Structured Review

Stratagene pcr strategy
<t>HIV-1</t> mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step <t>PCR</t> amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
Pcr Strategy, supplied by Stratagene, used in various techniques. Bioz Stars score: 89/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcr strategy/product/Stratagene
Average 89 stars, based on 19 article reviews
Price from $9.99 to $1999.99
pcr strategy - by Bioz Stars, 2020-07
89/100 stars

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1) Product Images from "High-throughput profiling of point mutations across the HIV-1 genome"

Article Title: High-throughput profiling of point mutations across the HIV-1 genome

Journal: Retrovirology

doi: 10.1186/s12977-014-0124-6

HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.
Figure Legend Snippet: HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.

Techniques Used: Mutagenesis, Next-Generation Sequencing, Sample Prep, Polymerase Chain Reaction, Amplification, Concentration Assay

Related Articles

Clone Assay:

Article Title: Replication Stalling at Friedreich's Ataxia (GAA)n Repeats In Vivo
Article Snippet: .. A starting (GAA)57 · (TTC)57 repeat was generated from complementary oligonucleotides d(GAA)10 and d(TTC)10 , using the PCR strategy described in reference , and cloned into the Eco RV site of pBluescript SK(−) plasmid (Stratagene). .. Plasmid pYES-TTC57 was obtained by inserting the blunt-ended Eco R1- Hin dIII fragment from pBluescript-GAA57 into the blunt-ended Xho I-site of pYES+.

Article Title: Fatty acids regulate perilipin5 in muscle by activating PPAR? [S]
Article Snippet: .. The full-length mouse Plin5 promoter (−2324/+244) was amplified by a PCR strategy described previously , cloned into pPCR-Script (Stratagene), digested out using Hind III, and inserted into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). .. Site-directed mutagenesis of the DR-1 element was performed with PCR as described previously ( ).

Amplification:

Article Title: High-throughput profiling of point mutations across the HIV-1 genome
Article Snippet: .. Viral mutant library preparation To generate the HIV-1 mutant library we designed a PCR strategy utilizing the HIV-1 proviral DNA plasmid pNL4-3 as template and the error-prone polymerase Mutazyme II (Strategene) to generate the point mutations during PCR amplification. ..

Article Title: Fatty acids regulate perilipin5 in muscle by activating PPAR? [S]
Article Snippet: .. The full-length mouse Plin5 promoter (−2324/+244) was amplified by a PCR strategy described previously , cloned into pPCR-Script (Stratagene), digested out using Hind III, and inserted into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). .. Site-directed mutagenesis of the DR-1 element was performed with PCR as described previously ( ).

Article Title: Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism
Article Snippet: .. Construction of the N-terminally GFP-tagged PTHR (GFP N-PTHR) was performed following a PCR strategy using pfu DNA polymerase (Stratagene). cDNA encoding the N-terminal fragment of human PTHR (corresponding to amino acids 1-60) was amplified with primers containing restriction sites for EcoRI (5′) and BamHI (3′). .. EGFP's cDNA from Clontech was amplified without stop codon with primers containing restriction sites for BamHI (5′) and KpnI (3′).

Mutagenesis:

Article Title: Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor
Article Snippet: .. Point mutations in the VFTM of human T1R2 were made by the same overlapping PCR strategy or by site-directed mutagenesis (Stratagene). .. The integrity of all DNA constructs was confirmed by sequencing.

Article Title: A Thioester Substrate Binds to the Enzyme Arthrobacter Thioesterase in Two Ionization States; Evidence from Raman Difference Spectroscopy
Article Snippet: .. [ ] Mutagenesis was carried out using a PCR strategy [ ] based on the WT-Arthio/pET-23b plasmid as template, commercial primers, and with the PCR kit supplied by Stratagene, and the Power Block IITM System thermal cycler manufactured by ERICOMP. .. PCR-amplified DNAs were cloned into pET-23b vector (Novagen) for expression in E. coli BL21(DE3).

