qiaquick pcr purification kit  (Qiagen)


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    QIAquick PCR Purification Kit
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    Catalog Number:
    28104
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    Structured Review

    Qiagen qiaquick pcr purification kit
    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, <t>PCR</t> amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the <t>QIAquick</t> PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

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    Images

    1) Product Images from "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism"

    Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-4-21

    Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
    Figure Legend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

    Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

    2) Product Images from "Use of a restriction enzyme-digested PCR product as substrate for helicase assays"

    Article Title: Use of a restriction enzyme-digested PCR product as substrate for helicase assays

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni009

    Substrates for helicase assays generated by PCR. A PCR product and its restriction fragment derivatives are shown. PCR reactions were performed as described in Materials and Methods with 32 P-labeled primer, and products were purified using the QIAquick PCR purification kit (Qiagen) (lane 1) and digest with PstI (lane 2), EcoRI (lane 3) and SmaI (lane 4) restriction enzymes.
    Figure Legend Snippet: Substrates for helicase assays generated by PCR. A PCR product and its restriction fragment derivatives are shown. PCR reactions were performed as described in Materials and Methods with 32 P-labeled primer, and products were purified using the QIAquick PCR purification kit (Qiagen) (lane 1) and digest with PstI (lane 2), EcoRI (lane 3) and SmaI (lane 4) restriction enzymes.

    Techniques Used: Generated, Polymerase Chain Reaction, Labeling, Purification

    3) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Figure Legend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Techniques Used: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    4) Product Images from "Improved Protocols for Illumina Sequencing"

    Article Title: Improved Protocols for Illumina Sequencing

    Journal:

    doi: 10.1002/0471142905.hg1802s62

    PCR cleanup. We prepared a paired-end PhiX library using conditions that would promote the formation of adapter and primer dimers and unextended PCR primers. After PCR, we divided the library in two: half was purified using a QIAquick spin column, as in the standard Illumina protocol (left), whereas the other half was purified using AMPure SPRI beads (right). Gels are shown after staining and excision of the gel slice corresponding to the desired size range of fragments. Image adapted with permission from Macmillan Publishers Ltd. .
    Figure Legend Snippet: PCR cleanup. We prepared a paired-end PhiX library using conditions that would promote the formation of adapter and primer dimers and unextended PCR primers. After PCR, we divided the library in two: half was purified using a QIAquick spin column, as in the standard Illumina protocol (left), whereas the other half was purified using AMPure SPRI beads (right). Gels are shown after staining and excision of the gel slice corresponding to the desired size range of fragments. Image adapted with permission from Macmillan Publishers Ltd. .

    Techniques Used: Polymerase Chain Reaction, Purification, Staining

    5) Product Images from "Rapid Phenotypic Characterization Method for Herpes Simplex Virus and Varicella-Zoster Virus Thymidine Kinases To Screen for Acyclovir-Resistant Viral Infection"

    Article Title: Rapid Phenotypic Characterization Method for Herpes Simplex Virus and Varicella-Zoster Virus Thymidine Kinases To Screen for Acyclovir-Resistant Viral Infection

    Journal:

    doi:

    Sensitivity of PCR to HSV-1 TK (A), HSV-2 TK (B), and VZV TK (C) genes. The sample DNA, calculated to correspond to 10, 1, 0.1, 0.01, and 0.001 virus-infected cell, was added to 100 μl of PCR mixture, and PCR was carried out under the conditions described in Materials and Methods. After purification of PCR products with a QIAquick PCR Purification Kit, 1 μl of the total 30-μl DNA suspension was analyzed on agarose gels. The amount of VZV TK gene in 1 μl of purified PCR products, amplified from 0.1 infected cell, was measured to be 40 ng by absorbance at 260 nm (panel C, lane 3).
    Figure Legend Snippet: Sensitivity of PCR to HSV-1 TK (A), HSV-2 TK (B), and VZV TK (C) genes. The sample DNA, calculated to correspond to 10, 1, 0.1, 0.01, and 0.001 virus-infected cell, was added to 100 μl of PCR mixture, and PCR was carried out under the conditions described in Materials and Methods. After purification of PCR products with a QIAquick PCR Purification Kit, 1 μl of the total 30-μl DNA suspension was analyzed on agarose gels. The amount of VZV TK gene in 1 μl of purified PCR products, amplified from 0.1 infected cell, was measured to be 40 ng by absorbance at 260 nm (panel C, lane 3).

    Techniques Used: Polymerase Chain Reaction, Infection, Purification, Amplification

    6) Product Images from "Improved Protocols for Illumina Sequencing"

    Article Title: Improved Protocols for Illumina Sequencing

    Journal:

    doi: 10.1002/0471142905.hg1802s62

    PCR cleanup. We prepared a paired-end PhiX library using conditions that would promote the formation of adapter and primer dimers and unextended PCR primers. After PCR, we divided the library in two: half was purified using a QIAquick spin column, as in the standard Illumina protocol (left), whereas the other half was purified using AMPure SPRI beads (right). Gels are shown after staining and excision of the gel slice corresponding to the desired size range of fragments. Image adapted with permission from Macmillan Publishers Ltd. .
    Figure Legend Snippet: PCR cleanup. We prepared a paired-end PhiX library using conditions that would promote the formation of adapter and primer dimers and unextended PCR primers. After PCR, we divided the library in two: half was purified using a QIAquick spin column, as in the standard Illumina protocol (left), whereas the other half was purified using AMPure SPRI beads (right). Gels are shown after staining and excision of the gel slice corresponding to the desired size range of fragments. Image adapted with permission from Macmillan Publishers Ltd. .

    Techniques Used: Polymerase Chain Reaction, Purification, Staining

    7) Product Images from "Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application"

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4371-5

    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
    Figure Legend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Techniques Used: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    8) Product Images from "Improved Protocols for Illumina Sequencing"

    Article Title: Improved Protocols for Illumina Sequencing

    Journal:

    doi: 10.1002/0471142905.hg1802s62

    PCR cleanup. We prepared a paired-end PhiX library using conditions that would promote the formation of adapter and primer dimers and unextended PCR primers. After PCR, we divided the library in two: half was purified using a QIAquick spin column, as in the standard Illumina protocol (left), whereas the other half was purified using AMPure SPRI beads (right). Gels are shown after staining and excision of the gel slice corresponding to the desired size range of fragments. Image adapted with permission from Macmillan Publishers Ltd. .
    Figure Legend Snippet: PCR cleanup. We prepared a paired-end PhiX library using conditions that would promote the formation of adapter and primer dimers and unextended PCR primers. After PCR, we divided the library in two: half was purified using a QIAquick spin column, as in the standard Illumina protocol (left), whereas the other half was purified using AMPure SPRI beads (right). Gels are shown after staining and excision of the gel slice corresponding to the desired size range of fragments. Image adapted with permission from Macmillan Publishers Ltd. .

    Techniques Used: Polymerase Chain Reaction, Purification, Staining

    9) Product Images from "Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase"

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    Journal:

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Agarose gel analysis of DNA fragments generated during the MURA process. The Serratia sp. phospholipase gene (0.96 kb) was prepared by PCR (lane 1). The template DNA was digested with DNase I and the digests were purified (lane 2) and subjected to the MURA PCR as described in Materials and Methods; the resultant DNA fragments reassembled with a unidirectional primer (lane 3) were purified by using a Qiaquick purification kit. Lane M, 100-bp ladder DNA size marker.
    Figure Legend Snippet: Agarose gel analysis of DNA fragments generated during the MURA process. The Serratia sp. phospholipase gene (0.96 kb) was prepared by PCR (lane 1). The template DNA was digested with DNase I and the digests were purified (lane 2) and subjected to the MURA PCR as described in Materials and Methods; the resultant DNA fragments reassembled with a unidirectional primer (lane 3) were purified by using a Qiaquick purification kit. Lane M, 100-bp ladder DNA size marker.

    Techniques Used: Agarose Gel Electrophoresis, Generated, Polymerase Chain Reaction, Purification, Marker

    10) Product Images from "Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase"

    Article Title: Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

    Journal:

    doi: 10.1128/AEM.68.12.6146-6151.2002

    Agarose gel analysis of DNA fragments generated during the MURA process. The Serratia sp. phospholipase gene (0.96 kb) was prepared by PCR (lane 1). The template DNA was digested with DNase I and the digests were purified (lane 2) and subjected to the MURA PCR as described in Materials and Methods; the resultant DNA fragments reassembled with a unidirectional primer (lane 3) were purified by using a Qiaquick purification kit. Lane M, 100-bp ladder DNA size marker.
    Figure Legend Snippet: Agarose gel analysis of DNA fragments generated during the MURA process. The Serratia sp. phospholipase gene (0.96 kb) was prepared by PCR (lane 1). The template DNA was digested with DNase I and the digests were purified (lane 2) and subjected to the MURA PCR as described in Materials and Methods; the resultant DNA fragments reassembled with a unidirectional primer (lane 3) were purified by using a Qiaquick purification kit. Lane M, 100-bp ladder DNA size marker.

