Structured Review

MACHEREY NAGEL pcr cleanup kit
ARID3B interacts with the KSHV genome in a lytic reactivation-dependent manner. (A) <t>DNA</t> affinity assays performed according to the method in reference 25 ; biotinylated <t>PCR</t> products spanning oriLyt left of KSHV (GenBank accession number NC_009333.1 ) and control DNA amplified from the RTA coding region were bound to streptavidin-Dynabeads and incubated with lysates from reactivated 293T rKSHV.219 cells expressing FLAG-ARID3B. This suggested that ARID3B bound these sequences with preference for the A/T-rich region. IB, immunoblotting. (B) TREx-BCBL1-RTA cells were treated with doxycycline for 18 h (w/Dox) to reactivate the lytic cycle or were left untreated (w/o Dox). Samples were subjected to ChIP with an antibody to ARID3B or normal rabbit IgG and primers specific for sequences in the A/T-rich region of oriLyt or the RTA coding region. The percentage of output versus input DNA was calculated and is presented relative to normal rabbit IgG values from unreactivated (w/o Dox) cells (set to 1). Data represent two biological replicates (i.e., independent ChIPs) and two technical replicates per ChIP from a single experiment, and values are given as the mean ± standard deviation. Student t tests were used to determine statistical significance.
Pcr Cleanup Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcr cleanup kit/product/MACHEREY NAGEL
Average 95 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcr cleanup kit - by Bioz Stars, 2021-06
95/100 stars

Images

1) Product Images from "ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle"

Article Title: ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle

Journal: Journal of Virology

doi: 10.1128/JVI.03262-15

ARID3B interacts with the KSHV genome in a lytic reactivation-dependent manner. (A) DNA affinity assays performed according to the method in reference 25 ; biotinylated PCR products spanning oriLyt left of KSHV (GenBank accession number NC_009333.1 ) and control DNA amplified from the RTA coding region were bound to streptavidin-Dynabeads and incubated with lysates from reactivated 293T rKSHV.219 cells expressing FLAG-ARID3B. This suggested that ARID3B bound these sequences with preference for the A/T-rich region. IB, immunoblotting. (B) TREx-BCBL1-RTA cells were treated with doxycycline for 18 h (w/Dox) to reactivate the lytic cycle or were left untreated (w/o Dox). Samples were subjected to ChIP with an antibody to ARID3B or normal rabbit IgG and primers specific for sequences in the A/T-rich region of oriLyt or the RTA coding region. The percentage of output versus input DNA was calculated and is presented relative to normal rabbit IgG values from unreactivated (w/o Dox) cells (set to 1). Data represent two biological replicates (i.e., independent ChIPs) and two technical replicates per ChIP from a single experiment, and values are given as the mean ± standard deviation. Student t tests were used to determine statistical significance.
Figure Legend Snippet: ARID3B interacts with the KSHV genome in a lytic reactivation-dependent manner. (A) DNA affinity assays performed according to the method in reference 25 ; biotinylated PCR products spanning oriLyt left of KSHV (GenBank accession number NC_009333.1 ) and control DNA amplified from the RTA coding region were bound to streptavidin-Dynabeads and incubated with lysates from reactivated 293T rKSHV.219 cells expressing FLAG-ARID3B. This suggested that ARID3B bound these sequences with preference for the A/T-rich region. IB, immunoblotting. (B) TREx-BCBL1-RTA cells were treated with doxycycline for 18 h (w/Dox) to reactivate the lytic cycle or were left untreated (w/o Dox). Samples were subjected to ChIP with an antibody to ARID3B or normal rabbit IgG and primers specific for sequences in the A/T-rich region of oriLyt or the RTA coding region. The percentage of output versus input DNA was calculated and is presented relative to normal rabbit IgG values from unreactivated (w/o Dox) cells (set to 1). Data represent two biological replicates (i.e., independent ChIPs) and two technical replicates per ChIP from a single experiment, and values are given as the mean ± standard deviation. Student t tests were used to determine statistical significance.

