qiagen qiaquick columns  (Qiagen)

 
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    Name:
    QIAquick PCR Purification Kit
    Description:
    For purification of up to 10 μg PCR products 100 bp to 10 kb Kit contents Qiagen QIAquick PCR Purification Kit 50 rxns 30L Elution Volume 10g Binding Capacity Tube Format Manual Processing Silica Technology 100 bp to 10 kb Fragment 40mers Fragments Removed Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion Labeling Microinjection PCR and in vitro Transcription For Purification of up to 10μg PCR Products Includes 50 QIAquick Spin Columns Buffers 2mL Collection Tubes Benefits Up to 95 recovery of ready to use DNA Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
    Catalog Number:
    28104
    Price:
    117
    Category:
    QIAquick PCR Purification Kit
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    Structured Review

    Qiagen qiagen qiaquick columns
    QIAquick PCR Purification Kit
    For purification of up to 10 μg PCR products 100 bp to 10 kb Kit contents Qiagen QIAquick PCR Purification Kit 50 rxns 30L Elution Volume 10g Binding Capacity Tube Format Manual Processing Silica Technology 100 bp to 10 kb Fragment 40mers Fragments Removed Ideal for Sequencing Microarray Analysis Ligation and Transformation Restriction Digestion Labeling Microinjection PCR and in vitro Transcription For Purification of up to 10μg PCR Products Includes 50 QIAquick Spin Columns Buffers 2mL Collection Tubes Benefits Up to 95 recovery of ready to use DNA Cleanup of DNA up to 10 kb in three easy steps Gel loading dye for convenient sample analysis
    https://www.bioz.com/result/qiagen qiaquick columns/product/Qiagen
    Average 99 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    qiagen qiaquick columns - by Bioz Stars, 2020-09
    99/100 stars

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    Related Articles

    DNA Extraction:

    Article Title: Epigenetic Regulation of HYAL-1 Hyaluronidase Expression
    Article Snippet: .. Genomic DNA isolation kit, QIAquick® PCR purification kit, EpiTect®Bisulfite kit, and Effectene® transfection reagent were from Qiagen (Valencia, CA). .. MG132 was purchased from EMD Biosciences Inc. (San Diego, CA), and human genomic DNA was obtained from Clontech.

    Transfection:

    Article Title: Epigenetic Regulation of HYAL-1 Hyaluronidase Expression
    Article Snippet: .. Genomic DNA isolation kit, QIAquick® PCR purification kit, EpiTect®Bisulfite kit, and Effectene® transfection reagent were from Qiagen (Valencia, CA). .. MG132 was purchased from EMD Biosciences Inc. (San Diego, CA), and human genomic DNA was obtained from Clontech.

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes
    Article Snippet: .. The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific). .. After 16 hours, puromycin selection was performed for 48 hours.

    Polymerase Chain Reaction:

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination
    Article Snippet: .. Immunoprecipitated bead–DNA complex was treated with proteinase K for 3 h at 50 °C in elution buffer and hydroxymethylated DNA purified by using the Qiagen PCR clean-up kit (Qiaquick). ..

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: .. Amplicons were confirmed by agarose electrophoresis, and purified with PCR purification kit (QIAGEN Cat.28104). .. 200 ng DNA samples were denatured and annealed according to the protocol in Ran et al . and subjected to T7E1 digestion in a 20 ul reaction according to the manufacturer’s protocol (NEB cat.M0302).

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins
    Article Snippet: .. In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104). ..

    Article Title: Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma
    Article Snippet: .. ChIP DNA was purified using a Quick PCR Purification Kit (Qiagen). ..

    Article Title: Epigenetic Regulation of HYAL-1 Hyaluronidase Expression
    Article Snippet: .. Genomic DNA isolation kit, QIAquick® PCR purification kit, EpiTect®Bisulfite kit, and Effectene® transfection reagent were from Qiagen (Valencia, CA). .. MG132 was purchased from EMD Biosciences Inc. (San Diego, CA), and human genomic DNA was obtained from Clontech.