    Techniques Used: Agarose Gel Electrophoresis, Generated, Polymerase Chain Reaction, Purification, Marker

    11) Product Images from "Improved Protocols for Illumina Sequencing"

    Article Title: Improved Protocols for Illumina Sequencing

    Journal:

    doi: 10.1002/0471142905.hg1802s62

    PCR cleanup. We prepared a paired-end PhiX library using conditions that would promote the formation of adapter and primer dimers and unextended PCR primers. After PCR, we divided the library in two: half was purified using a QIAquick spin column, as in the standard Illumina protocol (left), whereas the other half was purified using AMPure SPRI beads (right). Gels are shown after staining and excision of the gel slice corresponding to the desired size range of fragments. Image adapted with permission from Macmillan Publishers Ltd. .
    Figure Legend Snippet: PCR cleanup. We prepared a paired-end PhiX library using conditions that would promote the formation of adapter and primer dimers and unextended PCR primers. After PCR, we divided the library in two: half was purified using a QIAquick spin column, as in the standard Illumina protocol (left), whereas the other half was purified using AMPure SPRI beads (right). Gels are shown after staining and excision of the gel slice corresponding to the desired size range of fragments. Image adapted with permission from Macmillan Publishers Ltd. .

    Techniques Used: Polymerase Chain Reaction, Purification, Staining

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    Article Snippet: 1 μl of the 4C library was used as template for PCR amplification using oligonucleotide primers adjacent to pairs of HindIII and DpnII sites on the long arm of budding yeast chromosome 5. .. Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification.

    Article Title: Excess of Rare Variants in Genes that are Key Epigenetic Regulators of Spermatogenesis in the Patients with Non-Obstructive Azoospermia
    Article Snippet: The adaptor-ligated DNA products were amplified using index tagged primers in order to tag DNA from different individuals. .. The amplified products were purified with QIAquick PCR purification kits (QIAGEN) and hybridized to the custom-designed capture array as per NimbleGen's Sequence Capture protocol for enrichment. .. Each enriched library was loaded on the HiSeq 2000 platform and pair-end reads with the lengths of 90 bp were generated.

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Poly(A)-containing mRNAs were purified using oligo(dT)-attached magnetic beads and transcriptome libraries were generated using an Illumina TruSeqTM RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification. .. The complete libraries were sequenced using the Illumina HiSeq2500 system (Illumina, San Diego, CA, USA).

    Article Title: Population history provides foundational knowledge for utilizing and developing native plant restoration materials. Population history provides foundational knowledge for utilizing and developing native plant restoration materials
    Article Snippet: Genomic DNA from individual plants was digested with Pst I and Msp I, followed by the ligation of Illumina adaptor sequences; each individual was barcoded four times using unique, 5–10 base pair barcodes. .. Ligation products were pooled, purified using QIAquick PCR kits (Qiagen), and amplified using 16 cycles of PCR with eight replicates. .. A Pippin Prep (Sage Science, Beverly, MA, USA) was used to size select amplicons from 400 to 500 base pairs.

    Article Title: A novel variant of torque teno virus 7 identified in patients with Kawasaki disease
    Article Snippet: Paragraph title: Whole transcriptome amplification of nucleic acids pooled from serum, NPA, feces, WB-DNA, and WB-cDNA ... The PCR product was cleaned using the QIAquick PCR Clean-up kit (QIAGEN) following the standard manufacturer’s protocols.

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). .. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400 bp prior to end-repair, 3′end adenylation and adapter ligation.

    Whole Genome Amplification:

    Article Title: A novel variant of torque teno virus 7 identified in patients with Kawasaki disease
    Article Snippet: WTA ligation buffer (6 μl), WTA ligation reagent (2 μl), WTA ligation enzyme 1 (1 μl), and WTA ligation enzyme 2 (1 μl), which are all provided in the REPLI-g Cell WGA and WTA Kit (QIAGEN), and the pooled nucleic acids (10 μl) were mixed and incubated at 22°C for 2 hours. .. The PCR product was cleaned using the QIAquick PCR Clean-up kit (QIAGEN) following the standard manufacturer’s protocols.

    Autoradiography:

    Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control
    Article Snippet: Following washes with wash buffer II (50 mM Tris-HCl, pH 7.8, 50 mM NaCl, 0.1% Nonidet-P-40 substituent, 10 mM imidazole, 5 mM 2-mercaptoethanol) protein–RNA complexes were then eluted in elution buffer (wash buffer II + 250 mM imidazole), precipitated with TCA or acetone, size-separated on NuPAGE 4-12% Bis–Tris gels, transferred to nitrocellulose membranes, visualized by autoradiography and appropriately sized complexes were excised: size of Hel2 + crosslinked RNA was excised in the first set of experiments, while a region of the size of Hel2-1-492 + crosslinked RNA to the size of wild-type Hel2 + crosslinked RNA was excised in the second set of experiments where mutants were analysed. .. Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen).

    Construct:

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Poly(A)-containing mRNAs were purified using oligo(dT)-attached magnetic beads and transcriptome libraries were generated using an Illumina TruSeqTM RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification. .. The complete libraries were sequenced using the Illumina HiSeq2500 system (Illumina, San Diego, CA, USA).

    Concentration Assay:

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: Sample mRNA that did not have a 260/280 ratio of at least 1.8 or that was less than 5 ng/µL concentration was not transcribed to cDNA. .. KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX).

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification. .. We used Agencourt AMPure XP beads (Beckman Coulter, Brea, CA) at a 0.8× ratio to remove adapter dimers after ligation and replaced the Illumina Phusion enzyme with the Kapa HiFi HotStart ready mix (Kapa Biosystems, Wilmington, MA).

    SYBR Green Assay:

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: A Mastercycler ep realplex qPCR system (Eppendorf, Hamburg, Germany) with QuantiTect SYBR Green PCR Master Mix (Clontech Laboratories, Mountain View, CA) was used to measure relative mRNA expression in VICs. .. KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX).

    Microarray:

    Article Title: Functional divergence of a global regulatory complex governing fungal filamentation
    Article Snippet: Paragraph title: Microarray profiling ... Labeled cDNA was purified with a QIAquick PCR Purification Kit (QIAGEN).

    Incubation:

    Article Title: Biosynthesis of polybrominated aromatic organic compounds by marine bacteria
    Article Snippet: The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen). .. Purified products were treated with DpnI exonuclease (NEB) to digest methylated pETDuet-bmp1–7 template.

    Article Title: An advanced enrichment method for rare somatic retroelement insertions sequencing
    Article Snippet: Ligated fragments were incubated for 2 hours with 3 U of restriction enzyme AluI in 1 × Y Tango buffer to decrease the number of chimeric molecules. .. Restriction products were purified using QIAquick PCR Purification Kit (Qiagen).

    Article Title: Functional divergence of a global regulatory complex governing fungal filamentation
    Article Snippet: Template RNA was degraded using 2.5 units RNase H (USB) and 1 μg RNase A (Pharmacia), incubated at 37°C for 15 min. .. Labeled cDNA was purified with a QIAquick PCR Purification Kit (QIAGEN).

    Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control
    Article Snippet: Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen). .. Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen).

    Article Title: A novel variant of torque teno virus 7 identified in patients with Kawasaki disease
    Article Snippet: Then, REPLI-g Midi reaction buffer (29 μl) and RFPLI-g Midi DNA polymerase (1 μl), also provided in the above kit, were added and incubated at: 30°C for 8 hours, 95°C for 5 minutes, then held on ice. .. The PCR product was cleaned using the QIAquick PCR Clean-up kit (QIAGEN) following the standard manufacturer’s protocols.

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Immunoprecipitated chromatin was eluted by two sequential incubations with 100 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65°C for 15 min. .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). .. At least 1 ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol.

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: DNA-protein complexes were de-crosslinked by adding 10 µl 5 M NaCl to the samples and incubating at 65 C overnight. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106). .. Quantitative PCR was then performed with input and immunoprecipitated DNA using primers directed to the RAD51 promoter (F: 5′-CCCCCGGCATAAAGTTTGA-3′; R: 5′-GCTTTCAGAATTCCCGCCA-3′ , ), or IGX1A gene desert (QIAGEN, cat#GPH100001C(-)01 A) and Power SYBR according to manufacturer’s instructions, and analysed using a CFX96 Real-Time PCR machine.

    Expressing:

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: Primer efficiencies and relative expression ratios were calculated using the REST 2009 program. .. KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX).

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
    Article Snippet: Paragraph title: Cloning of immunoglobulin variable domain regions into a dual promoter expression plasmid ... F-PCR products were digested with FastDigest NotI and AscI (Thermo Fisher Cat# ER0595 and ER1891, respectively) at 37°C for 20 min, followed by inactivation at 80°C for 5 min and column purification (Qiagen/QiaQuick PCR Purification Cat# 28106).

    Western Blot:

    Article Title: A novel variant of torque teno virus 7 identified in patients with Kawasaki disease
    Article Snippet: Paragraph title: Whole transcriptome amplification of nucleic acids pooled from serum, NPA, feces, WB-DNA, and WB-cDNA ... The PCR product was cleaned using the QIAquick PCR Clean-up kit (QIAGEN) following the standard manufacturer’s protocols.