Techniques Used: Polymerase Chain Reaction, Amplification, Incubation, Expressing, Chromatin Immunoprecipitation, Standard Deviation

2) Product Images from "Data on the optimization of an archaea-specific probe-based qPCR assay"

Article Title: Data on the optimization of an archaea-specific probe-based qPCR assay

Journal: Data in Brief

doi: 10.1016/j.dib.2020.106610

The detection of amplification by qPCR from A) genomic DNA and B) 16S rRNA gene amplicon, using annealing temperature 57 °C. In A) the orange bars show the amplification detected using fluorescent dye (Sybr) and the blue bars show the detection using a probe specific to archaeal 16S rRNA genes from 200 pg genomic DNA. In B) the detection was tested using a PCR amplicon as template in order to have equal number of targets in all reactions. The DNA concentration/reaction in B was approximately 0.01 pg/reaction and the probe was used for detection.
Figure Legend Snippet: The detection of amplification by qPCR from A) genomic DNA and B) 16S rRNA gene amplicon, using annealing temperature 57 °C. In A) the orange bars show the amplification detected using fluorescent dye (Sybr) and the blue bars show the detection using a probe specific to archaeal 16S rRNA genes from 200 pg genomic DNA. In B) the detection was tested using a PCR amplicon as template in order to have equal number of targets in all reactions. The DNA concentration/reaction in B was approximately 0.01 pg/reaction and the probe was used for detection.

Techniques Used: Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Concentration Assay

3) Product Images from "Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp."

Article Title: Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp.

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00485-19

Verification of the integration of pOSV802 in S. coelicolor M145, S. lividans TK23, and S. albus J1074 chromosomes. (A) Principle of the PCR verification of the integration of the pOSV801 to pOSV812 vectors in the Streptomyces chromosomes (PCR 1 and PCR 2) (PCR 3, PCR verification before excision of modules 1 to 3). (B) PCR fragments obtained by PCR 1 ( attL region; expected sizes, 913 bp for M145 and TK23, 888 bp for J1074) and by PCR 2 ( attR region; expected sizes, 911 bp for M145 and TK23, 907 bp for J1074) on the three Streptomyces strains bearing pOSV802. No PCR amplification is expected when the genomic DNA of the wild-type Streptomyces strains is used as the matrix. MW corresponds to the molecular weight ladder (Thermo Scientific GeneRuler DNA ladder mix).
Figure Legend Snippet: Verification of the integration of pOSV802 in S. coelicolor M145, S. lividans TK23, and S. albus J1074 chromosomes. (A) Principle of the PCR verification of the integration of the pOSV801 to pOSV812 vectors in the Streptomyces chromosomes (PCR 1 and PCR 2) (PCR 3, PCR verification before excision of modules 1 to 3). (B) PCR fragments obtained by PCR 1 ( attL region; expected sizes, 913 bp for M145 and TK23, 888 bp for J1074) and by PCR 2 ( attR region; expected sizes, 911 bp for M145 and TK23, 907 bp for J1074) on the three Streptomyces strains bearing pOSV802. No PCR amplification is expected when the genomic DNA of the wild-type Streptomyces strains is used as the matrix. MW corresponds to the molecular weight ladder (Thermo Scientific GeneRuler DNA ladder mix).

Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight

Related Articles

Purification:

Article Title: Drought Stress Results in a Compartment-Specific Restructuring of the Rice Root-Associated Microbiomes
Article Snippet: After running a 1% agarose gel to verify proper amplification, libraries were cleaned with AmPure XP magnetic beads (Beckman Coulter, Inc.), quantified (Qubit dsDNA HS assay kit; Thermo Fisher Scientific), and pooled in equimolar concentrations. .. Pooled libraries were then concentrated, gel purified (Nucleoscopic gel and PCR cleanup kit; Macherey-Nagel), quality checked (BioAnalyzer HS DNA kit; Agilent Technologies), and submitted for 2- by 250-bp Miseq sequencing (Illumina) to the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center (supported by NIH Shared Instrumentation Grant 1S10OD010786-01). .. Sequence processing for 16S.