Article Title: High-throughput profiling of point mutations across the HIV-1 genome
Article Snippet: .. Viral mutant library preparation To generate the HIV-1 mutant library we designed a PCR strategy utilizing the HIV-1 proviral DNA plasmid pNL4-3 as template and the error-prone polymerase Mutazyme II (Strategene) to generate the point mutations during PCR amplification. ..

Article Title: Functional variants in HCN4 and CACNA1H may contribute to genetic generalized epilepsy
Article Snippet: .. Site‐directed mutagenesis in HCN4 and Cav3.2 was performed using overlap PCR strategy or Quick Change kit (Stratagene). .. In vitro synthesis of HCN4 wild type (WT) and mutant cRNAs was performed using the mMessage mMachine T7 transcription kit (Ambion).

Blocking Assay:

Article Title: A Thioester Substrate Binds to the Enzyme Arthrobacter Thioesterase in Two Ionization States; Evidence from Raman Difference Spectroscopy
Article Snippet: .. [ ] Mutagenesis was carried out using a PCR strategy [ ] based on the WT-Arthio/pET-23b plasmid as template, commercial primers, and with the PCR kit supplied by Stratagene, and the Power Block IITM System thermal cycler manufactured by ERICOMP. .. PCR-amplified DNAs were cloned into pET-23b vector (Novagen) for expression in E. coli BL21(DE3).

Generated:

Article Title: Replication Stalling at Friedreich's Ataxia (GAA)n Repeats In Vivo
Article Snippet: .. A starting (GAA)57 · (TTC)57 repeat was generated from complementary oligonucleotides d(GAA)10 and d(TTC)10 , using the PCR strategy described in reference , and cloned into the Eco RV site of pBluescript SK(−) plasmid (Stratagene). .. Plasmid pYES-TTC57 was obtained by inserting the blunt-ended Eco R1- Hin dIII fragment from pBluescript-GAA57 into the blunt-ended Xho I-site of pYES+.

Article Title: P1 and P2 protein heterodimer binding to the P0 protein of Saccharomyces cerevisiae is relatively non-specific and a source of ribosomal heterogeneity
Article Snippet: .. The constructs were generated by an overlapping PCR strategy (Quickchange II site-directed mutagenesis kit: Stratagene) using BS-P0 as the template and the appropriate oligonucleotide primers ( Supplementary Table S3 ). .. The RPP0 gene was removed from BS-P0 as a BamHI–XhoI DNA fragment of 2.8–2.6 kbp, depending on the specific mutations incorporated, and inserted into either the BamHI–SalI sites of pFL36(LEU2) or the EcoRI–SalI sites of pFL37(HIS3), pFL38(URA3) and pFL39(TRP1) , depending on the genetic markers available in the strain to be transformed.

Luciferase:

Article Title: Fatty acids regulate perilipin5 in muscle by activating PPAR? [S]
Article Snippet: .. The full-length mouse Plin5 promoter (−2324/+244) was amplified by a PCR strategy described previously , cloned into pPCR-Script (Stratagene), digested out using Hind III, and inserted into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). .. Site-directed mutagenesis of the DR-1 element was performed with PCR as described previously ( ).

Construct:

Article Title: P1 and P2 protein heterodimer binding to the P0 protein of Saccharomyces cerevisiae is relatively non-specific and a source of ribosomal heterogeneity
Article Snippet: .. The constructs were generated by an overlapping PCR strategy (Quickchange II site-directed mutagenesis kit: Stratagene) using BS-P0 as the template and the appropriate oligonucleotide primers ( Supplementary Table S3 ). .. The RPP0 gene was removed from BS-P0 as a BamHI–XhoI DNA fragment of 2.8–2.6 kbp, depending on the specific mutations incorporated, and inserted into either the BamHI–SalI sites of pFL36(LEU2) or the EcoRI–SalI sites of pFL37(HIS3), pFL38(URA3) and pFL39(TRP1) , depending on the genetic markers available in the strain to be transformed.