    Transformation Assay:

    Article Title: Biosynthesis of polybrominated aromatic organic compounds by marine bacteria
    Article Snippet: The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen). .. Purified products were treated with DpnI exonuclease (NEB) to digest methylated pETDuet-bmp1–7 template.

    Article Title: Distinct genetic difference between the Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from North Borneo and Peninsular Malaysia
    Article Snippet: PCR products were purified by QIAquick PCR purification Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. .. Plasmids of recombinant clones harbouring the PkDBPαII fragment were sent to a commercial laboratory for DNA sequencing.

    Ligation:

    Article Title: An advanced enrichment method for rare somatic retroelement insertions sequencing
    Article Snippet: The 10 μ l ligation mixture contained 50 pmoles of each st19BH and st20BH adapters, 10 U of T4 DNA ligase in a T4 reaction buffer (both Promega) and digested genomic DNA. .. Restriction products were purified using QIAquick PCR Purification Kit (Qiagen).

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
    Article Snippet: F-PCR products were digested with FastDigest NotI and AscI (Thermo Fisher Cat# ER0595 and ER1891, respectively) at 37°C for 20 min, followed by inactivation at 80°C for 5 min and column purification (Qiagen/QiaQuick PCR Purification Cat# 28106). .. P1316 is a derivative of the pTT3 expression vector ( ) and consists of (5’ to 3’): a CMV promoter, the mouse V kappa leader sequence, a NotI restriction site, an insert consisting of VL /joining fragment/VH , and the mouse IgG1 CH sequence amplified from mouse genomic DNA, flanked by AscI and XbaI restriction sites ( ; ).

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification. .. Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification.

    Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control
    Article Snippet: Dephosphorylation, 3’-linker ligation (0.5 U/μl T4 RNA ligase 1, 5 U/μl T4 RNA ligase 2, tr, KQ), re-phosphorylation with γ-32 P-ATP and non-labelled ATP and 5’-linker ligation were then performed on the Ni-NTA, always in the presence of 1 U/μl RNaseIN RNase inhibitor. .. Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen).

    Article Title: Population history provides foundational knowledge for utilizing and developing native plant restoration materials. Population history provides foundational knowledge for utilizing and developing native plant restoration materials
    Article Snippet: Genomic DNA from individual plants was digested with Pst I and Msp I, followed by the ligation of Illumina adaptor sequences; each individual was barcoded four times using unique, 5–10 base pair barcodes. .. Ligation products were pooled, purified using QIAquick PCR kits (Qiagen), and amplified using 16 cycles of PCR with eight replicates. .. A Pippin Prep (Sage Science, Beverly, MA, USA) was used to size select amplicons from 400 to 500 base pairs.

    Article Title: A novel variant of torque teno virus 7 identified in patients with Kawasaki disease
    Article Snippet: WTA ligation buffer (6 μl), WTA ligation reagent (2 μl), WTA ligation enzyme 1 (1 μl), and WTA ligation enzyme 2 (1 μl), which are all provided in the REPLI-g Cell WGA and WTA Kit (QIAGEN), and the pooled nucleic acids (10 μl) were mixed and incubated at 22°C for 2 hours. .. The PCR product was cleaned using the QIAquick PCR Clean-up kit (QIAGEN) following the standard manufacturer’s protocols.

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). .. At least 1 ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol.

    Protease Inhibitor:

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: After incubation, the beads were washed 7× with wash buffer (50 mM HEPES pH 7.6, 500 mM LiCl, 1 mM EDTA pH 8.0, 1% NP-40, 0.7% Na-Deoxycholate, 1× protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 50 mM NaCl). .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA).

    Cell Culture:

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: Paragraph title: Yeast cell culture and 4C protocol ... Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification.

    Generated:

    Article Title: MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1
    Article Snippet: A Bioruptor generated soluble chromatin (Cosmo Bio USA, Carlsbad, CA) with DNA fragment sizes of 200 to 800 bp. .. We purified DNA fragments with the QIAquick PCR purification kit (Qiagen).

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Poly(A)-containing mRNAs were purified using oligo(dT)-attached magnetic beads and transcriptome libraries were generated using an Illumina TruSeqTM RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification.

    DNA HS Assay:

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Immunoprecipitated chromatin was eluted by two sequential incubations with 100 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65°C for 15 min. .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). .. At least 1 ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol.

    DNA Sequencing:

    Article Title: Distinct genetic difference between the Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from North Borneo and Peninsular Malaysia
    Article Snippet: PCR products were purified by QIAquick PCR purification Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. .. The purified PCR products were then ligated into cloning vector pGEM-T® (Promega Corp, USA) and transformed into Escherichia coli TOP10F’.

    Article Title: Association of Intact dupA (dupA1) rather than dupA1 cluster with duodenal ulcer in Indian population
    Article Snippet: Paragraph title: DNA sequencing and phylogenetic analysis ... The amplified products were purified using the QIAquick PCR Purification Kit (QIAGEN).

    Article Title: Simian malaria in the Brazilian Atlantic forest: first description of natural infection of capuchin monkeys (Cebinae subfamily) by Plasmodium simium
    Article Snippet: Electrophoresis was performed in a room specific for amplified DNA, with appropriated sets of pipettes and plugged pipettes tips. .. For DNA sequencing, PCR products were purified using Purification Kit QIAquick (Qiagen) following manufacturer’s procedure. .. Around 3 ng of purified PCR products were amplified using 2.0 μM of each primer (forward or reverse of species-specific primers of second reaction) and 1 μL of Big Dye terminator kit in a program of: 96°C for 1 min, 35 cycles of 96°C for 15 sec, the temperature of primer annealing for 15 sec and 60°C for 15 sec.

    Sequencing:

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: Paragraph title: RNA Isolation, qPCR, and Sequencing ... KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX).

    Article Title: Understanding the cryptic introgression and mixed ancestry of Red Junglefowl in India
    Article Snippet: Paragraph title: Microsatellite genotyping and sequencing of D-loop region ... Thermocycling conditions of PCR consisted of an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 seconds, an annealing step at 52°C for 20 seconds, and an extension at 72°C for 60 seconds and finished by a final extension at 72°C for 10 min. PCR products were purified using the QIAquick PCR purification kit (QIAGEN, GmbH, Germany) according to the manufacturer’s protocol.

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
    Article Snippet: F-PCR products were digested with FastDigest NotI and AscI (Thermo Fisher Cat# ER0595 and ER1891, respectively) at 37°C for 20 min, followed by inactivation at 80°C for 5 min and column purification (Qiagen/QiaQuick PCR Purification Cat# 28106). .. The P1316 plasmid was also NotI/AscI digested and gel purified (Qiagen/QiaQuick Gel Extraction Cat# 28706).

    Article Title: Association of Intact dupA (dupA1) rather than dupA1 cluster with duodenal ulcer in Indian population
    Article Snippet: Primer walking method was used for the sequencing of full length of dupA gene. .. The amplified products were purified using the QIAquick PCR Purification Kit (QIAGEN).

    Article Title: Excess of Rare Variants in Genes that are Key Epigenetic Regulators of Spermatogenesis in the Patients with Non-Obstructive Azoospermia
    Article Snippet: The adaptor-ligated DNA products were amplified using index tagged primers in order to tag DNA from different individuals. .. The amplified products were purified with QIAquick PCR purification kits (QIAGEN) and hybridized to the custom-designed capture array as per NimbleGen's Sequence Capture protocol for enrichment. .. Each enriched library was loaded on the HiSeq 2000 platform and pair-end reads with the lengths of 90 bp were generated.

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Paragraph title: 4.3. RNA Extraction and Sequencing ... A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification.

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA).

    Article Title: Simian malaria in the Brazilian Atlantic forest: first description of natural infection of capuchin monkeys (Cebinae subfamily) by Plasmodium simium
    Article Snippet: Paragraph title: Sequencing of PCR-amplified DNA ... For DNA sequencing, PCR products were purified using Purification Kit QIAquick (Qiagen) following manufacturer’s procedure.

    Sonication:

    Article Title: Excess of Rare Variants in Genes that are Key Epigenetic Regulators of Spermatogenesis in the Patients with Non-Obstructive Azoospermia
    Article Snippet: Genomic DNA was extracted and randomly fragmented by sonication to an average size of 250 bp. .. The amplified products were purified with QIAquick PCR purification kits (QIAGEN) and hybridized to the custom-designed capture array as per NimbleGen's Sequence Capture protocol for enrichment.

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: The equivalent of 10 × 107 cells sonicated extract was used for each ChIP experiment for both cell lines. .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA).

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: In parallel, crosslinked cells were then thawed, lysed for 20 mins in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl), then 400–1000 bp fragments of chromatin were produced by sonication in a water bath sonicator (4 C, Misonix ultrasound liquid processor) for 13 cycles of 30 sec on, 30 sec off at 65 Amps. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106).