Article Title: Association of metformin administration with gut microbiome dysbiosis in healthy volunteers
Article Snippet: Each primer contained IonXpress adapter sequence and a unique barcode sequence. .. The amplified PCR products were purified using NucleoMag magnetic beads (Macherey-Nagel, Düren, Germany), and their quantity and quality were evaluated with the Agilent 2100 Bioanalyzer DNA High Sensitivity chip (Agilent Technologies, Santa Clara, CA, USA). .. Sequencing of the amplicon libraries was performed with Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific; Ion 318 Chip Kit v2, Ion PGM Hi-Q Sequencing Kit, minimal sequencing depth per sample– 250 000 reads) according to the instructions of the manufacturer.

Article Title: EEF1A1 deacetylation enables transcriptional activation of remyelination
Article Snippet: Sonicated chromatin was incubated overnight a 4 °C on a rotating wheel, and Magna ChIP™ Protein A + Protein G Magnetic Beads (Millipore) were added and incubated for 2 h at 4 °C. .. Magnetic beads were washed, immunoprecipitated samples were eluted in elution buffer (50 mM Tris, 10 mM EDTA, 1% SDS), crosslinking was reversed at 65 °C for 8–16 h and after RNase and proteinase K treatment, the DNA was purified using a PCR clean-up kit (Macherey-Nagel). .. Quantitative real-time PCR analyses were performed with an ABI 7000 Sequence Detection System (Applied Biosystems) using FastStart SYBR Green Master (Sigma), according to the manufacturer’s recommendations.

Article Title: ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle
Article Snippet: Elution of the chromatin-antibody complexes was carried out by incubation with 150 μl freshly prepared elution buffer (100 mM NaHCO3 , 1% SDS) containing 1.5 μl proteinase K (Roche; catalog no. 03 115 887 001) at 62°C for 2 h, followed by a 10-min incubation step at 95°C. .. DNA was purified using the NucleoSpin gel and PCR cleanup kit from Macherey-Nagel (catalog no. 740609) according to their DNA cleanup protocol for samples containing SDS. .. DNA was eluted with 90 μl buffer NE, and 5 μl of the DNA solution was used as the template DNA for qPCR using primers specific for the A/T-rich region of oriLyt left (forward primer, 5′-CCCTCCTTTGTTTTCCGGAAG-3′; reverse primer, 5′CTCATCGGGCCCTATTATAAAG-3′) and the RTA coding region (forward primer, 5′-GTCTACCTTCCGAGGATTATGG-3′; reverse primer, 5′-GATTCTGGCATGAGACCGCTTC-3′).

Article Title: Large-scale nuclear remodeling and transcriptional deregulation occur on both derivative chromosomes after Mantle Cell Lymphoma chromosomal translocation
Article Snippet: The non-solubilized material was removed by centrifugation at 15,000g for 10 min. .. The desired size of chromatin fragments (100-500 bp) was monitored by electrophoresis in a 1% agarose gel after reverse crosslinking (dilution 1:4 in water and adding 0.250M of NaCl keeping at 65° overnight in a thermalcycler), treatment with 100µg/ml RNase A (37° for 15 minutes) and 50µg/ml proteinase K and purification using the PCR cleanup kit (Macherey-Nagel). .. Chromatin immunoprecipitation was performed as following: 25 µg of chromatin solution was incubated at 4° overnight with 25µl of the PrG-Dynabeads (Thermo Scientific),3µg of antibody (rabbit-anti Nucleolin, Sigma-Aldrich), ChIP Buffer I (Active Motif, La Hulpe, Belgium), 1X PIC and water.

Polymerase Chain Reaction:

Article Title: Drought Stress Results in a Compartment-Specific Restructuring of the Rice Root-Associated Microbiomes
Article Snippet: After running a 1% agarose gel to verify proper amplification, libraries were cleaned with AmPure XP magnetic beads (Beckman Coulter, Inc.), quantified (Qubit dsDNA HS assay kit; Thermo Fisher Scientific), and pooled in equimolar concentrations. .. Pooled libraries were then concentrated, gel purified (Nucleoscopic gel and PCR cleanup kit; Macherey-Nagel), quality checked (BioAnalyzer HS DNA kit; Agilent Technologies), and submitted for 2- by 250-bp Miseq sequencing (Illumina) to the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center (supported by NIH Shared Instrumentation Grant 1S10OD010786-01). .. Sequence processing for 16S.

