously .

Article Title: Importin ?1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1
Article Snippet: .. Bacterial expression vectors for various GST (glutathione S-transferase)–Zac1 fusions were constructed by inserting the appropriate PCR fragments into the EcoRI/XhoI sites of pGEX-4T1 (GE Healthcare). .. HeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% charcoal/dextran-treated foetal bovine serum.

Article Title: Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis
Article Snippet: .. DNA sequences of PCR fragments obtained from purified DGGE bands and pure cultures were determined by the dideoxy chain termination method with an AutoRead sequencing kit (Amersham Pharmacia Biotech), a LI-COR system (MWG-Biotech), and IRD 800-labeled primers L1seq or HDA2seq and 616V, respectively. ..

Genomic Sequencing:

Article Title: The Caenorhabditis elegans unc-49 Locus Encodes Multiple Subunits of a Heteromultimeric GABA Receptor
Article Snippet: .. Genomic sequencing was performed on genomic PCR fragments corresponding to UNC-49B by using the ThermoSequenase cycle sequencing kit (Amersham Pharmacia, Piscataway, NJ). .. The S65C variant of GFP containing three introns (1997 Fire vector kit) was cloned into the T21C12ΔMlu construct such that GFP was inserted, in frame, into the large intracellular loop of one subunit, whereas the other subunits were wild type.

Construct:

Article Title: Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis
Article Snippet: .. Bacterial expression vectors for various GST-p53 or -Grail fusions were constructed by inserting the appropriate PCR fragments into the Eco RI– Xho I sites of pGEX-4T1 (GE HealthCare, Waukesha, WI, USA). .. Reporters for pG13-, p21 -, Mdm2 -, PUMA -, and Bax -LUC have been described previously., Cell transfections were performed using Fugene 6 (Roche, Basel, Switzerland) according to the manufacturer's instructions.

Article Title: Checkpoint suppressor 1 suppresses transcriptional activity of ERα and breast cancer cell proliferation via deacetylase SIRT1
Article Snippet: .. GST-tagged full-length and truncated CHES1 were constructed using similar methods by cloning PCR fragments into pGEX-4T-3 (Amersham Pharmacia, UK). .. Myc-CHES1 was obtained by using pcDNA3.1-Myc expression vector.

Article Title: Importin ?1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1
Article Snippet: .. Bacterial expression vectors for various GST (glutathione S-transferase)–Zac1 fusions were constructed by inserting the appropriate PCR fragments into the EcoRI/XhoI sites of pGEX-4T1 (GE Healthcare). .. HeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% charcoal/dextran-treated foetal bovine serum.

Purification:

Article Title: Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis
Article Snippet: .. DNA sequences of PCR fragments obtained from purified DGGE bands and pure cultures were determined by the dideoxy chain termination method with an AutoRead sequencing kit (Amersham Pharmacia Biotech), a LI-COR system (MWG-Biotech), and IRD 800-labeled primers L1seq or HDA2seq and 616V, respectively. ..

Concentration Assay:

Article Title: Expression of an Expansin Is Associated with Endosperm Weakening during Tomato Seed Germination 1
Article Snippet: .. The PCR fragments were used to screen a cDNA library prepared from whole gib-1 seeds imbibed in 100 μ m GA4+7 for 24 h. The cDNAs were labeled with enhanced chemiluminescence (ECL) nucleic acid labeling reagents (ECL kit, Amersham Life Science, Arlington Heights, IL) and were added to prehybridization solution at a final concentration of 10 ng/mL. .. Prehybridization was for 30 min at 42°C and hybridization was for 3 h at 42°C.

Generated:

Article Title: Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids
Article Snippet: .. The probes were 32 P -labeled PCR fragments of mfat-1 sequence, generated using the Random Prime Labeling System Redi PrimeTMII (GE Healthcare, Piscataway, USA). .. Northern blot analysis was performed as described previously .

cDNA Library Assay:

Article Title: Expression of an Expansin Is Associated with Endosperm Weakening during Tomato Seed Germination 1
Article Snippet: .. The PCR fragments were used to screen a cDNA library prepared from whole gib-1 seeds imbibed in 100 μ m GA4+7 for 24 h. The cDNAs were labeled with enhanced chemiluminescence (ECL) nucleic acid labeling reagents (ECL kit, Amersham Life Science, Arlington Heights, IL) and were added to prehybridization solution at a final concentration of 10 ng/mL. .. Prehybridization was for 30 min at 42°C and hybridization was for 3 h at 42°C.