    Recombinant:

    Article Title: Distinct genetic difference between the Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from North Borneo and Peninsular Malaysia
    Article Snippet: PCR products were purified by QIAquick PCR purification Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. .. PCR products were purified by QIAquick PCR purification Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.

    ChIP-sequencing:

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification. .. In preparation for sequencing, the DNA samples were end repaired, poly-A tailed and Single End Adapters (Illumina, San Diego, CA) were ligated.

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Paragraph title: Chromatin immunoprecipitation for ChIP-seq ... The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA).

    DNA Extraction:

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: DNA-protein complexes were de-crosslinked by adding 10 µl 5 M NaCl to the samples and incubating at 65 C overnight. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106). .. Quantitative PCR was then performed with input and immunoprecipitated DNA using primers directed to the RAD51 promoter (F: 5′-CCCCCGGCATAAAGTTTGA-3′; R: 5′-GCTTTCAGAATTCCCGCCA-3′ , ), or IGX1A gene desert (QIAGEN, cat#GPH100001C(-)01 A) and Power SYBR according to manufacturer’s instructions, and analysed using a CFX96 Real-Time PCR machine.

    BAC Assay:

    Article Title: Positional cloning of rp2 QTL associates the P450 genes CYP6Z1,CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vectorAnopheles funestus
    Article Snippet: In order to carry out a fine-scale mapping of the rp2 QTL, SNPs were identified in all the genes detected in the BAC clone and in other genes spanning therp2 QTL boundaries in the 2L chromosome such as the glutathione-s-transferaseGSTe2 , the cuticular protein genes CPLC5, CPLC9 and CPLC8 , the P450s CYP9M1, CYP303A1 and CYP4H18 , and AGAP007980 and AGAP009073. .. PCR products were purified using the QIaquick PCR purification kit (Qiagen, Hilden, Germany) and directly sequenced on both strands.

    Magnetic Beads:

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Poly(A)-containing mRNAs were purified using oligo(dT)-attached magnetic beads and transcriptome libraries were generated using an Illumina TruSeqTM RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification.

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0.5% BSA PBS overnight at 4°C. .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA).

    Mutagenesis:

    Article Title: Biosynthesis of polybrominated aromatic organic compounds by marine bacteria
    Article Snippet: Individual deletions of bmp2 and bmp5 were performed by PCR mutagenesis using PrimeStar Max DNA Polymerase standard protocol (Clontech) with pETDuet-bmp1–7 as the template. .. The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen).

    Isolation:

    Article Title: Functional divergence of a global regulatory complex governing fungal filamentation
    Article Snippet: RNA was isolated using the QIAGEN RNeasy kit and RNasefree DNase (QIAGEN). .. Labeled cDNA was purified with a QIAquick PCR Purification Kit (QIAGEN).

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: Paragraph title: RNA Isolation, qPCR, and Sequencing ... KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX).

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Immunoprecipitated chromatin was eluted by two sequential incubations with 100 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65°C for 15 min. .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). .. At least 1 ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol.

    Size-exclusion Chromatography:

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: In parallel, crosslinked cells were then thawed, lysed for 20 mins in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl), then 400–1000 bp fragments of chromatin were produced by sonication in a water bath sonicator (4 C, Misonix ultrasound liquid processor) for 13 cycles of 30 sec on, 30 sec off at 65 Amps. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106).

    Labeling:

    Article Title: Functional divergence of a global regulatory complex governing fungal filamentation
    Article Snippet: Template RNA was degraded using 2.5 units RNase H (USB) and 1 μg RNase A (Pharmacia), incubated at 37°C for 15 min. .. Labeled cDNA was purified with a QIAquick PCR Purification Kit (QIAGEN). .. The hybridization was carried out with DIG Easy Hyb Solution (Roche Diagnostics) containing 0.45% salmon sperm DNA and 0.45% yeast tRNA at 42°C for 24 hours in a SlideBooster Hyb chamber SB 800 (Advalytix, Brunnthal, Germany) with regular microagitation.

    Purification:

    Article Title: Biosynthesis of polybrominated aromatic organic compounds by marine bacteria
    Article Snippet: Primers were designed with the motif 5′-[20 nucleotide overlap] [35 nucleotide primer region]-3′ ( ). .. The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen). .. Purified products were treated with DpnI exonuclease (NEB) to digest methylated pETDuet-bmp1–7 template.

    Article Title: Distinct genetic difference between the Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from North Borneo and Peninsular Malaysia
    Article Snippet: The PCR product with an expected size of 1,053 bp was analysed on a 1% agarose gel. .. PCR products were purified by QIAquick PCR purification Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. .. The purified PCR products were then ligated into cloning vector pGEM-T® (Promega Corp, USA) and transformed into Escherichia coli TOP10F’.

    Article Title: MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1
    Article Snippet: RNaseA and proteinase K were added for 1h at 37°C. .. We purified DNA fragments with the QIAquick PCR purification kit (Qiagen). .. Input chromatin was used as a positive control in RT-PCR with the primers: GPH1005991(-)01A for MAF binding sites on SOX9 promoter and GPH100001C(-)01A for a chromosome 12 promoter desert as a negative control (SABiosciences).

    Article Title: An advanced enrichment method for rare somatic retroelement insertions sequencing
    Article Snippet: Ligated fragments were incubated for 2 hours with 3 U of restriction enzyme AluI in 1 × Y Tango buffer to decrease the number of chimeric molecules. .. Restriction products were purified using QIAquick PCR Purification Kit (Qiagen). .. DNA amplification for library preparation was performed in two subsequent suppression PCR steps.

    Article Title: Functional divergence of a global regulatory complex governing fungal filamentation
    Article Snippet: Template RNA was degraded using 2.5 units RNase H (USB) and 1 μg RNase A (Pharmacia), incubated at 37°C for 15 min. .. Labeled cDNA was purified with a QIAquick PCR Purification Kit (QIAGEN). .. The hybridization was carried out with DIG Easy Hyb Solution (Roche Diagnostics) containing 0.45% salmon sperm DNA and 0.45% yeast tRNA at 42°C for 24 hours in a SlideBooster Hyb chamber SB 800 (Advalytix, Brunnthal, Germany) with regular microagitation.

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: All mRNA targets and primers are listed in . .. KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX). .. Gelatin and hyaluronan substrate zymography was performed using hand cast 10% polyacrylamide gels containing either 0.2% w/v gelatin or 0.17 mg/ mL 1700 kDa sodium hyaluronate.

    Article Title: Understanding the cryptic introgression and mixed ancestry of Red Junglefowl in India
    Article Snippet: The PCRs were conducted in 10 μl reaction—0.25 mM MgCl2 , 1 μM each of forward and reverse primers, 0.25 mM dNTPs and 1 unit Taq polymerase. .. Thermocycling conditions of PCR consisted of an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 seconds, an annealing step at 52°C for 20 seconds, and an extension at 72°C for 60 seconds and finished by a final extension at 72°C for 10 min. PCR products were purified using the QIAquick PCR purification kit (QIAGEN, GmbH, Germany) according to the manufacturer’s protocol. .. Direct sequencing of a HV1 segment of the D-loop region was performed using two internal primers CR-for ( 5′-TCTATATTCCACATTTCTC-3′) a nd CR-rev ( 5′-GCGAGCATAACCAAATGG-3′ ).

    Article Title: Relationship between Gut Microbiota and Phosphorus Metabolism in Hemodialysis Patients: A Preliminary Exploration
    Article Snippet: The PCR procedure was set by the following cycle conditions: 94°C for 5 min, then 15 cycles at 94°C for 45 s, 55°C for 45 s, 72°C for 45 s followed by a final step of 72°C for 10 min, then stored at 4°C. .. After the PCR was completed, the products were purified using the QIAquick PCR purification kit (Qiagen Valencia, CA, USA). .. The 454-pyrosequencing data were analyzed in a well-designed pipeline.

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
    Article Snippet: PCR conditions were: 95°C for 2 min; 11 cycles of 95°C for 45 s, 63°C for 30 s, 72°C for 5 min; 7 cycles of 95°C for 45 s, 62°C for 30 s with 1°C decrement per cycle, 72°C for 5 min, 95°C for 45 s; 26 cycles of 56°C for 30 s, 72°C for 5 min; 72°C for 15 min. .. F-PCR products were digested with FastDigest NotI and AscI (Thermo Fisher Cat# ER0595 and ER1891, respectively) at 37°C for 20 min, followed by inactivation at 80°C for 5 min and column purification (Qiagen/QiaQuick PCR Purification Cat# 28106). .. The P1316 plasmid was also NotI/AscI digested and gel purified (Qiagen/QiaQuick Gel Extraction Cat# 28706).

    Article Title: Association of Intact dupA (dupA1) rather than dupA1 cluster with duodenal ulcer in Indian population
    Article Snippet: Primer jhp0919R was located from 326 bp to 350 of ORF jhp0919 of strain J99. .. The amplified products were purified using the QIAquick PCR Purification Kit (QIAGEN). .. The purified PCR product was quantified on gel.