Polymerase Chain Reaction:

Article Title: Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor
Article Snippet: .. Point mutations in the VFTM of human T1R2 were made by the same overlapping PCR strategy or by site-directed mutagenesis (Stratagene). .. The integrity of all DNA constructs was confirmed by sequencing.

Article Title: A Thioester Substrate Binds to the Enzyme Arthrobacter Thioesterase in Two Ionization States; Evidence from Raman Difference Spectroscopy
Article Snippet: .. [ ] Mutagenesis was carried out using a PCR strategy [ ] based on the WT-Arthio/pET-23b plasmid as template, commercial primers, and with the PCR kit supplied by Stratagene, and the Power Block IITM System thermal cycler manufactured by ERICOMP. .. PCR-amplified DNAs were cloned into pET-23b vector (Novagen) for expression in E. coli BL21(DE3).

Article Title: Replication Stalling at Friedreich's Ataxia (GAA)n Repeats In Vivo
Article Snippet: .. A starting (GAA)57 · (TTC)57 repeat was generated from complementary oligonucleotides d(GAA)10 and d(TTC)10 , using the PCR strategy described in reference , and cloned into the Eco RV site of pBluescript SK(−) plasmid (Stratagene). .. Plasmid pYES-TTC57 was obtained by inserting the blunt-ended Eco R1- Hin dIII fragment from pBluescript-GAA57 into the blunt-ended Xho I-site of pYES+.

Article Title: High-throughput profiling of point mutations across the HIV-1 genome
Article Snippet: .. Viral mutant library preparation To generate the HIV-1 mutant library we designed a PCR strategy utilizing the HIV-1 proviral DNA plasmid pNL4-3 as template and the error-prone polymerase Mutazyme II (Strategene) to generate the point mutations during PCR amplification. ..

Article Title: Fatty acids regulate perilipin5 in muscle by activating PPAR? [S]
Article Snippet: .. The full-length mouse Plin5 promoter (−2324/+244) was amplified by a PCR strategy described previously , cloned into pPCR-Script (Stratagene), digested out using Hind III, and inserted into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). .. Site-directed mutagenesis of the DR-1 element was performed with PCR as described previously ( ).

Article Title: Functional variants in HCN4 and CACNA1H may contribute to genetic generalized epilepsy
Article Snippet: .. Site‐directed mutagenesis in HCN4 and Cav3.2 was performed using overlap PCR strategy or Quick Change kit (Stratagene). .. In vitro synthesis of HCN4 wild type (WT) and mutant cRNAs was performed using the mMessage mMachine T7 transcription kit (Ambion).

Article Title: Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism
Article Snippet: .. Construction of the N-terminally GFP-tagged PTHR (GFP N-PTHR) was performed following a PCR strategy using pfu DNA polymerase (Stratagene). cDNA encoding the N-terminal fragment of human PTHR (corresponding to amino acids 1-60) was amplified with primers containing restriction sites for EcoRI (5′) and BamHI (3′). .. EGFP's cDNA from Clontech was amplified without stop codon with primers containing restriction sites for BamHI (5′) and KpnI (3′).

Plasmid Preparation:

Article Title: A Thioester Substrate Binds to the Enzyme Arthrobacter Thioesterase in Two Ionization States; Evidence from Raman Difference Spectroscopy
Article Snippet: .. [ ] Mutagenesis was carried out using a PCR strategy [ ] based on the WT-Arthio/pET-23b plasmid as template, commercial primers, and with the PCR kit supplied by Stratagene, and the Power Block IITM System thermal cycler manufactured by ERICOMP. .. PCR-amplified DNAs were cloned into pET-23b vector (Novagen) for expression in E. coli BL21(DE3).

