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Promega wizard sv gel and pcr clean up system
Wizard Sv Gel And Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/wizard sv gel and pcr clean up system/product/Promega
Average 97 stars, based on 101 article reviews
Price from $9.99 to $1999.99
wizard sv gel and pcr clean up system - by Bioz Stars, 2020-04
97/100 stars

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Real-time Polymerase Chain Reaction:

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882). ..

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882). ..

Concentration Assay:

Article Title: Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays
Article Snippet: .. The 183 × 12 + 60 and 188 × 12 DNA templates were separated on 1% agarose gels, extracted using Promega Wizard SV Gel and PCR Clean-Up System (catalog no. A9281), and ligated at low DNA concentration (1 ng/μl) to ensure circle formation rather than ligation of multiple linear fragments as described ( ). .. Ligation mixtures were concentrated and washed in 10 m m Tris-HCl (pH 7.5) using Amicon Ultra-4 Centrifugal Filter Units with a molecular mass cutoff of 100 kDa.

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega). .. Sample concentration was measured with the dsDNA HS Quibit fluorometer system (Life Technologies) and labeled using the ECL direct labeling kit according to manufacturer’s instructions (no. RPN3005; GE Healthcare).

Amplification:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: The probe was generated by PCR amplification of a 238-bp sequence from the canonical repeat cloned into the pBluescript KS+ (Stratagene, La Jolla, CA) vector. .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega).

Agarose Gel Electrophoresis:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega). .. Sample concentration was measured with the dsDNA HS Quibit fluorometer system (Life Technologies) and labeled using the ECL direct labeling kit according to manufacturer’s instructions (no. RPN3005; GE Healthcare).

Southern Blot:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: Paragraph title: Southern blotting ... Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega).

Magnetic Beads:

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: Following overnight incubation with antibodies / beads, 20 μl of Protein A+G magnetic beads (Millipore-Sigma, Cat no. 16-663) were added to each tube for pulling down Rsc2-AID*−9Myc and Ace1-HaloTag and rotated at 4°C for 2 h. The immunoprecipitated material was washed twice with 1 ml of IP wash buffer I (50 mM HEPESKOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100); once with 1 ml of IP wash buffer II (IP wash buffer I with 500 mM NaCl), once with 1 ml of IP wash buffer III (10 mM Tris pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and once with 1 ml of 1 × TE. .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882).

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: Following overnight incubation with antibodies / beads, 20 μl of Protein A+G magnetic beads (Millipore-Sigma, Cat no. 16-663) were added to each tube for pulling down Rsc2-AID*−9Myc and Ace1-HaloTag and rotated at 4°C for 2 h. The immunoprecipitated material was washed twice with 1 ml of IP wash buffer I (50 mM HEPESKOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100); once with 1 ml of IP wash buffer II (IP wash buffer I with 500 mM NaCl), once with 1 ml of IP wash buffer III (10 mM Tris pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and once with 1 ml of 1 × TE. .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882).

Isolation:

Article Title: Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays
Article Snippet: For circular DNA preparation, the plasmids were grown in 2 liters of Miller's LB media (Invitrogen catalog no. 12795-084) and isolated using four QIAfilter Plasmid Maxi Kits (Qiagen catalog no. 12262) on four columns as described in Qiagen manual. .. The 183 × 12 + 60 and 188 × 12 DNA templates were separated on 1% agarose gels, extracted using Promega Wizard SV Gel and PCR Clean-Up System (catalog no. A9281), and ligated at low DNA concentration (1 ng/μl) to ensure circle formation rather than ligation of multiple linear fragments as described ( ).

Ligation:

Article Title: Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays
Article Snippet: .. The 183 × 12 + 60 and 188 × 12 DNA templates were separated on 1% agarose gels, extracted using Promega Wizard SV Gel and PCR Clean-Up System (catalog no. A9281), and ligated at low DNA concentration (1 ng/μl) to ensure circle formation rather than ligation of multiple linear fragments as described ( ). .. Ligation mixtures were concentrated and washed in 10 m m Tris-HCl (pH 7.5) using Amicon Ultra-4 Centrifugal Filter Units with a molecular mass cutoff of 100 kDa.

Incubation:

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: As input control, 100 μl of WCE (stored at −20°C) was mixed with 50 μl of elution buffer and incubated overnight at 65°C for decrosslinking. .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882).