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes
    Article Snippet: .. The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific). .. After 16 hours, puromycin selection was performed for 48 hours.

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits
    Article Snippet: .. Kit extractions We tested three different silica-column kits: Zymo ZR Viral DNA/RNA Kit (outdated protocol, D7021), Zymo Quick-DNA/RNA Kit (updated protocol, D7021), and the QIAquick PCR Purification Kit (28104, Qiagen). .. For all silica-column kits, fresh collection tubes were used after each spin and centrifugation speeds were set to 16,000 × g. Centrifugation was performed on either an Eppendorf 5415D centrifuge (Eppendorf, Hauppauge, NY, USA) or a Thermo Fisher Scientific AccuSpin Micro 17 R centrifuge (13–100–676).

    Article Title: Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins
    Article Snippet: .. After elution of immune complexes, they were heated at 65°C for 4 h to reverse crosslinks, and the DNA was recovered with a "QIAquick" PCR purification kit from Qiagen Inc. (Valencia, CA). .. Primary amplification of AR DNA was across the polymorphic 5' CAG repeat region as previously described [ ], using 27–35 cycles of PCR amplification with 10% of the immunoprecipitated material or 50 ng of input DNA in a 50 μl reaction volume.

    Purification:

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination
    Article Snippet: .. Immunoprecipitated bead–DNA complex was treated with proteinase K for 3 h at 50 °C in elution buffer and hydroxymethylated DNA purified by using the Qiagen PCR clean-up kit (Qiaquick). ..

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: .. Amplicons were confirmed by agarose electrophoresis, and purified with PCR purification kit (QIAGEN Cat.28104). .. 200 ng DNA samples were denatured and annealed according to the protocol in Ran et al . and subjected to T7E1 digestion in a 20 ul reaction according to the manufacturer’s protocol (NEB cat.M0302).

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins
    Article Snippet: .. In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104). ..

    Article Title: Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma
    Article Snippet: .. ChIP DNA was purified using a Quick PCR Purification Kit (Qiagen). ..

    Article Title: Epigenetic Regulation of HYAL-1 Hyaluronidase Expression
    Article Snippet: .. Genomic DNA isolation kit, QIAquick® PCR purification kit, EpiTect®Bisulfite kit, and Effectene® transfection reagent were from Qiagen (Valencia, CA). .. MG132 was purchased from EMD Biosciences Inc. (San Diego, CA), and human genomic DNA was obtained from Clontech.

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes
    Article Snippet: .. The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific). .. After 16 hours, puromycin selection was performed for 48 hours.

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits
    Article Snippet: .. Kit extractions We tested three different silica-column kits: Zymo ZR Viral DNA/RNA Kit (outdated protocol, D7021), Zymo Quick-DNA/RNA Kit (updated protocol, D7021), and the QIAquick PCR Purification Kit (28104, Qiagen). .. For all silica-column kits, fresh collection tubes were used after each spin and centrifugation speeds were set to 16,000 × g. Centrifugation was performed on either an Eppendorf 5415D centrifuge (Eppendorf, Hauppauge, NY, USA) or a Thermo Fisher Scientific AccuSpin Micro 17 R centrifuge (13–100–676).

    Article Title: Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins
    Article Snippet: .. After elution of immune complexes, they were heated at 65°C for 4 h to reverse crosslinks, and the DNA was recovered with a "QIAquick" PCR purification kit from Qiagen Inc. (Valencia, CA). .. Primary amplification of AR DNA was across the polymorphic 5' CAG repeat region as previously described [ ], using 27–35 cycles of PCR amplification with 10% of the immunoprecipitated material or 50 ng of input DNA in a 50 μl reaction volume.

    Electrophoresis:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: .. Amplicons were confirmed by agarose electrophoresis, and purified with PCR purification kit (QIAGEN Cat.28104). .. 200 ng DNA samples were denatured and annealed according to the protocol in Ran et al . and subjected to T7E1 digestion in a 20 ul reaction according to the manufacturer’s protocol (NEB cat.M0302).