Labeling:

Article Title: Expression of an Expansin Is Associated with Endosperm Weakening during Tomato Seed Germination 1
Article Snippet: .. The PCR fragments were used to screen a cDNA library prepared from whole gib-1 seeds imbibed in 100 μ m GA4+7 for 24 h. The cDNAs were labeled with enhanced chemiluminescence (ECL) nucleic acid labeling reagents (ECL kit, Amersham Life Science, Arlington Heights, IL) and were added to prehybridization solution at a final concentration of 10 ng/mL. .. Prehybridization was for 30 min at 42°C and hybridization was for 3 h at 42°C.

Article Title: Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids
Article Snippet: .. The probes were 32 P -labeled PCR fragments of mfat-1 sequence, generated using the Random Prime Labeling System Redi PrimeTMII (GE Healthcare, Piscataway, USA). .. Northern blot analysis was performed as described previously .

Expressing:

Article Title: Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis
Article Snippet: .. Bacterial expression vectors for various GST-p53 or -Grail fusions were constructed by inserting the appropriate PCR fragments into the Eco RI– Xho I sites of pGEX-4T1 (GE HealthCare, Waukesha, WI, USA). .. Reporters for pG13-, p21 -, Mdm2 -, PUMA -, and Bax -LUC have been described previously., Cell transfections were performed using Fugene 6 (Roche, Basel, Switzerland) according to the manufacturer's instructions.

Article Title: Importin ?1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1
Article Snippet: .. Bacterial expression vectors for various GST (glutathione S-transferase)–Zac1 fusions were constructed by inserting the appropriate PCR fragments into the EcoRI/XhoI sites of pGEX-4T1 (GE Healthcare). .. HeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% charcoal/dextran-treated foetal bovine serum.

Sequencing:

Article Title: The Caenorhabditis elegans unc-49 Locus Encodes Multiple Subunits of a Heteromultimeric GABA Receptor
Article Snippet: .. Genomic sequencing was performed on genomic PCR fragments corresponding to UNC-49B by using the ThermoSequenase cycle sequencing kit (Amersham Pharmacia, Piscataway, NJ). .. The S65C variant of GFP containing three introns (1997 Fire vector kit) was cloned into the T21C12ΔMlu construct such that GFP was inserted, in frame, into the large intracellular loop of one subunit, whereas the other subunits were wild type.

Article Title: Handmade Cloned Transgenic Sheep Rich in Omega-3 Fatty Acids
Article Snippet: .. The probes were 32 P -labeled PCR fragments of mfat-1 sequence, generated using the Random Prime Labeling System Redi PrimeTMII (GE Healthcare, Piscataway, USA). .. Northern blot analysis was performed as described previously .

Article Title: Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis
Article Snippet: .. DNA sequences of PCR fragments obtained from purified DGGE bands and pure cultures were determined by the dideoxy chain termination method with an AutoRead sequencing kit (Amersham Pharmacia Biotech), a LI-COR system (MWG-Biotech), and IRD 800-labeled primers L1seq or HDA2seq and 616V, respectively. ..

Denaturing Gradient Gel Electrophoresis:

Article Title: Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis
Article Snippet: .. DNA sequences of PCR fragments obtained from purified DGGE bands and pure cultures were determined by the dideoxy chain termination method with an AutoRead sequencing kit (Amersham Pharmacia Biotech), a LI-COR system (MWG-Biotech), and IRD 800-labeled primers L1seq or HDA2seq and 616V, respectively. ..

Plasmid Preparation:

Article Title: FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)
Article Snippet: .. PCR fragments of the FaCCR and FaPOD cut with Bam HI and Sma I were subcloned into a Bam HI-Sma I cut pGEX-4T-1 vector (GE Healthcare, Munich, Germany), in frame with a coding region for an N-terminal GST (glutathione S-transferase) tag. .. In addition, the full-length coding region of FaCAD was constructed in pET-29a(+) (Novagen, Darmstadt, Germany) as PCR template DNA.