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: As input control, 100 μl of WCE (stored at −20°C) was mixed with 50 μl of elution buffer and incubated overnight at 65°C for decrosslinking. .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882).

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega). .. Labeled and 100-bp ladder probes (no N3231L; New England Biolabs) were added to blots at concentrations of 20 ng/ml and 10 ng/ml, respectively, in hybridization buffer and incubated overnight at 42°.

Labeling:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega). .. Sample concentration was measured with the dsDNA HS Quibit fluorometer system (Life Technologies) and labeled using the ECL direct labeling kit according to manufacturer’s instructions (no. RPN3005; GE Healthcare).

Purification:

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882). ..

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882). ..

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega). .. Sample concentration was measured with the dsDNA HS Quibit fluorometer system (Life Technologies) and labeled using the ECL direct labeling kit according to manufacturer’s instructions (no. RPN3005; GE Healthcare).

SYBR Green Assay:

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882). ..

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882). ..

Immunoprecipitation:

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: Following overnight incubation with antibodies / beads, 20 μl of Protein A+G magnetic beads (Millipore-Sigma, Cat no. 16-663) were added to each tube for pulling down Rsc2-AID*−9Myc and Ace1-HaloTag and rotated at 4°C for 2 h. The immunoprecipitated material was washed twice with 1 ml of IP wash buffer I (50 mM HEPESKOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100); once with 1 ml of IP wash buffer II (IP wash buffer I with 500 mM NaCl), once with 1 ml of IP wash buffer III (10 mM Tris pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and once with 1 ml of 1 × TE. .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882).

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: Following overnight incubation with antibodies / beads, 20 μl of Protein A+G magnetic beads (Millipore-Sigma, Cat no. 16-663) were added to each tube for pulling down Rsc2-AID*−9Myc and Ace1-HaloTag and rotated at 4°C for 2 h. The immunoprecipitated material was washed twice with 1 ml of IP wash buffer I (50 mM HEPESKOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100); once with 1 ml of IP wash buffer II (IP wash buffer I with 500 mM NaCl), once with 1 ml of IP wash buffer III (10 mM Tris pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) and once with 1 ml of 1 × TE. .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882).

Generated:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: The probe was generated by PCR amplification of a 238-bp sequence from the canonical repeat cloned into the pBluescript KS+ (Stratagene, La Jolla, CA) vector. .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega).

Polymerase Chain Reaction:

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882). ..

Article Title: Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays
Article Snippet: .. The 183 × 12 + 60 and 188 × 12 DNA templates were separated on 1% agarose gels, extracted using Promega Wizard SV Gel and PCR Clean-Up System (catalog no. A9281), and ligated at low DNA concentration (1 ng/μl) to ensure circle formation rather than ligation of multiple linear fragments as described ( ). .. Ligation mixtures were concentrated and washed in 10 m m Tris-HCl (pH 7.5) using Amicon Ultra-4 Centrifugal Filter Units with a molecular mass cutoff of 100 kDa.

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: .. DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882). ..

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega). .. Sample concentration was measured with the dsDNA HS Quibit fluorometer system (Life Technologies) and labeled using the ECL direct labeling kit according to manufacturer’s instructions (no. RPN3005; GE Healthcare).

Clone Assay:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: The probe was generated by PCR amplification of a 238-bp sequence from the canonical repeat cloned into the pBluescript KS+ (Stratagene, La Jolla, CA) vector. .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega).

Sequencing:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: The probe was generated by PCR amplification of a 238-bp sequence from the canonical repeat cloned into the pBluescript KS+ (Stratagene, La Jolla, CA) vector. .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega).

Molecular Cloning:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: The DNA was transferred overnight to a Hybond-N+ membrane (no. RPN203B; GE Healthcare) via capillary action, according to the protocol in Molecular Cloning: A Laboratory Manual ( ). .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega).

Chromatin Immunoprecipitation:

Article Title: Single-Molecule Analysis Reveals Linked Cycles Of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast
Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) ... DNA from all the samples (IPs and Inputs) were purified using Wizard SV Gel and PCR clean up system (Promega, Cat no. A9282) according to the manufacturer’s instructions and eluted in 50 μl of elution buffer. qPCR was performed in duplicate using a Bio-Rad CFX96 machine in 20 μl SYBR Green reaction mixture (Bio-Rad, USA, Cat no. 170-8882).