    Article Title: Positional cloning of rp2 QTL associates the P450 genes CYP6Z1,CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vectorAnopheles funestus
    Article Snippet: The PCR were carried out using 10 pmol of each primers and 30 ng of genomic DNA as template in 25 ml reactions. .. PCR products were purified using the QIaquick PCR purification kit (Qiagen, Hilden, Germany) and directly sequenced on both strands. .. Sequences for each gene were analysed to detect the polymorphic sites manually using BioEdit and as sequence differences in multiple alignments using ClustalW ( ).

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: 1 μl of the 4C library was used as template for PCR amplification using oligonucleotide primers adjacent to pairs of HindIII and DpnII sites on the long arm of budding yeast chromosome 5. .. Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification. .. In preparation for sequencing, the DNA samples were end repaired, poly-A tailed and Single End Adapters (Illumina, San Diego, CA) were ligated.

    Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control
    Article Snippet: RT was performed with indexed primers (Supplementary Table ); primers were digested with Exonuclease I (Thermo Scientific, 2 × 30 min with addition of 40 U ExoI before each incubation, followed by heat denaturation at 80 °C for 10 min). .. Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen). .. After quantification (Qubit, Thermo Scientific), libraries in the first set of experiments (wild-type Hel2-HTP) were pooled and sequenced in a single run on HiSeq2500 (Edinburgh Genomics), in fast mode, on two lanes, with 100 bp read length, using custom primers to read out the indices.

    Article Title: Excess of Rare Variants in Genes that are Key Epigenetic Regulators of Spermatogenesis in the Patients with Non-Obstructive Azoospermia
    Article Snippet: The adaptor-ligated DNA products were amplified using index tagged primers in order to tag DNA from different individuals. .. The amplified products were purified with QIAquick PCR purification kits (QIAGEN) and hybridized to the custom-designed capture array as per NimbleGen's Sequence Capture protocol for enrichment. .. Each enriched library was loaded on the HiSeq 2000 platform and pair-end reads with the lengths of 90 bp were generated.

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Poly(A)-containing mRNAs were purified using oligo(dT)-attached magnetic beads and transcriptome libraries were generated using an Illumina TruSeqTM RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification. .. The complete libraries were sequenced using the Illumina HiSeq2500 system (Illumina, San Diego, CA, USA).

    Article Title: Population history provides foundational knowledge for utilizing and developing native plant restoration materials. Population history provides foundational knowledge for utilizing and developing native plant restoration materials
    Article Snippet: Genomic DNA from individual plants was digested with Pst I and Msp I, followed by the ligation of Illumina adaptor sequences; each individual was barcoded four times using unique, 5–10 base pair barcodes. .. Ligation products were pooled, purified using QIAquick PCR kits (Qiagen), and amplified using 16 cycles of PCR with eight replicates. .. A Pippin Prep (Sage Science, Beverly, MA, USA) was used to size select amplicons from 400 to 500 base pairs.

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Immunoprecipitated chromatin was eluted by two sequential incubations with 100 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65°C for 15 min. .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). .. At least 1 ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol.

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: DNA-protein complexes were de-crosslinked by adding 10 µl 5 M NaCl to the samples and incubating at 65 C overnight. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106). .. Quantitative PCR was then performed with input and immunoprecipitated DNA using primers directed to the RAD51 promoter (F: 5′-CCCCCGGCATAAAGTTTGA-3′; R: 5′-GCTTTCAGAATTCCCGCCA-3′ , ), or IGX1A gene desert (QIAGEN, cat#GPH100001C(-)01 A) and Power SYBR according to manufacturer’s instructions, and analysed using a CFX96 Real-Time PCR machine.

    Article Title: Simian malaria in the Brazilian Atlantic forest: first description of natural infection of capuchin monkeys (Cebinae subfamily) by Plasmodium simium
    Article Snippet: Electrophoresis was performed in a room specific for amplified DNA, with appropriated sets of pipettes and plugged pipettes tips. .. For DNA sequencing, PCR products were purified using Purification Kit QIAquick (Qiagen) following manufacturer’s procedure. .. Around 3 ng of purified PCR products were amplified using 2.0 μM of each primer (forward or reverse of species-specific primers of second reaction) and 1 μL of Big Dye terminator kit in a program of: 96°C for 1 min, 35 cycles of 96°C for 15 sec, the temperature of primer annealing for 15 sec and 60°C for 15 sec.

    Polymerase Chain Reaction:

    Article Title: Biosynthesis of polybrominated aromatic organic compounds by marine bacteria
    Article Snippet: Primers were designed with the motif 5′-[20 nucleotide overlap] [35 nucleotide primer region]-3′ ( ). .. The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen). .. Purified products were treated with DpnI exonuclease (NEB) to digest methylated pETDuet-bmp1–7 template.

    Article Title: Distinct genetic difference between the Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from North Borneo and Peninsular Malaysia
    Article Snippet: The PCR product with an expected size of 1,053 bp was analysed on a 1% agarose gel. .. PCR products were purified by QIAquick PCR purification Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. .. The purified PCR products were then ligated into cloning vector pGEM-T® (Promega Corp, USA) and transformed into Escherichia coli TOP10F’.

    Article Title: MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1
    Article Snippet: RNaseA and proteinase K were added for 1h at 37°C. .. We purified DNA fragments with the QIAquick PCR purification kit (Qiagen). .. Input chromatin was used as a positive control in RT-PCR with the primers: GPH1005991(-)01A for MAF binding sites on SOX9 promoter and GPH100001C(-)01A for a chromosome 12 promoter desert as a negative control (SABiosciences).

    Article Title: An advanced enrichment method for rare somatic retroelement insertions sequencing
    Article Snippet: Ligated fragments were incubated for 2 hours with 3 U of restriction enzyme AluI in 1 × Y Tango buffer to decrease the number of chimeric molecules. .. Restriction products were purified using QIAquick PCR Purification Kit (Qiagen). .. DNA amplification for library preparation was performed in two subsequent suppression PCR steps.

    Article Title: Functional divergence of a global regulatory complex governing fungal filamentation
    Article Snippet: Template RNA was degraded using 2.5 units RNase H (USB) and 1 μg RNase A (Pharmacia), incubated at 37°C for 15 min. .. Labeled cDNA was purified with a QIAquick PCR Purification Kit (QIAGEN). .. The hybridization was carried out with DIG Easy Hyb Solution (Roche Diagnostics) containing 0.45% salmon sperm DNA and 0.45% yeast tRNA at 42°C for 24 hours in a SlideBooster Hyb chamber SB 800 (Advalytix, Brunnthal, Germany) with regular microagitation.

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: All mRNA targets and primers are listed in . .. KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX). .. Gelatin and hyaluronan substrate zymography was performed using hand cast 10% polyacrylamide gels containing either 0.2% w/v gelatin or 0.17 mg/ mL 1700 kDa sodium hyaluronate.

    Article Title: Understanding the cryptic introgression and mixed ancestry of Red Junglefowl in India
    Article Snippet: The PCRs were conducted in 10 μl reaction—0.25 mM MgCl2 , 1 μM each of forward and reverse primers, 0.25 mM dNTPs and 1 unit Taq polymerase. .. Thermocycling conditions of PCR consisted of an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 seconds, an annealing step at 52°C for 20 seconds, and an extension at 72°C for 60 seconds and finished by a final extension at 72°C for 10 min. PCR products were purified using the QIAquick PCR purification kit (QIAGEN, GmbH, Germany) according to the manufacturer’s protocol. .. Direct sequencing of a HV1 segment of the D-loop region was performed using two internal primers CR-for ( 5′-TCTATATTCCACATTTCTC-3′) a nd CR-rev ( 5′-GCGAGCATAACCAAATGG-3′ ).

    Article Title: Relationship between Gut Microbiota and Phosphorus Metabolism in Hemodialysis Patients: A Preliminary Exploration
    Article Snippet: The PCR procedure was set by the following cycle conditions: 94°C for 5 min, then 15 cycles at 94°C for 45 s, 55°C for 45 s, 72°C for 45 s followed by a final step of 72°C for 10 min, then stored at 4°C. .. After the PCR was completed, the products were purified using the QIAquick PCR purification kit (Qiagen Valencia, CA, USA). .. The 454-pyrosequencing data were analyzed in a well-designed pipeline.

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
    Article Snippet: PCR conditions were: 95°C for 2 min; 11 cycles of 95°C for 45 s, 63°C for 30 s, 72°C for 5 min; 7 cycles of 95°C for 45 s, 62°C for 30 s with 1°C decrement per cycle, 72°C for 5 min, 95°C for 45 s; 26 cycles of 56°C for 30 s, 72°C for 5 min; 72°C for 15 min. .. F-PCR products were digested with FastDigest NotI and AscI (Thermo Fisher Cat# ER0595 and ER1891, respectively) at 37°C for 20 min, followed by inactivation at 80°C for 5 min and column purification (Qiagen/QiaQuick PCR Purification Cat# 28106). .. The P1316 plasmid was also NotI/AscI digested and gel purified (Qiagen/QiaQuick Gel Extraction Cat# 28706).