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    GE Healthcare pcr amplified cdna fragments
    Characterization of androgen-responsive non-coding intronic RNAs . Selected intronic transcripts extended by <t>RACE-PCR</t> (A-D, red boxes) or gene-specific PCR (E, yellow box) are shown. Extended unspliced antisense intronic transcript fragments were mapped to the genomic sequence (A-E, gray lines) relative to the respective spliced protein-coding transcript expressed in the opposite strand (A-E, green arrows). Double-stranded <t>cDNA</t> clones spotted on the microarrays are represented as blue boxes (A-E). A previously described antisense unspliced intronic transcript mapping to GAS6 locus is shown (E, black arrow). Chromosome coordinates and the length of extended intronic transcript fragments are indicated. (F) Strand-specific multi-tissue northern blot using probes complementary to the antisense intronic transcript mapped in the GAS6 locus.
    Pcr Amplified Cdna Fragments, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare pcr fragments
    Relative expression profiles of down- (A) or up-regulated (B) monolignol genes in F . × ananassa cv. Elsanta . Total RNA was isolated from a single untreated fruit (wild type; WT), control fruits (pBI-intron), down-regulated fruits (pBI- FaCCRi , pBI- FaCADi , and pBI- FaPODi ) (A) , and up-regulated fruits (pBI- <t>FaCCR</t> , pBI- FaCAD , and pBI- FaPOD ) (B) . Expression levels of all samples were monitored by <t>qRT-PCR</t> with specific primers for target genes ( FaCCR . FaCAD , and FaPOD ) and an interspac er gene. The latter was used as an internal control for normalization. For each box-plot graph, one of the WT group was used as the reference (set to one) and each group contained five biological replicates. The Wilcoxon-Mann-Whitney U -test was used for a non-parametric comparison of two groups from pBI-intron and fruits infiltrated with different constructs. Values indicate statistically decreased and increased levels ( P
    Pcr Fragments, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare restriction enzyme digestion ctnnb1 exon 3 pcr fragments
    Analytical restriction enzyme cleavage analysis . (A), <t>CTNNB1</t> <t>exon</t> 3 <t>PCR</t> fragments (176 bp) were digested with Xmn I or Nla III that cuts only wild-type or only S37A mutant sequences, respectively. Three out of the nine parathyroid adenomas with S37A mutation were identified in our previous study [10]. Nla III cuts also outside of codon 37, close to the fragment end (23 bp). U; uncleaved CTNNB1 exon 3 PCR fragment. (B), CTNNB1 exon 3 was PCR amplified from a 1:1 mixture of constitutional DNA and tumor DNA with the S37A mutation. The fragment was analyzed by restriction enzyme digestions as in (A).
    Restriction Enzyme Digestion Ctnnb1 Exon 3 Pcr Fragments, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare pcr amplified cdna fragment
    Cloning of Rap2a GTPase binding domain (RBD) from mouse Ral guanine nucleotide dissociation stimulator (RalGDS). ( a , left ) Total RNA was obtained from mouse embryonic fibroblasts (MEFs) and RAW264.7 cells and subsequently transcribed into <t>cDNA</t> that was used as template in <t>RT-PCR</t> reactions. PCR reactions were fracionated on 1.2% agarose gel. Expected size of the amplicons: 401-bp. Lanes 1–4: cDNA amplicons from MEFs (1–2) and RAW264.7 cells (3–4). (a, right ) The band from lane 3 was excised from gel, purified and cloned into a pCR2.1 vector, used to transform Stbl3 E. coli cells. Lanes 5–6: PCR from pCR2.1 plasmid containing RBD-RalGDS insert. ( b ) Plasmid pGEX-6P-1 was digested with Bam HI and Eco RI and purified (lanes 1 and 2), and fractionated on 1% agarose gel. ( c ) RBD-RalGDS inserts that were digested and purified from pCR2.1 plasmids were ligated into Bam HI and Eco RI sites of pGEX-6P-1 and used to transform Stbl3 cells. Shown is a representative ethidium bromide-stained 1.2% agarose gel of a PCR screening of Stbl3 E. coli colonies for the presence of insert where lanes 1 and 2 show positive ones. Lanes 3–6: negative colonies. ( d ) Positive colonies that were then grown in bacteria liquid medium were further processed for plasmid miniprep, followed by digestion with Bam HI and Eco RI and fractionated on 1.2% agarose gel to reveal for the presence of the insert. Shown is a negative image of the photodocumented gel. Lanes 1, 4 and 7 show the expected insert of 401-bp. Lanes 2, 5 and 8: linearized pGEX-6P-1-RalGDS-RBD plasmids. Lanes 3, 6 and 9: undigested pGEX-6P-1-RalGDS-RBD plasmids. Plasmids were sent for sequencing. Agarose gels in the panels were run in TAE 1X, stained with ethidium bromide and photodocumented. DNA marker (lanes M): 1kb plus DNA ladder (Invitrogen).
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    Characterization of androgen-responsive non-coding intronic RNAs . Selected intronic transcripts extended by RACE-PCR (A-D, red boxes) or gene-specific PCR (E, yellow box) are shown. Extended unspliced antisense intronic transcript fragments were mapped to the genomic sequence (A-E, gray lines) relative to the respective spliced protein-coding transcript expressed in the opposite strand (A-E, green arrows). Double-stranded cDNA clones spotted on the microarrays are represented as blue boxes (A-E). A previously described antisense unspliced intronic transcript mapping to GAS6 locus is shown (E, black arrow). Chromosome coordinates and the length of extended intronic transcript fragments are indicated. (F) Strand-specific multi-tissue northern blot using probes complementary to the antisense intronic transcript mapped in the GAS6 locus.