Plasmid Preparation:

Article Title: Nucleosome spacing periodically modulates nucleosome chain folding and DNA topology in circular nucleosome arrays
Article Snippet: Typical yield was 1 mg of plasmid DNA.). .. The 183 × 12 + 60 and 188 × 12 DNA templates were separated on 1% agarose gels, extracted using Promega Wizard SV Gel and PCR Clean-Up System (catalog no. A9281), and ligated at low DNA concentration (1 ng/μl) to ensure circle formation rather than ligation of multiple linear fragments as described ( ).

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: The vector-specific primers, T3 and T7, were used to amplify the probe ( ). .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega).

Hybridization:

Article Title: Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster
Article Snippet: The membrane was prehybridized in ECL Gold hybridization buffer (no RPN3006; GE Healthcare) in a Hybaid hybridization oven (Analytical Instruments) at 42° for 30 min. .. Product was run on a 0.5% agarose gel (SeaKem LE Agarose; Lonza) and the bands were cut and gel purified using the Wizard SV Gel and PCR Clean-Up System (no. A9282; Promega).

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    Promega pcr clean up system
    Establishment of conditional Ssu72-knockout DT40 cell lines. (A) Schematic representations of the chicken Ssu72 genomic fragment, knockout constructs, and configuration of the targeted alleles. Exons are shown as black boxes (E1–5), and the location of the 5′ probe used for Southern blotting is shown as a grey box. The double-headed arrows above the genes indicate length (in nucleotides). The XbaI and EcoRI restriction sites are indicated by vertical lines labeled X and R, respectively. (B) Southern blot analysis of wild-type (WT), heterozygous mutant (B15), homozygous mutant (P1, P2, P3), and unanticipated rearranged mutant (P10) clones. Genomic <t>DNA</t> obtained from each clone was digested with Xba I and Eco RI, and then hybridized with the 5′ probe shown in panel A. (C) <t>RT-PCR</t> analysis of the wild-type and mutant clones using primer pairs specific for the indicated gene. (D) Immunoblotting analysis of DT40 P3 (−/−) whole-cell extracts treated with Dox for the indicated times, using the indicated antibodies. Western blotting of β-actin was used as to confirm equal protein loading.
    Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up system/product/Promega
    Average 99 stars, based on 326 article reviews
    Price from $9.99 to $1999.99
    pcr clean up system - by Bioz Stars, 2020-04
    99/100 stars
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    Establishment of conditional Ssu72-knockout DT40 cell lines. (A) Schematic representations of the chicken Ssu72 genomic fragment, knockout constructs, and configuration of the targeted alleles. Exons are shown as black boxes (E1–5), and the location of the 5′ probe used for Southern blotting is shown as a grey box. The double-headed arrows above the genes indicate length (in nucleotides). The XbaI and EcoRI restriction sites are indicated by vertical lines labeled X and R, respectively. (B) Southern blot analysis of wild-type (WT), heterozygous mutant (B15), homozygous mutant (P1, P2, P3), and unanticipated rearranged mutant (P10) clones. Genomic DNA obtained from each clone was digested with Xba I and Eco RI, and then hybridized with the 5′ probe shown in panel A. (C) RT-PCR analysis of the wild-type and mutant clones using primer pairs specific for the indicated gene. (D) Immunoblotting analysis of DT40 P3 (−/−) whole-cell extracts treated with Dox for the indicated times, using the indicated antibodies. Western blotting of β-actin was used as to confirm equal protein loading.

    Journal: PLoS ONE

    Article Title: Vertebrate Ssu72 Regulates and Coordinates 3′-End Formation of RNAs Transcribed by RNA Polymerase II

    doi: 10.1371/journal.pone.0106040

    Figure Lengend Snippet: Establishment of conditional Ssu72-knockout DT40 cell lines. (A) Schematic representations of the chicken Ssu72 genomic fragment, knockout constructs, and configuration of the targeted alleles. Exons are shown as black boxes (E1–5), and the location of the 5′ probe used for Southern blotting is shown as a grey box. The double-headed arrows above the genes indicate length (in nucleotides). The XbaI and EcoRI restriction sites are indicated by vertical lines labeled X and R, respectively. (B) Southern blot analysis of wild-type (WT), heterozygous mutant (B15), homozygous mutant (P1, P2, P3), and unanticipated rearranged mutant (P10) clones. Genomic DNA obtained from each clone was digested with Xba I and Eco RI, and then hybridized with the 5′ probe shown in panel A. (C) RT-PCR analysis of the wild-type and mutant clones using primer pairs specific for the indicated gene. (D) Immunoblotting analysis of DT40 P3 (−/−) whole-cell extracts treated with Dox for the indicated times, using the indicated antibodies. Western blotting of β-actin was used as to confirm equal protein loading.