    Immunoprecipitation:

    Article Title: Specific functions of TET1 and TET2 in regulating mesenchymal cell lineage determination
    Article Snippet: .. Immunoprecipitated bead–DNA complex was treated with proteinase K for 3 h at 50 °C in elution buffer and hydroxymethylated DNA purified by using the Qiagen PCR clean-up kit (Qiaquick). ..

    Incubation:

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins
    Article Snippet: .. In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104). ..

    Expressing:

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes
    Article Snippet: .. The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific). .. After 16 hours, puromycin selection was performed for 48 hours.

    Knock-In:

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes
    Article Snippet: .. The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific). .. After 16 hours, puromycin selection was performed for 48 hours.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Streamlined procedure for gene knockouts using all-in-one adenoviral CRISPR-Cas9
    Article Snippet: .. Amplicons were confirmed by agarose electrophoresis, and purified with PCR purification kit (QIAGEN Cat.28104). .. 200 ng DNA samples were denatured and annealed according to the protocol in Ran et al . and subjected to T7E1 digestion in a 20 ul reaction according to the manufacturer’s protocol (NEB cat.M0302).

    Chromatin Immunoprecipitation:

    Article Title: Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma
    Article Snippet: .. ChIP DNA was purified using a Quick PCR Purification Kit (Qiagen). ..

    Plasmid Preparation:

    Article Title: Orphan CpG islands boost the regulatory activity of poised enhancers and dictate the responsiveness of their target genes
    Article Snippet: .. The resulting PCR product was purified using QIAgen PCR purification columns (28104, QIAgen). mESC were transfected with the sgRNA-Cas9 expressing vector and the knock-in donor using Lipofectamine according to the manufacturer protocol (Thermo Scientific). .. After 16 hours, puromycin selection was performed for 48 hours.

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    Qiagen pcr purification column
    Impact of Xrcc4 deletion on Tus/ Ter -induced and I-SceI-induced HR. A , Schematic of 6x Ter -HR reporter and HR repair products of Tus- Ter -induced fork stalling. Green box: wt GFP . Grey boxes: mutant GFP . Open ovals A and B: 5’ and 3’ artificial RFP exons. 5’Tr- GFP : 5’-truncated GFP . Orange triangle: 6x Ter element array. Navy blue line: I-SceI endonuclease cut site. STGC, LTGC: short tract and long tract gene conversion HR repair outcomes. LTGC generates wt RFP through RNA splicing (red filled ovals). B , Xrcc4 gene structure in Xrcc4 fl/fl ES cells. Xrcc4 Δ / Δ allele lacks exon 3. Black triangles: loxP sites. Grey boxes: Xrcc4 Exons 2–4. Location and direction of Exon3 genotyping primers a, a’, and b as indicated by arrows. Gel: <t>PCR</t> products for Xrcc4 fl/fl ES clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13. C , RT <t>qPCR</t> analysis of Xrcc4 expression in Xrcc4 fl/fl or Xrcc4 Δ / Δ clones. Xrcc4 expression normalized to GAPDH and displayed as fold difference from Xrcc4 fl/fl clone 8 of the same experiment (x = -2 ΔΔCt , with ΔΔCt = [Ct Xrcc4 -Ct Gapdh ]-[Ct Xrcc4 -Ct GAPDH ]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV Xrcc4 2 + SDEV GAPDH 2 ]). Xrcc4 abundance by Western blot in Xrcc4 fl/fl clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13 cell protein extracts. D , Representative primary FACS data for two Xrcc4 fl/fl and two Xrcc4 Δ/Δ 6x Ter -HR reporter clones, as indicated, transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. FACS plots produced from pooled data of duplicate samples from three independent experiments. Numbers represent percentages. E , Frequencies of Tus/ Ter -induced and I-SceI-induced repair in five independently derived Xrcc4 fl/fl (orange triangles, red squares) or Xrcc4 Δ/Δ (blue diamonds, navy blue circles) 6x Ter . Error bars: standard error of the mean (s.e.m.). One-way ANOVA (Analysis of Variance) test comparing trend in HR between five Xrcc4 fl/fl and five Xrcc4 Δ/Δ clones: Tus-induced HR, total HR, p = 0.0017; STGC, p = 0.0015; LTGC, p = 0.7142; LTGC/(Total HR), p = 0.2636. I-SceI-induced HR, total HR, p
    Pcr Purification Column, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 649 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr purification column/product/Qiagen
    Average 99 stars, based on 649 article reviews
    Price from $9.99 to $1999.99
    pcr purification column - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