    Journal: BMC Biology

    Article Title: Androgen responsive intronic non-coding RNAs

    doi: 10.1186/1741-7007-5-4

    Figure Lengend Snippet: Characterization of androgen-responsive non-coding intronic RNAs . Selected intronic transcripts extended by RACE-PCR (A-D, red boxes) or gene-specific PCR (E, yellow box) are shown. Extended unspliced antisense intronic transcript fragments were mapped to the genomic sequence (A-E, gray lines) relative to the respective spliced protein-coding transcript expressed in the opposite strand (A-E, green arrows). Double-stranded cDNA clones spotted on the microarrays are represented as blue boxes (A-E). A previously described antisense unspliced intronic transcript mapping to GAS6 locus is shown (E, black arrow). Chromosome coordinates and the length of extended intronic transcript fragments are indicated. (F) Strand-specific multi-tissue northern blot using probes complementary to the antisense intronic transcript mapped in the GAS6 locus.

    Article Snippet: Microarray experiments The microarray platform used in this study was constructed using PCR-amplified cDNA fragments derived from ORESTES clones [ ], and spotted onto Type 7 STAR glass slides (GE Healthcare, Piscataway, NJ, USA) as previously described [ ].

    Techniques: Polymerase Chain Reaction, Sequencing, Clone Assay, Northern Blot

    Relative expression profiles of down- (A) or up-regulated (B) monolignol genes in F . × ananassa cv. Elsanta . Total RNA was isolated from a single untreated fruit (wild type; WT), control fruits (pBI-intron), down-regulated fruits (pBI- FaCCRi , pBI- FaCADi , and pBI- FaPODi ) (A) , and up-regulated fruits (pBI- FaCCR , pBI- FaCAD , and pBI- FaPOD ) (B) . Expression levels of all samples were monitored by qRT-PCR with specific primers for target genes ( FaCCR . FaCAD , and FaPOD ) and an interspac er gene. The latter was used as an internal control for normalization. For each box-plot graph, one of the WT group was used as the reference (set to one) and each group contained five biological replicates. The Wilcoxon-Mann-Whitney U -test was used for a non-parametric comparison of two groups from pBI-intron and fruits infiltrated with different constructs. Values indicate statistically decreased and increased levels ( P

    Journal: Frontiers in Plant Science

    Article Title: FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)

    doi: 10.3389/fpls.2014.00518

    Figure Lengend Snippet: Relative expression profiles of down- (A) or up-regulated (B) monolignol genes in F . × ananassa cv. Elsanta . Total RNA was isolated from a single untreated fruit (wild type; WT), control fruits (pBI-intron), down-regulated fruits (pBI- FaCCRi , pBI- FaCADi , and pBI- FaPODi ) (A) , and up-regulated fruits (pBI- FaCCR , pBI- FaCAD , and pBI- FaPOD ) (B) . Expression levels of all samples were monitored by qRT-PCR with specific primers for target genes ( FaCCR . FaCAD , and FaPOD ) and an interspac er gene. The latter was used as an internal control for normalization. For each box-plot graph, one of the WT group was used as the reference (set to one) and each group contained five biological replicates. The Wilcoxon-Mann-Whitney U -test was used for a non-parametric comparison of two groups from pBI-intron and fruits infiltrated with different constructs. Values indicate statistically decreased and increased levels ( P

    Article Snippet: PCR fragments of the FaCCR and FaPOD cut with Bam HI and Sma I were subcloned into a Bam HI-Sma I cut pGEX-4T-1 vector (GE Healthcare, Munich, Germany), in frame with a coding region for an N-terminal GST (glutathione S-transferase) tag.