    Article Title: Association of Intact dupA (dupA1) rather than dupA1 cluster with duodenal ulcer in Indian population
    Article Snippet: Primer jhp0919R was located from 326 bp to 350 of ORF jhp0919 of strain J99. .. The amplified products were purified using the QIAquick PCR Purification Kit (QIAGEN). .. The purified PCR product was quantified on gel.

    Article Title: Positional cloning of rp2 QTL associates the P450 genes CYP6Z1,CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vectorAnopheles funestus
    Article Snippet: The PCR were carried out using 10 pmol of each primers and 30 ng of genomic DNA as template in 25 ml reactions. .. PCR products were purified using the QIaquick PCR purification kit (Qiagen, Hilden, Germany) and directly sequenced on both strands. .. Sequences for each gene were analysed to detect the polymorphic sites manually using BioEdit and as sequence differences in multiple alignments using ClustalW ( ).

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: 1 μl of the 4C library was used as template for PCR amplification using oligonucleotide primers adjacent to pairs of HindIII and DpnII sites on the long arm of budding yeast chromosome 5. .. Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification. .. In preparation for sequencing, the DNA samples were end repaired, poly-A tailed and Single End Adapters (Illumina, San Diego, CA) were ligated.

    Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control
    Article Snippet: RT was performed with indexed primers (Supplementary Table ); primers were digested with Exonuclease I (Thermo Scientific, 2 × 30 min with addition of 40 U ExoI before each incubation, followed by heat denaturation at 80 °C for 10 min). .. Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen). .. After quantification (Qubit, Thermo Scientific), libraries in the first set of experiments (wild-type Hel2-HTP) were pooled and sequenced in a single run on HiSeq2500 (Edinburgh Genomics), in fast mode, on two lanes, with 100 bp read length, using custom primers to read out the indices.

    Article Title: Excess of Rare Variants in Genes that are Key Epigenetic Regulators of Spermatogenesis in the Patients with Non-Obstructive Azoospermia
    Article Snippet: The adaptor-ligated DNA products were amplified using index tagged primers in order to tag DNA from different individuals. .. The amplified products were purified with QIAquick PCR purification kits (QIAGEN) and hybridized to the custom-designed capture array as per NimbleGen's Sequence Capture protocol for enrichment. .. Each enriched library was loaded on the HiSeq 2000 platform and pair-end reads with the lengths of 90 bp were generated.

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Poly(A)-containing mRNAs were purified using oligo(dT)-attached magnetic beads and transcriptome libraries were generated using an Illumina TruSeqTM RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification. .. The complete libraries were sequenced using the Illumina HiSeq2500 system (Illumina, San Diego, CA, USA).

    Article Title: Population history provides foundational knowledge for utilizing and developing native plant restoration materials. Population history provides foundational knowledge for utilizing and developing native plant restoration materials
    Article Snippet: Genomic DNA from individual plants was digested with Pst I and Msp I, followed by the ligation of Illumina adaptor sequences; each individual was barcoded four times using unique, 5–10 base pair barcodes. .. Ligation products were pooled, purified using QIAquick PCR kits (Qiagen), and amplified using 16 cycles of PCR with eight replicates. .. A Pippin Prep (Sage Science, Beverly, MA, USA) was used to size select amplicons from 400 to 500 base pairs.

    Article Title: A novel variant of torque teno virus 7 identified in patients with Kawasaki disease
    Article Snippet: Then, REPLI-g Midi reaction buffer (29 μl) and RFPLI-g Midi DNA polymerase (1 μl), also provided in the above kit, were added and incubated at: 30°C for 8 hours, 95°C for 5 minutes, then held on ice. .. The PCR product was cleaned using the QIAquick PCR Clean-up kit (QIAGEN) following the standard manufacturer’s protocols. .. We prepared DNA libraries for sequencing using the TruSeq PCR-free DNA Library Preparation Kit (Illumina, San Diego, CA).

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Immunoprecipitated chromatin was eluted by two sequential incubations with 100 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65°C for 15 min. .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). .. At least 1 ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol.

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: DNA-protein complexes were de-crosslinked by adding 10 µl 5 M NaCl to the samples and incubating at 65 C overnight. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106). .. Quantitative PCR was then performed with input and immunoprecipitated DNA using primers directed to the RAD51 promoter (F: 5′-CCCCCGGCATAAAGTTTGA-3′; R: 5′-GCTTTCAGAATTCCCGCCA-3′ , ), or IGX1A gene desert (QIAGEN, cat#GPH100001C(-)01 A) and Power SYBR according to manufacturer’s instructions, and analysed using a CFX96 Real-Time PCR machine.

    Article Title: Simian malaria in the Brazilian Atlantic forest: first description of natural infection of capuchin monkeys (Cebinae subfamily) by Plasmodium simium
    Article Snippet: Electrophoresis was performed in a room specific for amplified DNA, with appropriated sets of pipettes and plugged pipettes tips. .. For DNA sequencing, PCR products were purified using Purification Kit QIAquick (Qiagen) following manufacturer’s procedure. .. Around 3 ng of purified PCR products were amplified using 2.0 μM of each primer (forward or reverse of species-specific primers of second reaction) and 1 μL of Big Dye terminator kit in a program of: 96°C for 1 min, 35 cycles of 96°C for 15 sec, the temperature of primer annealing for 15 sec and 60°C for 15 sec.

    De-Phosphorylation Assay:

    Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control
    Article Snippet: Dephosphorylation, 3’-linker ligation (0.5 U/μl T4 RNA ligase 1, 5 U/μl T4 RNA ligase 2, tr, KQ), re-phosphorylation with γ-32 P-ATP and non-labelled ATP and 5’-linker ligation were then performed on the Ni-NTA, always in the presence of 1 U/μl RNaseIN RNase inhibitor. .. Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen).

    Gel Extraction:

    Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control
    Article Snippet: RT was performed with indexed primers (Supplementary Table ); primers were digested with Exonuclease I (Thermo Scientific, 2 × 30 min with addition of 40 U ExoI before each incubation, followed by heat denaturation at 80 °C for 10 min). .. Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen). .. After quantification (Qubit, Thermo Scientific), libraries in the first set of experiments (wild-type Hel2-HTP) were pooled and sequenced in a single run on HiSeq2500 (Edinburgh Genomics), in fast mode, on two lanes, with 100 bp read length, using custom primers to read out the indices.

    cDNA Library Assay:

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Poly(A)-containing mRNAs were purified using oligo(dT)-attached magnetic beads and transcriptome libraries were generated using an Illumina TruSeqTM RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification. .. The complete libraries were sequenced using the Illumina HiSeq2500 system (Illumina, San Diego, CA, USA).

    Agarose Gel Electrophoresis:

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: 1 μl of the 4C library was used as template for PCR amplification using oligonucleotide primers adjacent to pairs of HindIII and DpnII sites on the long arm of budding yeast chromosome 5. .. Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification. .. In preparation for sequencing, the DNA samples were end repaired, poly-A tailed and Single End Adapters (Illumina, San Diego, CA) were ligated.

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: RNA quality was examined using agarose gel electrophoresis and NanoDrop spectrophotometry (ThermoFisher Scientific, Waltham, MA, USA). .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: DNA-protein complexes were de-crosslinked by adding 10 µl 5 M NaCl to the samples and incubating at 65 C overnight. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat28106). .. Quantitative PCR was then performed with input and immunoprecipitated DNA using primers directed to the RAD51 promoter (F: 5′-CCCCCGGCATAAAGTTTGA-3′; R: 5′-GCTTTCAGAATTCCCGCCA-3′ , ), or IGX1A gene desert (QIAGEN, cat#GPH100001C(-)01 A) and Power SYBR according to manufacturer’s instructions, and analysed using a CFX96 Real-Time PCR machine.

    Chromatin Immunoprecipitation:

    Article Title: MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1
    Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) ... We purified DNA fragments with the QIAquick PCR purification kit (Qiagen).

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Paragraph title: Chromatin immunoprecipitation for ChIP-seq ... The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA).

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) and quantitative PCR ... For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106).

    Plasmid Preparation:

    Article Title: Biosynthesis of polybrominated aromatic organic compounds by marine bacteria
    Article Snippet: The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen). .. The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen).

    Article Title: A toolbox of IgG subclass-switched recombinant monoclonal antibodies for enhanced multiplex immunolabeling of brain
    Article Snippet: Paragraph title: Cloning of immunoglobulin variable domain regions into a dual promoter expression plasmid ... F-PCR products were digested with FastDigest NotI and AscI (Thermo Fisher Cat# ER0595 and ER1891, respectively) at 37°C for 20 min, followed by inactivation at 80°C for 5 min and column purification (Qiagen/QiaQuick PCR Purification Cat# 28106).

    Real-time Polymerase Chain Reaction:

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: All mRNA targets and primers are listed in . .. KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX). .. Gelatin and hyaluronan substrate zymography was performed using hand cast 10% polyacrylamide gels containing either 0.2% w/v gelatin or 0.17 mg/ mL 1700 kDa sodium hyaluronate.

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) and quantitative PCR ... For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106).