Article Title: Replication Stalling at Friedreich's Ataxia (GAA)n Repeats In Vivo
Article Snippet: .. A starting (GAA)57 · (TTC)57 repeat was generated from complementary oligonucleotides d(GAA)10 and d(TTC)10 , using the PCR strategy described in reference , and cloned into the Eco RV site of pBluescript SK(−) plasmid (Stratagene). .. Plasmid pYES-TTC57 was obtained by inserting the blunt-ended Eco R1- Hin dIII fragment from pBluescript-GAA57 into the blunt-ended Xho I-site of pYES+.

Article Title: High-throughput profiling of point mutations across the HIV-1 genome
Article Snippet: .. Viral mutant library preparation To generate the HIV-1 mutant library we designed a PCR strategy utilizing the HIV-1 proviral DNA plasmid pNL4-3 as template and the error-prone polymerase Mutazyme II (Strategene) to generate the point mutations during PCR amplification. ..

Article Title: Fatty acids regulate perilipin5 in muscle by activating PPAR? [S]
Article Snippet: .. The full-length mouse Plin5 promoter (−2324/+244) was amplified by a PCR strategy described previously , cloned into pPCR-Script (Stratagene), digested out using Hind III, and inserted into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). .. Site-directed mutagenesis of the DR-1 element was performed with PCR as described previously ( ).

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    Stratagene quick change pcr mutagenesis strategies
    The KRAB–KAP-1 repression system targets <t>SETDB1</t> and enhances H3-K9 methylation and HP1 recruitment to promoters of transcriptionally silenced genes. ( A ) Schematic representation of a two-plasmid system used to create a stably integrated luciferase transgene in NIH/3T3 cells that is regulated by a heterologous KRAB repressor protein. Numbered arrow sets represent the relative position of <t>PCR</t> primers used for PCR amplification of DNA retained by ChIP. ( B ) Two single-cell subclones containing the heterologous KRAB–PAX3–HBD transcriptional repressor and the integrated luciferase transgene, which is either expressed (cl-49) or stably silenced (cl-74) following hormone treatment. Luciferase activities were measured in subconfluent populations of cells and reported as relative light units per milligram of protein. ( C ) ChIP experiments showing the colocalization of KAP-1 and SETDB1 at the TK promoter region of the luciferase transgene in the cells where transcription of the luciferase gene has been stably silenced (cl-74). Formaldehyde cross-linked chromatin from cl-49 and cl-74 cells was immunoprecipitated with either affinity-purified KAP-1 or SETDB1 IgG. An equal amount of promoter sequence in cl-49 and cl-74 nucleosomal preparations was determined by PCR from 1% of the input chromatin. PCR-amplified DNA fragments are illustrated in A . cl-2 represents a negative control cell line. ( D ) ChIPs of cross-linked chromatin with KAP-1, SETDB1, HP1α, and MeK9 antiserum as in C . Bold numbers below each lane represent quantitation of amplified DNA, expressed as percentage of signal intensity for the amplified input DNA.
    Quick Change Pcr Mutagenesis Strategies, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quick change pcr mutagenesis strategies/product/Stratagene
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    quick change pcr mutagenesis strategies - by Bioz Stars, 2020-07
    85/100 stars
      Buy from Supplier

    90
    Stratagene pcr based strategy
    Changes in <t>PGC-1α</t> expression and acetylation after chronic exercise. A : Real-time <t>PCR</t> analyses of PGC-1 α mRNA expression in the skeletal muscle of C57BL/6J wild-type mice (black columns) and ob / ob mice (gray columns). All data are normalized per 18S rRNA. B : Densitometry of protein data. Homogenates of skeletal muscle were probed with an antibody detecting PGC-1α (90 kDa). Blots were also probed with GAPDH as a loading control. C : Densitometry of immunoprecipitation (IP) experiments performed on skeletal muscle lysates, using PGC-1α for precipitation and an antibody directed against acetyl-lysine or PGC-1α for detection. Data are given as acetylated PGC-1α per total PGC-1α in these samples. All data are means ± SEM. * P
    Pcr Based Strategy, supplied by Stratagene, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based strategy/product/Stratagene
    Average 90 stars, based on 22 article reviews
    Price from $9.99 to $1999.99
    pcr based strategy - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