    Techniques: Expressing, Isolation, Quantitative RT-PCR, MANN-WHITNEY, Construct

    Relative expression profiles of monolignol biosynthesis genes in vegetative tissues, flowers, and fruit developmental stages of F . × ananassa cv. Elsanta . Total RNA was isolated from (A) fruit developmental stages at small green (SG), green white (GW), white (W), turning (T), and red (R) after pollination. (B) Expression levels of vegetative tissues (leaf; stem; runner; root), flower (F) and fruit developmental stages (SG, GW, W, T, and R) were monitored by qRT-PCR. FaCCR . FaCAD . FaPOD and FaPOD27 (Ring et al., 2013 ) were target genes. The white fruit was used as the reference with one for each graph. Values are mean ± SE of 5–6 replicates from two sets of cDNAs and are shown as relative changes.

    Journal: Frontiers in Plant Science

    Article Title: FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)

    doi: 10.3389/fpls.2014.00518

    Figure Lengend Snippet: Relative expression profiles of monolignol biosynthesis genes in vegetative tissues, flowers, and fruit developmental stages of F . × ananassa cv. Elsanta . Total RNA was isolated from (A) fruit developmental stages at small green (SG), green white (GW), white (W), turning (T), and red (R) after pollination. (B) Expression levels of vegetative tissues (leaf; stem; runner; root), flower (F) and fruit developmental stages (SG, GW, W, T, and R) were monitored by qRT-PCR. FaCCR . FaCAD . FaPOD and FaPOD27 (Ring et al., 2013 ) were target genes. The white fruit was used as the reference with one for each graph. Values are mean ± SE of 5–6 replicates from two sets of cDNAs and are shown as relative changes.

    Article Snippet: PCR fragments of the FaCCR and FaPOD cut with Bam HI and Sma I were subcloned into a Bam HI-Sma I cut pGEX-4T-1 vector (GE Healthcare, Munich, Germany), in frame with a coding region for an N-terminal GST (glutathione S-transferase) tag.

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    Relative expression profiles of phenylpropanoid biosynthesis genes of F . × ananassa cv. Elsanta in response to Agrobacterium . Expression levels in control (gray column) and agroinfiltrated fruits (black column) were monitored by qRT-PCR at different time points (1, 3, 6, 12, 24, 48, and 96 h). FaPAL . FaCHS, FaCCR . FaCAD . FaPOD , and FaPOD27 were target genes. The control fruit (1 h) was used as the reference with one for each graph. Values are mean ± SE of 2–3 replicates from one fruit and are shown as relative changes.

    Journal: Frontiers in Plant Science

    Article Title: FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)

    doi: 10.3389/fpls.2014.00518

    Figure Lengend Snippet: Relative expression profiles of phenylpropanoid biosynthesis genes of F . × ananassa cv. Elsanta in response to Agrobacterium . Expression levels in control (gray column) and agroinfiltrated fruits (black column) were monitored by qRT-PCR at different time points (1, 3, 6, 12, 24, 48, and 96 h). FaPAL . FaCHS, FaCCR . FaCAD . FaPOD , and FaPOD27 were target genes. The control fruit (1 h) was used as the reference with one for each graph. Values are mean ± SE of 2–3 replicates from one fruit and are shown as relative changes.

    Article Snippet: PCR fragments of the FaCCR and FaPOD cut with Bam HI and Sma I were subcloned into a Bam HI-Sma I cut pGEX-4T-1 vector (GE Healthcare, Munich, Germany), in frame with a coding region for an N-terminal GST (glutathione S-transferase) tag.

    Techniques: Expressing, Quantitative RT-PCR

    Analytical restriction enzyme cleavage analysis . (A), CTNNB1 exon 3 PCR fragments (176 bp) were digested with Xmn I or Nla III that cuts only wild-type or only S37A mutant sequences, respectively. Three out of the nine parathyroid adenomas with S37A mutation were identified in our previous study [10]. Nla III cuts also outside of codon 37, close to the fragment end (23 bp). U; uncleaved CTNNB1 exon 3 PCR fragment. (B), CTNNB1 exon 3 was PCR amplified from a 1:1 mixture of constitutional DNA and tumor DNA with the S37A mutation. The fragment was analyzed by restriction enzyme digestions as in (A).