Article Title: Association of metformin administration with gut microbiome dysbiosis in healthy volunteers
Article Snippet: Each primer contained IonXpress adapter sequence and a unique barcode sequence. .. The amplified PCR products were purified using NucleoMag magnetic beads (Macherey-Nagel, Düren, Germany), and their quantity and quality were evaluated with the Agilent 2100 Bioanalyzer DNA High Sensitivity chip (Agilent Technologies, Santa Clara, CA, USA). .. Sequencing of the amplicon libraries was performed with Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific; Ion 318 Chip Kit v2, Ion PGM Hi-Q Sequencing Kit, minimal sequencing depth per sample– 250 000 reads) according to the instructions of the manufacturer.

Article Title: EEF1A1 deacetylation enables transcriptional activation of remyelination
Article Snippet: Sonicated chromatin was incubated overnight a 4 °C on a rotating wheel, and Magna ChIP™ Protein A + Protein G Magnetic Beads (Millipore) were added and incubated for 2 h at 4 °C. .. Magnetic beads were washed, immunoprecipitated samples were eluted in elution buffer (50 mM Tris, 10 mM EDTA, 1% SDS), crosslinking was reversed at 65 °C for 8–16 h and after RNase and proteinase K treatment, the DNA was purified using a PCR clean-up kit (Macherey-Nagel). .. Quantitative real-time PCR analyses were performed with an ABI 7000 Sequence Detection System (Applied Biosystems) using FastStart SYBR Green Master (Sigma), according to the manufacturer’s recommendations.

Article Title: Multi-contact 4C: long-molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies.
Article Snippet: .. We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. .. We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites.

Article Title: Combined Analysis of Methylation and Gene Expression Profiles in Separate Compartments of Small Bowel Mucosa Identified Celiac Disease Patients’ Signatures
Article Snippet: Na-bisulfite-treated DNA samples were amplified by three different probes provided with the KAPA2G Fast HotStartReadyMix PCR Kit (KAPABIOSYSTEMS®).The amplified samples were analysed by electrophoresis on a 2% agarose gel. .. DNAclean upThe NucleoMag®96 PCR clean-up kit from MACHEREY-NAGEL® was used to purify the samples. .. SequencingThe processed samples were sent for next-generation high-throughput sequencing on an Illumina Nextera platform.

Article Title: ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle
Article Snippet: Elution of the chromatin-antibody complexes was carried out by incubation with 150 μl freshly prepared elution buffer (100 mM NaHCO3 , 1% SDS) containing 1.5 μl proteinase K (Roche; catalog no. 03 115 887 001) at 62°C for 2 h, followed by a 10-min incubation step at 95°C. .. DNA was purified using the NucleoSpin gel and PCR cleanup kit from Macherey-Nagel (catalog no. 740609) according to their DNA cleanup protocol for samples containing SDS. .. DNA was eluted with 90 μl buffer NE, and 5 μl of the DNA solution was used as the template DNA for qPCR using primers specific for the A/T-rich region of oriLyt left (forward primer, 5′-CCCTCCTTTGTTTTCCGGAAG-3′; reverse primer, 5′CTCATCGGGCCCTATTATAAAG-3′) and the RTA coding region (forward primer, 5′-GTCTACCTTCCGAGGATTATGG-3′; reverse primer, 5′-GATTCTGGCATGAGACCGCTTC-3′).