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    Impact of Xrcc4 deletion on Tus/ Ter -induced and I-SceI-induced HR. A , Schematic of 6x Ter -HR reporter and HR repair products of Tus- Ter -induced fork stalling. Green box: wt GFP . Grey boxes: mutant GFP . Open ovals A and B: 5’ and 3’ artificial RFP exons. 5’Tr- GFP : 5’-truncated GFP . Orange triangle: 6x Ter element array. Navy blue line: I-SceI endonuclease cut site. STGC, LTGC: short tract and long tract gene conversion HR repair outcomes. LTGC generates wt RFP through RNA splicing (red filled ovals). B , Xrcc4 gene structure in Xrcc4 fl/fl ES cells. Xrcc4 Δ / Δ allele lacks exon 3. Black triangles: loxP sites. Grey boxes: Xrcc4 Exons 2–4. Location and direction of Exon3 genotyping primers a, a’, and b as indicated by arrows. Gel: PCR products for Xrcc4 fl/fl ES clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13. C , RT qPCR analysis of Xrcc4 expression in Xrcc4 fl/fl or Xrcc4 Δ / Δ clones. Xrcc4 expression normalized to GAPDH and displayed as fold difference from Xrcc4 fl/fl clone 8 of the same experiment (x = -2 ΔΔCt , with ΔΔCt = [Ct Xrcc4 -Ct Gapdh ]-[Ct Xrcc4 -Ct GAPDH ]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV Xrcc4 2 + SDEV GAPDH 2 ]). Xrcc4 abundance by Western blot in Xrcc4 fl/fl clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13 cell protein extracts. D , Representative primary FACS data for two Xrcc4 fl/fl and two Xrcc4 Δ/Δ 6x Ter -HR reporter clones, as indicated, transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. FACS plots produced from pooled data of duplicate samples from three independent experiments. Numbers represent percentages. E , Frequencies of Tus/ Ter -induced and I-SceI-induced repair in five independently derived Xrcc4 fl/fl (orange triangles, red squares) or Xrcc4 Δ/Δ (blue diamonds, navy blue circles) 6x Ter . Error bars: standard error of the mean (s.e.m.). One-way ANOVA (Analysis of Variance) test comparing trend in HR between five Xrcc4 fl/fl and five Xrcc4 Δ/Δ clones: Tus-induced HR, total HR, p = 0.0017; STGC, p = 0.0015; LTGC, p = 0.7142; LTGC/(Total HR), p = 0.2636. I-SceI-induced HR, total HR, p

    Journal: PLoS Genetics

    Article Title: Rad51 recruitment and exclusion of non-homologous end joining during homologous recombination at a Tus/Ter mammalian replication fork barrier