    Journal: Molecular Cancer

    Article Title: Stabilizing mutation of CTNNB1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients

    doi: 10.1186/1476-4598-7-53

    Figure Lengend Snippet: Analytical restriction enzyme cleavage analysis . (A), CTNNB1 exon 3 PCR fragments (176 bp) were digested with Xmn I or Nla III that cuts only wild-type or only S37A mutant sequences, respectively. Three out of the nine parathyroid adenomas with S37A mutation were identified in our previous study [10]. Nla III cuts also outside of codon 37, close to the fragment end (23 bp). U; uncleaved CTNNB1 exon 3 PCR fragment. (B), CTNNB1 exon 3 was PCR amplified from a 1:1 mixture of constitutional DNA and tumor DNA with the S37A mutation. The fragment was analyzed by restriction enzyme digestions as in (A).

    Article Snippet: Restriction Enzyme Digestion CTNNB1 exon 3 PCR fragments were purified using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Europe GmbH, Uppsala, Sweden) and cleaved with Xma I or Nla III according to instructions by the manufacturer (New England Biolabs, Inc., Beverly, MA).

    Techniques: Polymerase Chain Reaction, Mutagenesis, Amplification

    Representative results of direct DNA sequencing of CTNNB1 exon 3 . Constitutional DNA from blood (left panel) and parathyroid adenoma (right panel) of the same pHPT patient.

    Journal: Molecular Cancer

    Article Title: Stabilizing mutation of CTNNB1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients

    doi: 10.1186/1476-4598-7-53

    Figure Lengend Snippet: Representative results of direct DNA sequencing of CTNNB1 exon 3 . Constitutional DNA from blood (left panel) and parathyroid adenoma (right panel) of the same pHPT patient.

    Article Snippet: Restriction Enzyme Digestion CTNNB1 exon 3 PCR fragments were purified using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Europe GmbH, Uppsala, Sweden) and cleaved with Xma I or Nla III according to instructions by the manufacturer (New England Biolabs, Inc., Beverly, MA).

    Techniques: DNA Sequencing

    Cloning of Rap2a GTPase binding domain (RBD) from mouse Ral guanine nucleotide dissociation stimulator (RalGDS). ( a , left ) Total RNA was obtained from mouse embryonic fibroblasts (MEFs) and RAW264.7 cells and subsequently transcribed into cDNA that was used as template in RT-PCR reactions. PCR reactions were fracionated on 1.2% agarose gel. Expected size of the amplicons: 401-bp. Lanes 1–4: cDNA amplicons from MEFs (1–2) and RAW264.7 cells (3–4). (a, right ) The band from lane 3 was excised from gel, purified and cloned into a pCR2.1 vector, used to transform Stbl3 E. coli cells. Lanes 5–6: PCR from pCR2.1 plasmid containing RBD-RalGDS insert. ( b ) Plasmid pGEX-6P-1 was digested with Bam HI and Eco RI and purified (lanes 1 and 2), and fractionated on 1% agarose gel. ( c ) RBD-RalGDS inserts that were digested and purified from pCR2.1 plasmids were ligated into Bam HI and Eco RI sites of pGEX-6P-1 and used to transform Stbl3 cells. Shown is a representative ethidium bromide-stained 1.2% agarose gel of a PCR screening of Stbl3 E. coli colonies for the presence of insert where lanes 1 and 2 show positive ones. Lanes 3–6: negative colonies. ( d ) Positive colonies that were then grown in bacteria liquid medium were further processed for plasmid miniprep, followed by digestion with Bam HI and Eco RI and fractionated on 1.2% agarose gel to reveal for the presence of the insert. Shown is a negative image of the photodocumented gel. Lanes 1, 4 and 7 show the expected insert of 401-bp. Lanes 2, 5 and 8: linearized pGEX-6P-1-RalGDS-RBD plasmids. Lanes 3, 6 and 9: undigested pGEX-6P-1-RalGDS-RBD plasmids. Plasmids were sent for sequencing. Agarose gels in the panels were run in TAE 1X, stained with ethidium bromide and photodocumented. DNA marker (lanes M): 1kb plus DNA ladder (Invitrogen).