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    The KRAB–KAP-1 repression system targets SETDB1 and enhances H3-K9 methylation and HP1 recruitment to promoters of transcriptionally silenced genes. ( A ) Schematic representation of a two-plasmid system used to create a stably integrated luciferase transgene in NIH/3T3 cells that is regulated by a heterologous KRAB repressor protein. Numbered arrow sets represent the relative position of PCR primers used for PCR amplification of DNA retained by ChIP. ( B ) Two single-cell subclones containing the heterologous KRAB–PAX3–HBD transcriptional repressor and the integrated luciferase transgene, which is either expressed (cl-49) or stably silenced (cl-74) following hormone treatment. Luciferase activities were measured in subconfluent populations of cells and reported as relative light units per milligram of protein. ( C ) ChIP experiments showing the colocalization of KAP-1 and SETDB1 at the TK promoter region of the luciferase transgene in the cells where transcription of the luciferase gene has been stably silenced (cl-74). Formaldehyde cross-linked chromatin from cl-49 and cl-74 cells was immunoprecipitated with either affinity-purified KAP-1 or SETDB1 IgG. An equal amount of promoter sequence in cl-49 and cl-74 nucleosomal preparations was determined by PCR from 1% of the input chromatin. PCR-amplified DNA fragments are illustrated in A . cl-2 represents a negative control cell line. ( D ) ChIPs of cross-linked chromatin with KAP-1, SETDB1, HP1α, and MeK9 antiserum as in C . Bold numbers below each lane represent quantitation of amplified DNA, expressed as percentage of signal intensity for the amplified input DNA.

    Journal: Genes & Development

    Article Title: SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins

    doi: 10.1101/gad.973302

    Figure Lengend Snippet: The KRAB–KAP-1 repression system targets SETDB1 and enhances H3-K9 methylation and HP1 recruitment to promoters of transcriptionally silenced genes. ( A ) Schematic representation of a two-plasmid system used to create a stably integrated luciferase transgene in NIH/3T3 cells that is regulated by a heterologous KRAB repressor protein. Numbered arrow sets represent the relative position of PCR primers used for PCR amplification of DNA retained by ChIP. ( B ) Two single-cell subclones containing the heterologous KRAB–PAX3–HBD transcriptional repressor and the integrated luciferase transgene, which is either expressed (cl-49) or stably silenced (cl-74) following hormone treatment. Luciferase activities were measured in subconfluent populations of cells and reported as relative light units per milligram of protein. ( C ) ChIP experiments showing the colocalization of KAP-1 and SETDB1 at the TK promoter region of the luciferase transgene in the cells where transcription of the luciferase gene has been stably silenced (cl-74). Formaldehyde cross-linked chromatin from cl-49 and cl-74 cells was immunoprecipitated with either affinity-purified KAP-1 or SETDB1 IgG. An equal amount of promoter sequence in cl-49 and cl-74 nucleosomal preparations was determined by PCR from 1% of the input chromatin. PCR-amplified DNA fragments are illustrated in A . cl-2 represents a negative control cell line. ( D ) ChIPs of cross-linked chromatin with KAP-1, SETDB1, HP1α, and MeK9 antiserum as in C . Bold numbers below each lane represent quantitation of amplified DNA, expressed as percentage of signal intensity for the amplified input DNA.

    Article Snippet: Amino acid substitutions in SETDB1 (R643V, CC 729, 731 LP, H1224K, C1226A, C1279Y) were created using Quick Change PCR mutagenesis strategies (Stratagene).