    doi: 10.1371/journal.pgen.1007486

    Figure Lengend Snippet: Impact of Xrcc4 deletion on Tus/ Ter -induced and I-SceI-induced HR. A , Schematic of 6x Ter -HR reporter and HR repair products of Tus- Ter -induced fork stalling. Green box: wt GFP . Grey boxes: mutant GFP . Open ovals A and B: 5’ and 3’ artificial RFP exons. 5’Tr- GFP : 5’-truncated GFP . Orange triangle: 6x Ter element array. Navy blue line: I-SceI endonuclease cut site. STGC, LTGC: short tract and long tract gene conversion HR repair outcomes. LTGC generates wt RFP through RNA splicing (red filled ovals). B , Xrcc4 gene structure in Xrcc4 fl/fl ES cells. Xrcc4 Δ / Δ allele lacks exon 3. Black triangles: loxP sites. Grey boxes: Xrcc4 Exons 2–4. Location and direction of Exon3 genotyping primers a, a’, and b as indicated by arrows. Gel: PCR products for Xrcc4 fl/fl ES clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13. C , RT qPCR analysis of Xrcc4 expression in Xrcc4 fl/fl or Xrcc4 Δ / Δ clones. Xrcc4 expression normalized to GAPDH and displayed as fold difference from Xrcc4 fl/fl clone 8 of the same experiment (x = -2 ΔΔCt , with ΔΔCt = [Ct Xrcc4 -Ct Gapdh ]-[Ct Xrcc4 -Ct GAPDH ]). Error-bars represent standard deviation of the ΔCt value (SDEV = √[SDEV Xrcc4 2 + SDEV GAPDH 2 ]). Xrcc4 abundance by Western blot in Xrcc4 fl/fl clones 8 and 39, and Xrcc4 Δ / Δ clones 11 and 13 cell protein extracts. D , Representative primary FACS data for two Xrcc4 fl/fl and two Xrcc4 Δ/Δ 6x Ter -HR reporter clones, as indicated, transfected with empty, 3xMyc-NLS Tus or 3xMyc-NLS I-SceI expression vectors. FACS plots produced from pooled data of duplicate samples from three independent experiments. Numbers represent percentages. E , Frequencies of Tus/ Ter -induced and I-SceI-induced repair in five independently derived Xrcc4 fl/fl (orange triangles, red squares) or Xrcc4 Δ/Δ (blue diamonds, navy blue circles) 6x Ter . Error bars: standard error of the mean (s.e.m.). One-way ANOVA (Analysis of Variance) test comparing trend in HR between five Xrcc4 fl/fl and five Xrcc4 Δ/Δ clones: Tus-induced HR, total HR, p = 0.0017; STGC, p = 0.0015; LTGC, p = 0.7142; LTGC/(Total HR), p = 0.2636. I-SceI-induced HR, total HR, p

    Article Snippet: DNA purified by Qiagen PCR Purification column (Qiagen, 28106) was analyzed by qPCR using an ABI Prism 7300 sequence detection system and SYBR Green (Applied Biosystems, 4368702).

    Techniques: Mutagenesis, Polymerase Chain Reaction, Clone Assay, Quantitative RT-PCR, Expressing, Standard Deviation, Western Blot, FACS, Transfection, Produced, Derivative Assay

    Comparison of selected treatments on the visualization of RPA-generated amplicons. a Results using primer pair TYL828F/TYL834R from crude extracts prepared in 0.5 N NaOH from Tomato yellow leaf curl virus (TYLCV)-infected tomato leaves and cleaned by heating to 65 °C for 15 min. Lane M: 50 bp ladder MW standard, size is indicated in kilobases (kb); Lane 1 fresh tissue; Lane 2 tissue kept frozen at −20 °C for 5 months; Lane 3 tissue kept frozen at −80 °C for 3 months; Lane 4 desiccated tissue maintained at room temperature for 15 years; Lane 5 non-inoculated tissue kept frozen at −80 °C for 3 months. b Detection of TYLCV from crude extracts prepared in 0.5 N NaOH and cleaned as follows: Lane 1 untreated; Lane 2 QIAquick PCR purification column; Lane 3 heated at 65 °C for 10 min; Lane 4 heated at 95 °C for 10 min; Lane 5 amplicon loading buffer contained 5 % SDS; Lane 6 amplicon loading buffer contained 10 % SDS; Lane 7 amplicon loading buffer contained 5 % formamide; Lane 8 amplicon loading buffer contained 15 % formamide. Ten μl of amplified product were loaded into each lane of the 1.5 % agarose gels and stained with ethidium bromide