    Journal: Data in Brief

    Article Title: Data in support of Rap2a GTPase expression, activation and effects in LPS-mediated innate immune response and NF-κB activation

    doi: 10.1016/j.dib.2019.103965

    Figure Lengend Snippet: Cloning of Rap2a GTPase binding domain (RBD) from mouse Ral guanine nucleotide dissociation stimulator (RalGDS). ( a , left ) Total RNA was obtained from mouse embryonic fibroblasts (MEFs) and RAW264.7 cells and subsequently transcribed into cDNA that was used as template in RT-PCR reactions. PCR reactions were fracionated on 1.2% agarose gel. Expected size of the amplicons: 401-bp. Lanes 1–4: cDNA amplicons from MEFs (1–2) and RAW264.7 cells (3–4). (a, right ) The band from lane 3 was excised from gel, purified and cloned into a pCR2.1 vector, used to transform Stbl3 E. coli cells. Lanes 5–6: PCR from pCR2.1 plasmid containing RBD-RalGDS insert. ( b ) Plasmid pGEX-6P-1 was digested with Bam HI and Eco RI and purified (lanes 1 and 2), and fractionated on 1% agarose gel. ( c ) RBD-RalGDS inserts that were digested and purified from pCR2.1 plasmids were ligated into Bam HI and Eco RI sites of pGEX-6P-1 and used to transform Stbl3 cells. Shown is a representative ethidium bromide-stained 1.2% agarose gel of a PCR screening of Stbl3 E. coli colonies for the presence of insert where lanes 1 and 2 show positive ones. Lanes 3–6: negative colonies. ( d ) Positive colonies that were then grown in bacteria liquid medium were further processed for plasmid miniprep, followed by digestion with Bam HI and Eco RI and fractionated on 1.2% agarose gel to reveal for the presence of the insert. Shown is a negative image of the photodocumented gel. Lanes 1, 4 and 7 show the expected insert of 401-bp. Lanes 2, 5 and 8: linearized pGEX-6P-1-RalGDS-RBD plasmids. Lanes 3, 6 and 9: undigested pGEX-6P-1-RalGDS-RBD plasmids. Plasmids were sent for sequencing. Agarose gels in the panels were run in TAE 1X, stained with ethidium bromide and photodocumented. DNA marker (lanes M): 1kb plus DNA ladder (Invitrogen).

    Article Snippet: PCR-amplified cDNA fragment of 401-bp comprising the RBD-RalGDS mRNA coding sequence was cloned into pCR2.1 vector, and then a Bam HI- Eco RI fragment was obtained by digestion, purified and finally subcloned in pGEX-6P-1 vector (GE Healthcare).

    Techniques: Clone Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Purification, Plasmid Preparation, Staining, Sequencing, Marker

    Expression analysis of Rap2a in LPS-treated RAW264.7 macrophages and human THP-1 monocytes. Non-quantitative RT-PCR and quantitative RT-qPCR analyses of Rap2a in RAW264.7 ( a, b ) and RAP2A in THP-1 ( c ) cells that were stimulated as indicated. Lower graph in ( a ) shows fold change expression after densitometric analysis of Rap2a:Gapdh ratio. Total RNA was extracted and subsequently transcribed into cDNA that was used as template in RT-qPCR reactions for detection of mRNAs. NT, not treated . NC, PCR negative control.

    Journal: Data in Brief

    Article Title: Data in support of Rap2a GTPase expression, activation and effects in LPS-mediated innate immune response and NF-κB activation

    doi: 10.1016/j.dib.2019.103965

    Figure Lengend Snippet: Expression analysis of Rap2a in LPS-treated RAW264.7 macrophages and human THP-1 monocytes. Non-quantitative RT-PCR and quantitative RT-qPCR analyses of Rap2a in RAW264.7 ( a, b ) and RAP2A in THP-1 ( c ) cells that were stimulated as indicated. Lower graph in ( a ) shows fold change expression after densitometric analysis of Rap2a:Gapdh ratio. Total RNA was extracted and subsequently transcribed into cDNA that was used as template in RT-qPCR reactions for detection of mRNAs. NT, not treated . NC, PCR negative control.

    Article Snippet: PCR-amplified cDNA fragment of 401-bp comprising the RBD-RalGDS mRNA coding sequence was cloned into pCR2.1 vector, and then a Bam HI- Eco RI fragment was obtained by digestion, purified and finally subcloned in pGEX-6P-1 vector (GE Healthcare).

    Techniques: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Negative Control