    Techniques: Methylation, Plasmid Preparation, Stable Transfection, Luciferase, Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation, Immunoprecipitation, Affinity Purification, Sequencing, Negative Control, Quantitation Assay

    GILZ is expressed upon stimulation with C3 exotoxin or toxin B. (A) HeLa cells were incubated with toxin B (50ng/ml) for indicated the time spans and immunoblots were performed from whole cell lysates using anti-Rac1 mAb102 recognizing only non-glucosylated Rac-1, and an anti-Rac1 mAb antibody recognizing total Rac1 as well as antibodies recognizing RhoB, actin or GILZ. (B) Immunoblot of GILZ and actin expression upon stimulation of HeLa cells with C2IN-C3lim (100 ng/mL) + C2IIa (200 ng/mL) for indicated time spans. (C) To explore the expression of additional GILZ isoforms, HeLa cells were transfected with 7.5 nM siRNA specific for GILZ or control siRNA for 48 h and subsequently either left untreated or stimulated with C. difficile toxin B (50 ng/ml) or 100 µM DEX for 4 h. Arrows mark three GILZ isoforms which were inhibited by the used GILZ siRNA. Note that only isoform 1 was induced by the used stimuli. (D) Cells were stimulated with toxin B for 2 or 4 h to determine GILZ mRNA expression by real-time RT-PCR. Mean + SD of 2 independent experiments normalized to untreated. (E) To assay transcriptional activity of the GILZ promoter cells were transfected with a luciferase reporter under control of a 2088 bp GILZ promoter and co-transfected with pCMV-ß-gal (for standardization) 24 h before infection with a Y. enterocolitica pYV + and various mutant strains or treatment with DEX or toxin B. Means + SD of 4 independent experiments normalized to untreated. Significant differences compared to untreated are indicated by asterisks (p

    Journal: PLoS ONE

    Article Title: Yersinia enterocolitica YopT and Clostridium difficile Toxin B Induce Expression of GILZ in Epithelial Cells

    doi: 10.1371/journal.pone.0040730

    Figure Lengend Snippet: GILZ is expressed upon stimulation with C3 exotoxin or toxin B. (A) HeLa cells were incubated with toxin B (50ng/ml) for indicated the time spans and immunoblots were performed from whole cell lysates using anti-Rac1 mAb102 recognizing only non-glucosylated Rac-1, and an anti-Rac1 mAb antibody recognizing total Rac1 as well as antibodies recognizing RhoB, actin or GILZ. (B) Immunoblot of GILZ and actin expression upon stimulation of HeLa cells with C2IN-C3lim (100 ng/mL) + C2IIa (200 ng/mL) for indicated time spans. (C) To explore the expression of additional GILZ isoforms, HeLa cells were transfected with 7.5 nM siRNA specific for GILZ or control siRNA for 48 h and subsequently either left untreated or stimulated with C. difficile toxin B (50 ng/ml) or 100 µM DEX for 4 h. Arrows mark three GILZ isoforms which were inhibited by the used GILZ siRNA. Note that only isoform 1 was induced by the used stimuli. (D) Cells were stimulated with toxin B for 2 or 4 h to determine GILZ mRNA expression by real-time RT-PCR. Mean + SD of 2 independent experiments normalized to untreated. (E) To assay transcriptional activity of the GILZ promoter cells were transfected with a luciferase reporter under control of a 2088 bp GILZ promoter and co-transfected with pCMV-ß-gal (for standardization) 24 h before infection with a Y. enterocolitica pYV + and various mutant strains or treatment with DEX or toxin B. Means + SD of 4 independent experiments normalized to untreated. Significant differences compared to untreated are indicated by asterisks (p

    Article Snippet: Point mutations were introduced into the plasmid p1940-Luc by the Quick change PCR based strategy (Stratagene, Cedar Creek, USA) to generate the additional constructs using the following phosphorylated primers: c-myc 1-mut, 5′-GAC GCA GCC GGC TCC TCC-3′, 5′-CCG GGG CGT CAG GGG CCA TGC-3; c-myc 2 mut, 5′GTC CAG GGA GTA TGA CAT GGG AG-3′, 5′-CCG GGC CCT CAC CAT CAC G-3′, Oct-1a, 5′-TGC ATG CCC CTG ACG CTG-3′, 5′-GGC GAG TCC TGT ACC GGG CTT TGT GG-3′; Oct-1b 5′ TGT ATT TCT TAT TTC TCT AGA AAT CAG CTC CAG-3′.