Article Title: Large-scale nuclear remodeling and transcriptional deregulation occur on both derivative chromosomes after Mantle Cell Lymphoma chromosomal translocation
Article Snippet: The non-solubilized material was removed by centrifugation at 15,000g for 10 min. .. The desired size of chromatin fragments (100-500 bp) was monitored by electrophoresis in a 1% agarose gel after reverse crosslinking (dilution 1:4 in water and adding 0.250M of NaCl keeping at 65° overnight in a thermalcycler), treatment with 100µg/ml RNase A (37° for 15 minutes) and 50µg/ml proteinase K and purification using the PCR cleanup kit (Macherey-Nagel). .. Chromatin immunoprecipitation was performed as following: 25 µg of chromatin solution was incubated at 4° overnight with 25µl of the PrG-Dynabeads (Thermo Scientific),3µg of antibody (rabbit-anti Nucleolin, Sigma-Aldrich), ChIP Buffer I (Active Motif, La Hulpe, Belgium), 1X PIC and water.

Sequencing:

Article Title: Drought Stress Results in a Compartment-Specific Restructuring of the Rice Root-Associated Microbiomes
Article Snippet: After running a 1% agarose gel to verify proper amplification, libraries were cleaned with AmPure XP magnetic beads (Beckman Coulter, Inc.), quantified (Qubit dsDNA HS assay kit; Thermo Fisher Scientific), and pooled in equimolar concentrations. .. Pooled libraries were then concentrated, gel purified (Nucleoscopic gel and PCR cleanup kit; Macherey-Nagel), quality checked (BioAnalyzer HS DNA kit; Agilent Technologies), and submitted for 2- by 250-bp Miseq sequencing (Illumina) to the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center (supported by NIH Shared Instrumentation Grant 1S10OD010786-01). .. Sequence processing for 16S.

Expressing:

Article Title: Drought Stress Results in a Compartment-Specific Restructuring of the Rice Root-Associated Microbiomes
Article Snippet: After running a 1% agarose gel to verify proper amplification, libraries were cleaned with AmPure XP magnetic beads (Beckman Coulter, Inc.), quantified (Qubit dsDNA HS assay kit; Thermo Fisher Scientific), and pooled in equimolar concentrations. .. Pooled libraries were then concentrated, gel purified (Nucleoscopic gel and PCR cleanup kit; Macherey-Nagel), quality checked (BioAnalyzer HS DNA kit; Agilent Technologies), and submitted for 2- by 250-bp Miseq sequencing (Illumina) to the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center (supported by NIH Shared Instrumentation Grant 1S10OD010786-01). .. Sequence processing for 16S.

Amplification:

Article Title: Association of metformin administration with gut microbiome dysbiosis in healthy volunteers
Article Snippet: Each primer contained IonXpress adapter sequence and a unique barcode sequence. .. The amplified PCR products were purified using NucleoMag magnetic beads (Macherey-Nagel, Düren, Germany), and their quantity and quality were evaluated with the Agilent 2100 Bioanalyzer DNA High Sensitivity chip (Agilent Technologies, Santa Clara, CA, USA). .. Sequencing of the amplicon libraries was performed with Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific; Ion 318 Chip Kit v2, Ion PGM Hi-Q Sequencing Kit, minimal sequencing depth per sample– 250 000 reads) according to the instructions of the manufacturer.

Magnetic Beads:

Article Title: Association of metformin administration with gut microbiome dysbiosis in healthy volunteers
Article Snippet: Each primer contained IonXpress adapter sequence and a unique barcode sequence. .. The amplified PCR products were purified using NucleoMag magnetic beads (Macherey-Nagel, Düren, Germany), and their quantity and quality were evaluated with the Agilent 2100 Bioanalyzer DNA High Sensitivity chip (Agilent Technologies, Santa Clara, CA, USA). .. Sequencing of the amplicon libraries was performed with Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific; Ion 318 Chip Kit v2, Ion PGM Hi-Q Sequencing Kit, minimal sequencing depth per sample– 250 000 reads) according to the instructions of the manufacturer.