    RNA Extraction:

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Paragraph title: 4.3. RNA Extraction and Sequencing ... A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification.

    Sample Prep:

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: Poly(A)-containing mRNAs were purified using oligo(dT)-attached magnetic beads and transcriptome libraries were generated using an Illumina TruSeqTM RNA Sample Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification.

    Electrophoresis:

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: 1 μl of the 4C library was used as template for PCR amplification using oligonucleotide primers adjacent to pairs of HindIII and DpnII sites on the long arm of budding yeast chromosome 5. .. Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification. .. In preparation for sequencing, the DNA samples were end repaired, poly-A tailed and Single End Adapters (Illumina, San Diego, CA) were ligated.

    Ethanol Precipitation:

    Article Title: Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control
    Article Snippet: Complexes were released from the membrane and proteins were degraded by digestion with Proteinase K; the resulting RNA was purified by phenol–chloroform–isoamyl alcohol extraction and ethanol precipitation. .. Twenty-one cycles of PCR were performed and libraries purified (QIAQuick PCR purification kit, Qiagen), size selected on 2.5% Metaphore agarose (Lonza) and gel-extracted using the MinElute Gel Extraction Kit (Qiagen).

    Next-Generation Sequencing:

    Article Title: Population history provides foundational knowledge for utilizing and developing native plant restoration materials. Population history provides foundational knowledge for utilizing and developing native plant restoration materials
    Article Snippet: Paragraph title: Next‐generation sequencing and data processing ... Ligation products were pooled, purified using QIAquick PCR kits (Qiagen), and amplified using 16 cycles of PCR with eight replicates.

    Chromosome Walking:

    Article Title: Association of Intact dupA (dupA1) rather than dupA1 cluster with duodenal ulcer in Indian population
    Article Snippet: Primer walking method was used for the sequencing of full length of dupA gene. .. The amplified products were purified using the QIAquick PCR Purification Kit (QIAGEN).

    Spectrophotometry:

    Article Title: Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds
    Article Snippet: The quantity and quality of mRNA was assessed using a Nanodrop 2000 UV–vis spectrophotometer (Thermo Fisher Scientific). .. KIAA1199 qPCR products were purified using a QIAquick PCR purification kit (Quiagen, Venlo, Netherlands) and sequenced by Lone Star Laboratories (Houston, TX).

    Article Title: Insights into Body Size Evolution: A Comparative Transcriptome Study on Three Species of Asian Sisoridae Catfish
    Article Snippet: RNA quality was examined using agarose gel electrophoresis and NanoDrop spectrophotometry (ThermoFisher Scientific, Waltham, MA, USA). .. A cDNA library was constructed and purified using a QiaQuick PCR extraction kit (Qiagen, Valencia, CA, USA) and then enriched via PCR amplification.

    Produced:

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: In parallel, crosslinked cells were then thawed, lysed for 20 mins in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl), then 400–1000 bp fragments of chromatin were produced by sonication in a water bath sonicator (4 C, Misonix ultrasound liquid processor) for 13 cycles of 30 sec on, 30 sec off at 65 Amps. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106).

    Immunoprecipitation:

    Article Title: MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1
    Article Snippet: Anti-MAF (Imgenex) and control mouse IgG (Santa Cruz) immunoprecipitated chromatin-protein complexes, and cross-linking reversed with 0.3 M NaCl at 65°C for 12 hours. .. We purified DNA fragments with the QIAquick PCR purification kit (Qiagen).

    Article Title: MIR sequences recruit zinc finger protein ZNF768 to expressed genes
    Article Snippet: Immunoprecipitated chromatin was eluted by two sequential incubations with 100 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65°C for 15 min. .. The two eluates were pooled and incubated at 65°C for 12 h to reverse-crosslink of chromatin, followed by treatment with RNase A (0.2 μg/ml) at 37°C for 2 h and proteinase K (0.2 μg/ml) at 55°C for 2 h. The DNA was isolated by phenol:chloroform:isoamylalcohol (25:24:1 pH 8.0) extraction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA).

    DNA Purification:

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation
    Article Snippet: 1 μl of the 4C library was used as template for PCR amplification using oligonucleotide primers adjacent to pairs of HindIII and DpnII sites on the long arm of budding yeast chromosome 5. .. Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis, the remainder was applied to QIAquick PCR Purification Kit (Qiagen, Netherlands) for DNA purification. .. In preparation for sequencing, the DNA samples were end repaired, poly-A tailed and Single End Adapters (Illumina, San Diego, CA) were ligated.

    Lysis:

    Article Title: CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma
    Article Snippet: In parallel, crosslinked cells were then thawed, lysed for 20 mins in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl), then 400–1000 bp fragments of chromatin were produced by sonication in a water bath sonicator (4 C, Misonix ultrasound liquid processor) for 13 cycles of 30 sec on, 30 sec off at 65 Amps. .. For DNA isolation, proteinase K solution (1 µl proteinase K, 5 µl 0.5 M EDTA, and 10 µl 1 M Tris-HCl) was added to each sample and incubated for 2 hours at 45 C, then purified using a QIAquick PCR purification column (QIAGEN, cat#28106).

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    Qiagen pcr purification kit
    HDAC1 and DNMT1 colocalize on the Cyp1a1 proximal promoter region (A) Schematic representation of the mouse Cyp1a1 promoter and the primer positions used to amplify specific regions in ChIP experiments. Position of AhRE motif clusters and primers (black arrows) relative to the transcriptional start site (blue arrow) numbered as +1 are indicated. A nucleosome (dashed circle), probably positioned over the promoter, is localized on the scheme in addition to other previously described elements where transcription factors interact with the promoter. (B) ChIP assays for HDAC1, DNMT1, AHR and RNA pol II and ReChIP assays for HDAC1 and DNMT1 were performed with chromatin from cells treated for 1.5 h with DMSO vehicle (lanes labeled D) or 5 μM B[ a ]P (lanes labeled B). The <t>DNA</t> was amplified by <t>PCR</t> using primers spanning the enhancer (−0.8 kbp) and the proximal promoter (−0.1 kbp) regions and PCR products were visualized by ethidium bromide staining after gel electrophoresis. (C) DNA was quantified by real-time PCR and normalized to inputs. (D) HDAC1- and DNMT1-imunoprecipitated DNA samples were used to compare the occupancy by HDAC1 and DNMT1 across the Cyp1a1 promoter in control and stimulated cells. DNA enrichment was evaluated by real-time PCR, normalized to inputs and compared to IgG non-specific immunoprecipitation. Results shown in gels are representative of three independent experiments and quantification data are means ± SD of three independent experiments.
    Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1826 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HDAC1 and DNMT1 colocalize on the Cyp1a1 proximal promoter region (A) Schematic representation of the mouse Cyp1a1 promoter and the primer positions used to amplify specific regions in ChIP experiments. Position of AhRE motif clusters and primers (black arrows) relative to the transcriptional start site (blue arrow) numbered as +1 are indicated. A nucleosome (dashed circle), probably positioned over the promoter, is localized on the scheme in addition to other previously described elements where transcription factors interact with the promoter. (B) ChIP assays for HDAC1, DNMT1, AHR and RNA pol II and ReChIP assays for HDAC1 and DNMT1 were performed with chromatin from cells treated for 1.5 h with DMSO vehicle (lanes labeled D) or 5 μM B[ a ]P (lanes labeled B). The DNA was amplified by PCR using primers spanning the enhancer (−0.8 kbp) and the proximal promoter (−0.1 kbp) regions and PCR products were visualized by ethidium bromide staining after gel electrophoresis. (C) DNA was quantified by real-time PCR and normalized to inputs. (D) HDAC1- and DNMT1-imunoprecipitated DNA samples were used to compare the occupancy by HDAC1 and DNMT1 across the Cyp1a1 promoter in control and stimulated cells. DNA enrichment was evaluated by real-time PCR, normalized to inputs and compared to IgG non-specific immunoprecipitation. Results shown in gels are representative of three independent experiments and quantification data are means ± SD of three independent experiments.

    Journal: Biochimica et biophysica acta

    Article Title: HDAC1 bound to the Cyp1a1 promoter blocks histone acetylation associated with Ah receptor-mediated transactivation

    doi: 10.1016/j.bbaexp.2007.07.002

    Figure Lengend Snippet: HDAC1 and DNMT1 colocalize on the Cyp1a1 proximal promoter region (A) Schematic representation of the mouse Cyp1a1 promoter and the primer positions used to amplify specific regions in ChIP experiments. Position of AhRE motif clusters and primers (black arrows) relative to the transcriptional start site (blue arrow) numbered as +1 are indicated. A nucleosome (dashed circle), probably positioned over the promoter, is localized on the scheme in addition to other previously described elements where transcription factors interact with the promoter. (B) ChIP assays for HDAC1, DNMT1, AHR and RNA pol II and ReChIP assays for HDAC1 and DNMT1 were performed with chromatin from cells treated for 1.5 h with DMSO vehicle (lanes labeled D) or 5 μM B[ a ]P (lanes labeled B). The DNA was amplified by PCR using primers spanning the enhancer (−0.8 kbp) and the proximal promoter (−0.1 kbp) regions and PCR products were visualized by ethidium bromide staining after gel electrophoresis. (C) DNA was quantified by real-time PCR and normalized to inputs. (D) HDAC1- and DNMT1-imunoprecipitated DNA samples were used to compare the occupancy by HDAC1 and DNMT1 across the Cyp1a1 promoter in control and stimulated cells. DNA enrichment was evaluated by real-time PCR, normalized to inputs and compared to IgG non-specific immunoprecipitation. Results shown in gels are representative of three independent experiments and quantification data are means ± SD of three independent experiments.