    Article Snippet: DNA fragments were purified using Wizard SV Gel and the PCR Clean-Up System (Promega), and subjected to RT-PCR using SYBR Premix Ex Taq II (TaKaRa) on an Mx3000P Real-time PCR system.

    Techniques: Knock-Out, Construct, Southern Blot, Labeling, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Western Blot

    Cay1 and Rap1 genetically interact to maintain telomere homeostasis A Telomere length analysis of Apa I-digested DNA from cay1 Δ trt1 Δ cells harvested at increasing generation doublings (gen). B Telomere length analysis of Apa I-digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control. C PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1-arrested by nitrogen starvation (G1). Genomic DNA was digested with Not I and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused). D Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains. E qRT–PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1 + mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t -test. ** P

    Journal: The EMBO Journal

    Article Title: Fission yeast Cactin restricts telomere transcription and elongation by controlling Rap1 levels

    doi: 10.15252/embj.201489559

    Figure Lengend Snippet: Cay1 and Rap1 genetically interact to maintain telomere homeostasis A Telomere length analysis of Apa I-digested DNA from cay1 Δ trt1 Δ cells harvested at increasing generation doublings (gen). B Telomere length analysis of Apa I-digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control. C PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1-arrested by nitrogen starvation (G1). Genomic DNA was digested with Not I and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused). D Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains. E qRT–PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1 + mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t -test. ** P

    Article Snippet: Immunoprecipitated chromatin was eluted in elution buffer (1% SDS, 100 mM sodium bicarbonate, 40 mg/ml RNase A) and incubated at 37°C for 1 h. Cross-links were reversed at 65°C for 16 h and DNA purified with the Wizard SV Gel and PCR Clean-Up System (Promega).

    Techniques: Northern Blot, Quantitative RT-PCR, Two Tailed Test

    Agarose gels showing the outcome of control PCR experiments. (A) DNA fragments of 5′ end of the myosin chitin synthase 1 ( mcs1 ) were amplified using primers CC-125 and CC-117 (see Table 1 ). In all three preparations, no PCR fragment was found in the absence of template (control), whereas strong bands of 707 bp appeared after PCR on total RNA preparations (lanes 3 and 4). These bands were not present when RNA which had been pre-treated with DNase I to remove contaminating genomic DNA (lanes 5 and 6). After transcribing this purified RNA into cDNA, PCR product of 585 bp appeared confirming the splicing of 122 bp predicted intron. The absence of 122 bp intron on the cDNA product was further confirmed by cDNA sequencing. Note that (1/10) and (1/50) indicate dilutions (1/10: 1 part RNA, 9 parts water; 1/50: 1 part RNA, 49 parts water). (B) Random amplification of yeast colonies with match maker PCR mix generated products with maximum sizes of 2000 bp in all three cDNA libraries (only IPO323_Yeasts shown). This suggests that entire open reading frames of proteins, up to ∼600–700 aa long, are represented in the library. Note that PCRs designed to amplify shorter fragments (585 bp and 1544 bp) of the chitin synthase gene mcs1 (5568 bp without introns) still produced positive bands (see main text). This suggests that fragments of larger genes are also represented in the libraries. (C) Primers were designed to amplify the entire open reading frame of the small GTPases rab7 (815 bp) and rab11 (807 bp) (see Table 1 , rab7 : primers SK-Sep-63 and SK-Sep-64; rab11 : primers SK-Sep-65 and SK-Sep-66). Both open reading frames were amplified from genomic DNA of IPO323. Smaller fragments (615 bp and 633 bp) were found after PCR reactions using cDNA from all three preparations (IPO323_Yeasts, IPO323_Hyphae, K4418_mixed). This corresponds with the predicted presence of introns in both genes ( rab7 : 815 bp; rab11 : 807 bp; see main text for more details) and further confirmed by DNA sequencing.