    Techniques: Incubation, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Activity Assay, Luciferase, Infection, Mutagenesis

    Changes in PGC-1α expression and acetylation after chronic exercise. A : Real-time PCR analyses of PGC-1 α mRNA expression in the skeletal muscle of C57BL/6J wild-type mice (black columns) and ob / ob mice (gray columns). All data are normalized per 18S rRNA. B : Densitometry of protein data. Homogenates of skeletal muscle were probed with an antibody detecting PGC-1α (90 kDa). Blots were also probed with GAPDH as a loading control. C : Densitometry of immunoprecipitation (IP) experiments performed on skeletal muscle lysates, using PGC-1α for precipitation and an antibody directed against acetyl-lysine or PGC-1α for detection. Data are given as acetylated PGC-1α per total PGC-1α in these samples. All data are means ± SEM. * P

    Journal: Diabetes

    Article Title: Mitochondrial Biogenesis and Peroxisome Proliferator-Activated Receptor-? Coactivator-1? (PGC-1?) Deacetylation by Physical Activity

    doi: 10.2337/db10-0331

    Figure Lengend Snippet: Changes in PGC-1α expression and acetylation after chronic exercise. A : Real-time PCR analyses of PGC-1 α mRNA expression in the skeletal muscle of C57BL/6J wild-type mice (black columns) and ob / ob mice (gray columns). All data are normalized per 18S rRNA. B : Densitometry of protein data. Homogenates of skeletal muscle were probed with an antibody detecting PGC-1α (90 kDa). Blots were also probed with GAPDH as a loading control. C : Densitometry of immunoprecipitation (IP) experiments performed on skeletal muscle lysates, using PGC-1α for precipitation and an antibody directed against acetyl-lysine or PGC-1α for detection. Data are given as acetylated PGC-1α per total PGC-1α in these samples. All data are means ± SEM. * P

    Article Snippet: Site-directed mutations at Thr177 and Ser538 in PGC-1α were introduced by a PCR-based strategy with the QuikChange site-directed mutagenesis kit (Stratagene).

    Techniques: Pyrolysis Gas Chromatography, Expressing, Real-time Polymerase Chain Reaction, Mouse Assay, Immunoprecipitation

    HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.

    Journal: Retrovirology

    Article Title: High-throughput profiling of point mutations across the HIV-1 genome

    doi: 10.1186/s12977-014-0124-6

    Figure Lengend Snippet: HIV-1 mutant library and NGS sample preparation. (A) Genomic region for each HIV-1 mutation library. (B) The two-step PCR amplicon approach for NGS sample preparation. Virion cDNA from each mutant viral population cell passage is used as template for a HIV-1 specific staggered PCR step that uses primers specific to the HIV-1 mutagenized region containing overhangs with a complex 10 ‘N’ nucleotide tag with two keto “K” or amino “M” nucleotide positions that identify the specific fragment and population, respectively. PCR products from step one are pooled, the amplicon molecule concentration is accurately measured, and then decreased for error correction. The pooled sample is then used as template for a second PCR using a single primer set containing the remainder of the Illumina adapter region for NGS.

    Article Snippet: Viral mutant library preparation To generate the HIV-1 mutant library we designed a PCR strategy utilizing the HIV-1 proviral DNA plasmid pNL4-3 as template and the error-prone polymerase Mutazyme II (Strategene) to generate the point mutations during PCR amplification.

    Techniques: Mutagenesis, Next-Generation Sequencing, Sample Prep, Polymerase Chain Reaction, Amplification, Concentration Assay