    Journal: Virology Journal

    Article Title: Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics

    doi: 10.1186/s12985-016-0504-8

    Figure Lengend Snippet: Comparison of selected treatments on the visualization of RPA-generated amplicons. a Results using primer pair TYL828F/TYL834R from crude extracts prepared in 0.5 N NaOH from Tomato yellow leaf curl virus (TYLCV)-infected tomato leaves and cleaned by heating to 65 °C for 15 min. Lane M: 50 bp ladder MW standard, size is indicated in kilobases (kb); Lane 1 fresh tissue; Lane 2 tissue kept frozen at −20 °C for 5 months; Lane 3 tissue kept frozen at −80 °C for 3 months; Lane 4 desiccated tissue maintained at room temperature for 15 years; Lane 5 non-inoculated tissue kept frozen at −80 °C for 3 months. b Detection of TYLCV from crude extracts prepared in 0.5 N NaOH and cleaned as follows: Lane 1 untreated; Lane 2 QIAquick PCR purification column; Lane 3 heated at 65 °C for 10 min; Lane 4 heated at 95 °C for 10 min; Lane 5 amplicon loading buffer contained 5 % SDS; Lane 6 amplicon loading buffer contained 10 % SDS; Lane 7 amplicon loading buffer contained 5 % formamide; Lane 8 amplicon loading buffer contained 15 % formamide. Ten μl of amplified product were loaded into each lane of the 1.5 % agarose gels and stained with ethidium bromide

    Article Snippet: Proteins were separated from the amplified DNA (“cleaned”) with either QIAquick PCR purification columns (Qiagen, Valencia, CA) or using one of the methods described below.

    Techniques: Recombinase Polymerase Amplification, Generated, Infection, Polymerase Chain Reaction, Purification, Amplification, Staining

    Reduced H3K27me3 binding is detected by ChIP-qPCR. (A) ChIP was performed using chromatin from KARPAS-422 cells treated with the EZH2 inhibitor CPI-360. qPCR using the positive control primer MYT1 showed reduced H3K27me3 occupancy in the presence of the inhibitor. (B) ChIP was performed using chromatin from PC9 cells treated with the EZH2 inhibitor GSK126. qPCR using the positive control primer MYT1 showed reduced H3K27me3 occupancy in cells treated with the inhibitor. ( C ) Libraries were generated from KARPAS-422 cells using 15 cycles of PCR amplification. Library DNA was diluted and qPCR was performed using positive control primers for MYT1 and CCND2 . ( D ) Libraries were generated from PC9 cells as described in (C) and library DNA was used for qPCR using positive control primers for MYT1 and CCND2 . All experiments are represented as the mean of two independent experiments with qPCRs performed in triplicate ±SD. The ACTB promoter served as a negative control for all experiments.

    Journal: PLoS ONE

    Article Title: An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition

    doi: 10.1371/journal.pone.0166438

    Figure Lengend Snippet: Reduced H3K27me3 binding is detected by ChIP-qPCR. (A) ChIP was performed using chromatin from KARPAS-422 cells treated with the EZH2 inhibitor CPI-360. qPCR using the positive control primer MYT1 showed reduced H3K27me3 occupancy in the presence of the inhibitor. (B) ChIP was performed using chromatin from PC9 cells treated with the EZH2 inhibitor GSK126. qPCR using the positive control primer MYT1 showed reduced H3K27me3 occupancy in cells treated with the inhibitor. ( C ) Libraries were generated from KARPAS-422 cells using 15 cycles of PCR amplification. Library DNA was diluted and qPCR was performed using positive control primers for MYT1 and CCND2 . ( D ) Libraries were generated from PC9 cells as described in (C) and library DNA was used for qPCR using positive control primers for MYT1 and CCND2 . All experiments are represented as the mean of two independent experiments with qPCRs performed in triplicate ±SD. The ACTB promoter served as a negative control for all experiments.

    Article Snippet: Eluted chromatin was treated with 10 mg of RNAse A at 37°C for 1 h followed by proteinase K at 50°C for 2 h. Samples were then de-crosslinked at 65°C for 5 h. Reverse crosslinked DNA was purified by a PCR purification column (Qiaquick PCR purification, Qiagen 28106), and eluted with 150 μl of buffer EB (10 mM Tris-HCl pH 8) for qPCR.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Generated, Polymerase Chain Reaction, Amplification, Negative Control