    Journal: Fungal Genetics and Biology

    Article Title: Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici

    doi: 10.1016/j.fgb.2015.03.023

    Figure Lengend Snippet: Agarose gels showing the outcome of control PCR experiments. (A) DNA fragments of 5′ end of the myosin chitin synthase 1 ( mcs1 ) were amplified using primers CC-125 and CC-117 (see Table 1 ). In all three preparations, no PCR fragment was found in the absence of template (control), whereas strong bands of 707 bp appeared after PCR on total RNA preparations (lanes 3 and 4). These bands were not present when RNA which had been pre-treated with DNase I to remove contaminating genomic DNA (lanes 5 and 6). After transcribing this purified RNA into cDNA, PCR product of 585 bp appeared confirming the splicing of 122 bp predicted intron. The absence of 122 bp intron on the cDNA product was further confirmed by cDNA sequencing. Note that (1/10) and (1/50) indicate dilutions (1/10: 1 part RNA, 9 parts water; 1/50: 1 part RNA, 49 parts water). (B) Random amplification of yeast colonies with match maker PCR mix generated products with maximum sizes of 2000 bp in all three cDNA libraries (only IPO323_Yeasts shown). This suggests that entire open reading frames of proteins, up to ∼600–700 aa long, are represented in the library. Note that PCRs designed to amplify shorter fragments (585 bp and 1544 bp) of the chitin synthase gene mcs1 (5568 bp without introns) still produced positive bands (see main text). This suggests that fragments of larger genes are also represented in the libraries. (C) Primers were designed to amplify the entire open reading frame of the small GTPases rab7 (815 bp) and rab11 (807 bp) (see Table 1 , rab7 : primers SK-Sep-63 and SK-Sep-64; rab11 : primers SK-Sep-65 and SK-Sep-66). Both open reading frames were amplified from genomic DNA of IPO323. Smaller fragments (615 bp and 633 bp) were found after PCR reactions using cDNA from all three preparations (IPO323_Yeasts, IPO323_Hyphae, K4418_mixed). This corresponds with the predicted presence of introns in both genes ( rab7 : 815 bp; rab11 : 807 bp; see main text for more details) and further confirmed by DNA sequencing.

    Article Snippet: The plasmid DNA was further purified by using Wizard® SV Gel and PCR Clean-Up System (Promega, Southampton, UK) and DNA sequencing was carried out at GATC biotech (GATC biotech, Cologne, Germany).

    Techniques: Polymerase Chain Reaction, Amplification, Purification, Sequencing, Generated, Produced, DNA Sequencing

    Identification of osKaR insertion site Arbitrary-primed PCR (AP-PCR) is a quick method to precisely identify the genomic region where a transposon has inserted. The following is specific for osKaR insertions: genomic DNA of the osKaR mutant is extracted (see Basic Protocol 1) and used for a semi-random PCR using the osKaR -specific primer oPCR1 and the random primer Deg3. The Deg3 primer consists of an 11-nucleotide random primer (in blue) with a 25-nucleotide specific tail (in red). The resulting PCR product is purified and used for a 2 nd PCR using the osKaR -specific primer Anchor1 and the Deg3-tail specific primer Deg4. The resulting PCR product is purified and DNA sequencing is performed using the osKaR -specific primer Anchor2.

    Journal: Current protocols in microbiology

    Article Title: Genetic Manipulation of Streptococcus pyogenes (The Group A Streptococcus, GAS)

    doi: 10.1002/9780471729259.mc09d03s30

    Figure Lengend Snippet: Identification of osKaR insertion site Arbitrary-primed PCR (AP-PCR) is a quick method to precisely identify the genomic region where a transposon has inserted. The following is specific for osKaR insertions: genomic DNA of the osKaR mutant is extracted (see Basic Protocol 1) and used for a semi-random PCR using the osKaR -specific primer oPCR1 and the random primer Deg3. The Deg3 primer consists of an 11-nucleotide random primer (in blue) with a 25-nucleotide specific tail (in red). The resulting PCR product is purified and used for a 2 nd PCR using the osKaR -specific primer Anchor1 and the Deg3-tail specific primer Deg4. The resulting PCR product is purified and DNA sequencing is performed using the osKaR -specific primer Anchor2.

    Article Snippet: Genomic DNA of GAS osKaR mutant (see Basic Protocol 1) Primers oPCR1, Deg3, Anchor1, Deg4 and Anchor2 (see recipe) Reagents and equipment for PCR Gel and PCR Clean-Up System kit (Wizard SV, Promega Cat. No. A9282)

    Techniques: Polymerase Chain Reaction, Mutagenesis, Purification, DNA Sequencing