Article Title: EEF1A1 deacetylation enables transcriptional activation of remyelination
Article Snippet: Sonicated chromatin was incubated overnight a 4 °C on a rotating wheel, and Magna ChIP™ Protein A + Protein G Magnetic Beads (Millipore) were added and incubated for 2 h at 4 °C. .. Magnetic beads were washed, immunoprecipitated samples were eluted in elution buffer (50 mM Tris, 10 mM EDTA, 1% SDS), crosslinking was reversed at 65 °C for 8–16 h and after RNase and proteinase K treatment, the DNA was purified using a PCR clean-up kit (Macherey-Nagel). .. Quantitative real-time PCR analyses were performed with an ABI 7000 Sequence Detection System (Applied Biosystems) using FastStart SYBR Green Master (Sigma), according to the manufacturer’s recommendations.

Chromatin Immunoprecipitation:

Article Title: Association of metformin administration with gut microbiome dysbiosis in healthy volunteers
Article Snippet: Each primer contained IonXpress adapter sequence and a unique barcode sequence. .. The amplified PCR products were purified using NucleoMag magnetic beads (Macherey-Nagel, Düren, Germany), and their quantity and quality were evaluated with the Agilent 2100 Bioanalyzer DNA High Sensitivity chip (Agilent Technologies, Santa Clara, CA, USA). .. Sequencing of the amplicon libraries was performed with Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific; Ion 318 Chip Kit v2, Ion PGM Hi-Q Sequencing Kit, minimal sequencing depth per sample– 250 000 reads) according to the instructions of the manufacturer.

Immunoprecipitation:

Article Title: EEF1A1 deacetylation enables transcriptional activation of remyelination
Article Snippet: Sonicated chromatin was incubated overnight a 4 °C on a rotating wheel, and Magna ChIP™ Protein A + Protein G Magnetic Beads (Millipore) were added and incubated for 2 h at 4 °C. .. Magnetic beads were washed, immunoprecipitated samples were eluted in elution buffer (50 mM Tris, 10 mM EDTA, 1% SDS), crosslinking was reversed at 65 °C for 8–16 h and after RNase and proteinase K treatment, the DNA was purified using a PCR clean-up kit (Macherey-Nagel). .. Quantitative real-time PCR analyses were performed with an ABI 7000 Sequence Detection System (Applied Biosystems) using FastStart SYBR Green Master (Sigma), according to the manufacturer’s recommendations.

Ligation:

Article Title: Multi-contact 4C: long-molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies.
Article Snippet: .. We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. .. We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites.

Electrophoresis:

Article Title: Large-scale nuclear remodeling and transcriptional deregulation occur on both derivative chromosomes after Mantle Cell Lymphoma chromosomal translocation
Article Snippet: The non-solubilized material was removed by centrifugation at 15,000g for 10 min. .. The desired size of chromatin fragments (100-500 bp) was monitored by electrophoresis in a 1% agarose gel after reverse crosslinking (dilution 1:4 in water and adding 0.250M of NaCl keeping at 65° overnight in a thermalcycler), treatment with 100µg/ml RNase A (37° for 15 minutes) and 50µg/ml proteinase K and purification using the PCR cleanup kit (Macherey-Nagel). .. Chromatin immunoprecipitation was performed as following: 25 µg of chromatin solution was incubated at 4° overnight with 25µl of the PrG-Dynabeads (Thermo Scientific),3µg of antibody (rabbit-anti Nucleolin, Sigma-Aldrich), ChIP Buffer I (Active Motif, La Hulpe, Belgium), 1X PIC and water.

Agarose Gel Electrophoresis:

Article Title: Large-scale nuclear remodeling and transcriptional deregulation occur on both derivative chromosomes after Mantle Cell Lymphoma chromosomal translocation
Article Snippet: The non-solubilized material was removed by centrifugation at 15,000g for 10 min. .. The desired size of chromatin fragments (100-500 bp) was monitored by electrophoresis in a 1% agarose gel after reverse crosslinking (dilution 1:4 in water and adding 0.250M of NaCl keeping at 65° overnight in a thermalcycler), treatment with 100µg/ml RNase A (37° for 15 minutes) and 50µg/ml proteinase K and purification using the PCR cleanup kit (Macherey-Nagel). .. Chromatin immunoprecipitation was performed as following: 25 µg of chromatin solution was incubated at 4° overnight with 25µl of the PrG-Dynabeads (Thermo Scientific),3µg of antibody (rabbit-anti Nucleolin, Sigma-Aldrich), ChIP Buffer I (Active Motif, La Hulpe, Belgium), 1X PIC and water.

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    MACHEREY NAGEL pcr cleanup kit
    ARID3B interacts with the KSHV genome in a lytic reactivation-dependent manner. (A) <t>DNA</t> affinity assays performed according to the method in reference 25 ; biotinylated <t>PCR</t> products spanning oriLyt left of KSHV (GenBank accession number NC_009333.1 ) and control DNA amplified from the RTA coding region were bound to streptavidin-Dynabeads and incubated with lysates from reactivated 293T rKSHV.219 cells expressing FLAG-ARID3B. This suggested that ARID3B bound these sequences with preference for the A/T-rich region. IB, immunoblotting. (B) TREx-BCBL1-RTA cells were treated with doxycycline for 18 h (w/Dox) to reactivate the lytic cycle or were left untreated (w/o Dox). Samples were subjected to ChIP with an antibody to ARID3B or normal rabbit IgG and primers specific for sequences in the A/T-rich region of oriLyt or the RTA coding region. The percentage of output versus input DNA was calculated and is presented relative to normal rabbit IgG values from unreactivated (w/o Dox) cells (set to 1). Data represent two biological replicates (i.e., independent ChIPs) and two technical replicates per ChIP from a single experiment, and values are given as the mean ± standard deviation. Student t tests were used to determine statistical significance.
    Pcr Cleanup Kit, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr cleanup kit/product/MACHEREY NAGEL
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr cleanup kit - by Bioz Stars, 2021-06
    95/100 stars
      Buy from Supplier

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    ARID3B interacts with the KSHV genome in a lytic reactivation-dependent manner. (A) DNA affinity assays performed according to the method in reference 25 ; biotinylated PCR products spanning oriLyt left of KSHV (GenBank accession number NC_009333.1 ) and control DNA amplified from the RTA coding region were bound to streptavidin-Dynabeads and incubated with lysates from reactivated 293T rKSHV.219 cells expressing FLAG-ARID3B. This suggested that ARID3B bound these sequences with preference for the A/T-rich region. IB, immunoblotting. (B) TREx-BCBL1-RTA cells were treated with doxycycline for 18 h (w/Dox) to reactivate the lytic cycle or were left untreated (w/o Dox). Samples were subjected to ChIP with an antibody to ARID3B or normal rabbit IgG and primers specific for sequences in the A/T-rich region of oriLyt or the RTA coding region. The percentage of output versus input DNA was calculated and is presented relative to normal rabbit IgG values from unreactivated (w/o Dox) cells (set to 1). Data represent two biological replicates (i.e., independent ChIPs) and two technical replicates per ChIP from a single experiment, and values are given as the mean ± standard deviation. Student t tests were used to determine statistical significance.

    Journal: Journal of Virology

    Article Title: ARID3B: a Novel Regulator of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle

    doi: 10.1128/JVI.03262-15

    Figure Lengend Snippet: ARID3B interacts with the KSHV genome in a lytic reactivation-dependent manner. (A) DNA affinity assays performed according to the method in reference 25 ; biotinylated PCR products spanning oriLyt left of KSHV (GenBank accession number NC_009333.1 ) and control DNA amplified from the RTA coding region were bound to streptavidin-Dynabeads and incubated with lysates from reactivated 293T rKSHV.219 cells expressing FLAG-ARID3B. This suggested that ARID3B bound these sequences with preference for the A/T-rich region. IB, immunoblotting. (B) TREx-BCBL1-RTA cells were treated with doxycycline for 18 h (w/Dox) to reactivate the lytic cycle or were left untreated (w/o Dox). Samples were subjected to ChIP with an antibody to ARID3B or normal rabbit IgG and primers specific for sequences in the A/T-rich region of oriLyt or the RTA coding region. The percentage of output versus input DNA was calculated and is presented relative to normal rabbit IgG values from unreactivated (w/o Dox) cells (set to 1). Data represent two biological replicates (i.e., independent ChIPs) and two technical replicates per ChIP from a single experiment, and values are given as the mean ± standard deviation. Student t tests were used to determine statistical significance.

    Article Snippet: DNA was purified using the NucleoSpin gel and PCR cleanup kit from Macherey-Nagel (catalog no. 740609) according to their DNA cleanup protocol for samples containing SDS.

    Techniques: Polymerase Chain Reaction, Amplification, Incubation, Expressing, Chromatin Immunoprecipitation, Standard Deviation

    The detection of amplification by qPCR from A) genomic DNA and B) 16S rRNA gene amplicon, using annealing temperature 57 °C. In A) the orange bars show the amplification detected using fluorescent dye (Sybr) and the blue bars show the detection using a probe specific to archaeal 16S rRNA genes from 200 pg genomic DNA. In B) the detection was tested using a PCR amplicon as template in order to have equal number of targets in all reactions. The DNA concentration/reaction in B was approximately 0.01 pg/reaction and the probe was used for detection.

    Journal: Data in Brief

    Article Title: Data on the optimization of an archaea-specific probe-based qPCR assay

    doi: 10.1016/j.dib.2020.106610

    Figure Lengend Snippet: The detection of amplification by qPCR from A) genomic DNA and B) 16S rRNA gene amplicon, using annealing temperature 57 °C. In A) the orange bars show the amplification detected using fluorescent dye (Sybr) and the blue bars show the detection using a probe specific to archaeal 16S rRNA genes from 200 pg genomic DNA. In B) the detection was tested using a PCR amplicon as template in order to have equal number of targets in all reactions. The DNA concentration/reaction in B was approximately 0.01 pg/reaction and the probe was used for detection.

    Article Snippet: The excised ca 800 bp PCR amplicon was purified from agarose using the NucleoSpin gel and PCR cleanup kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) and the DNA concentration of each preparate was set to approximately 10 pg/µL, of which 1 µL of 10−3 dilutions (i.e. 0.01 pg) were used for the qPCR test.

    Techniques: Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Concentration Assay

    Verification of the integration of pOSV802 in S. coelicolor M145, S. lividans TK23, and S. albus J1074 chromosomes. (A) Principle of the PCR verification of the integration of the pOSV801 to pOSV812 vectors in the Streptomyces chromosomes (PCR 1 and PCR 2) (PCR 3, PCR verification before excision of modules 1 to 3). (B) PCR fragments obtained by PCR 1 ( attL region; expected sizes, 913 bp for M145 and TK23, 888 bp for J1074) and by PCR 2 ( attR region; expected sizes, 911 bp for M145 and TK23, 907 bp for J1074) on the three Streptomyces strains bearing pOSV802. No PCR amplification is expected when the genomic DNA of the wild-type Streptomyces strains is used as the matrix. MW corresponds to the molecular weight ladder (Thermo Scientific GeneRuler DNA ladder mix).

    Journal: Applied and Environmental Microbiology

    Article Title: Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp.

    doi: 10.1128/AEM.00485-19

    Figure Lengend Snippet: Verification of the integration of pOSV802 in S. coelicolor M145, S. lividans TK23, and S. albus J1074 chromosomes. (A) Principle of the PCR verification of the integration of the pOSV801 to pOSV812 vectors in the Streptomyces chromosomes (PCR 1 and PCR 2) (PCR 3, PCR verification before excision of modules 1 to 3). (B) PCR fragments obtained by PCR 1 ( attL region; expected sizes, 913 bp for M145 and TK23, 888 bp for J1074) and by PCR 2 ( attR region; expected sizes, 911 bp for M145 and TK23, 907 bp for J1074) on the three Streptomyces strains bearing pOSV802. No PCR amplification is expected when the genomic DNA of the wild-type Streptomyces strains is used as the matrix. MW corresponds to the molecular weight ladder (Thermo Scientific GeneRuler DNA ladder mix).

    Article Snippet: DNA fragments were purified from agarose gels using the NucleoSpin gel and PCR cleanup kit from Macherey-Nagel.

    Techniques: Polymerase Chain Reaction, Amplification, Molecular Weight