    Article Snippet: Samples were then digested with proteinase K at 45°C for 1.5 h. DNA was purified by chromatography on QIAquick® columns (PCR purification kit, Qiagen), eluted in ddH2 O, and an aliquot was used for analysis by PCR.

    Techniques: Chromatin Immunoprecipitation, Labeling, Amplification, Polymerase Chain Reaction, Staining, Nucleic Acid Electrophoresis, Real-time Polymerase Chain Reaction, Immunoprecipitation

    AHR activation by B[ a ]P induces posttranslational modification of histones in the Cyp1a1 promoter (A) Hepa-1 cells were treated for 1.5 h with DMSO (lanes labeled D) or B[ a ]P (lanes labeled B). Cells were then used for ChIP analysis using specific antibodies raised against post-translationally modified histone amino acids. ChIP DNA was amplified by PCR using primers specific for the indicated regions. PCR products amplified with −3.2, −0.8 and −0.1 kbp primers were resolved by electrophoresis. Results shown are representative of three independent experiments. (B) Results of real time PCR amplification are expressed as percent of total input. Data are the means (± SD) of three independent experiments.

    Journal: Biochimica et biophysica acta

    Article Title: HDAC1 bound to the Cyp1a1 promoter blocks histone acetylation associated with Ah receptor-mediated transactivation

    doi: 10.1016/j.bbaexp.2007.07.002

    Figure Lengend Snippet: AHR activation by B[ a ]P induces posttranslational modification of histones in the Cyp1a1 promoter (A) Hepa-1 cells were treated for 1.5 h with DMSO (lanes labeled D) or B[ a ]P (lanes labeled B). Cells were then used for ChIP analysis using specific antibodies raised against post-translationally modified histone amino acids. ChIP DNA was amplified by PCR using primers specific for the indicated regions. PCR products amplified with −3.2, −0.8 and −0.1 kbp primers were resolved by electrophoresis. Results shown are representative of three independent experiments. (B) Results of real time PCR amplification are expressed as percent of total input. Data are the means (± SD) of three independent experiments.

    Article Snippet: Samples were then digested with proteinase K at 45°C for 1.5 h. DNA was purified by chromatography on QIAquick® columns (PCR purification kit, Qiagen), eluted in ddH2 O, and an aliquot was used for analysis by PCR.

    Techniques: Activation Assay, Modification, Labeling, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Electrophoresis, Real-time Polymerase Chain Reaction

    Nuak2 is a direct TGFβ target gene. A , cloning of the mouse Nuak2 promoter and enhancer. Shown is a schematic diagram of the mouse Nuak2 gene spanning the promoter sequences, the transcriptional start site ( TSS ), the first exon, and part of the first intronic sequence. B , ChIP assays using an antibody against endogenous SMAD2/SMAD3 ( S2/3 ) and amplification of genomic sequences corresponding to the Nuak2 intronic enhancer, the Pai1 promoter, and the β-globin ( Hbb ) control region in NMuMG cells stimulated with or without 5 ng/ml TGFβ for 1 h. Control immunoprecipitations with mouse IgG are also shown as reference. The amount of PCR-amplified DNA signal after ChIP is normalized against the equivalent PCR signal of the input chromatin prior to immunoprecipitation, and the relative ratios are shown in the diagrams as average values determined from triplicate determinations with their corresponding S.E. ( error bars ). C , the genomic fragments, depicted in A , that were cloned into the luciferase reporter are shown relative to the luciferase ( luc ) cDNA in the corresponding constructs. D , luciferase assay from HEK 293T cells transiently transfected with the Nuak2 enhancer construct and pcDNA3 control or constitutively active pcDNA3-ALK5TD. E , luciferase assay was performed using NMuMG cells transiently transfected with the Nuak2 1- or 2-kbp promoters and the Nuak2 intronic enhancer constructs with or without TGFβ (5 ng/ml) stimulation for 17 h. Each independent experiment was repeated at least three times. Asterisks depict differences compared with respective controls or between the conditions indicated with lines : *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transforming growth factor β (TGFβ) induces NUAK kinase expression to fine-tune its signaling output

    doi: 10.1074/jbc.RA118.004984

    Figure Lengend Snippet: Nuak2 is a direct TGFβ target gene. A , cloning of the mouse Nuak2 promoter and enhancer. Shown is a schematic diagram of the mouse Nuak2 gene spanning the promoter sequences, the transcriptional start site ( TSS ), the first exon, and part of the first intronic sequence. B , ChIP assays using an antibody against endogenous SMAD2/SMAD3 ( S2/3 ) and amplification of genomic sequences corresponding to the Nuak2 intronic enhancer, the Pai1 promoter, and the β-globin ( Hbb ) control region in NMuMG cells stimulated with or without 5 ng/ml TGFβ for 1 h. Control immunoprecipitations with mouse IgG are also shown as reference. The amount of PCR-amplified DNA signal after ChIP is normalized against the equivalent PCR signal of the input chromatin prior to immunoprecipitation, and the relative ratios are shown in the diagrams as average values determined from triplicate determinations with their corresponding S.E. ( error bars ). C , the genomic fragments, depicted in A , that were cloned into the luciferase reporter are shown relative to the luciferase ( luc ) cDNA in the corresponding constructs. D , luciferase assay from HEK 293T cells transiently transfected with the Nuak2 enhancer construct and pcDNA3 control or constitutively active pcDNA3-ALK5TD. E , luciferase assay was performed using NMuMG cells transiently transfected with the Nuak2 1- or 2-kbp promoters and the Nuak2 intronic enhancer constructs with or without TGFβ (5 ng/ml) stimulation for 17 h. Each independent experiment was repeated at least three times. Asterisks depict differences compared with respective controls or between the conditions indicated with lines : *, p

    Article Snippet: The precipitated complexes were washed five times with radioimmune precipitation assay washing buffer (50 mm HEPES-KOH, pH 7.0, 0.5 m LiCl, 1 mm EDTA, 0.7% (w/v) sodium deoxycholate, 1% Igepal CA630) and once with TE buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA), and DNA was eluted in 200 μl of elution buffer (lysis buffer without protease inhibitor mixture) after shaking at 65 °C for 6 h. For the ChIP input controls, 100 μl of sonicated cell lysate were diluted 4 times with elution buffer and treated at 65 °C for 6 h. Eluted DNA and input DNA were purified using a PCR purification kit (Qiagen AB, Sollentuna, Sweden) and were then analyzed by a qPCR assay using specific primers for the mouse Nuak2 intron-enhancer region (forward, 5′-TGAGAAACGACGGAGACAAGCTGCT-3′; reverse, 5′-GTCTGGAGGTTTTGCTGCAGGTCTG-3′), mouse Pai-1 enhancer (forward, 5′-GTCCAAGAGGAACGAGAACC-3′; reverse, 5′-GGCTTTGTAGGCTCTTGTGG-3′), and mouse Hbb (hemoglobin B) gene serving as a control genomic region (forward, 5′-CAACCTGCCCAGGGCCTCAC-3′; reverse, 5′-AGGCTGCTGTCTCTGGCCTGT-3′).

    Techniques: Clone Assay, Sequencing, Chromatin Immunoprecipitation, Amplification, Genomic Sequencing, Polymerase Chain Reaction, Immunoprecipitation, Luciferase, Construct, Transfection

    MORF2-REP profiles of Ethiopian T . evansi stocks and T . evansi and T . brucei reference strains. 1.5% agarose gel showing MORF2-REP minisatellite PCR amplicons. Lane M: 100 bp plus marker, lanes 1 to 14: Ethiopian T . evansi stocks MCAM/ET/2013/MU/01-02-04-05-06-07-08-09-10-11-13-14-15-17, lane 15: T . b . gambiense LiTat 1.3, lane 16: T . b . brucei AnTat 1.1 E lane 17: T . evansi type A (RoTat 1.2), lane 18: T . evansi type B (KETRI 2479), lane 19: T . equiperdum Dodola 940, lane 20: T . b . gambiense ABBA, lane N: negative control

    Journal: PLoS Neglected Tropical Diseases

    Article Title: New Trypanosoma evansi Type B Isolates from Ethiopian Dromedary Camels

    doi: 10.1371/journal.pntd.0004556

    Figure Lengend Snippet: MORF2-REP profiles of Ethiopian T . evansi stocks and T . evansi and T . brucei reference strains. 1.5% agarose gel showing MORF2-REP minisatellite PCR amplicons. Lane M: 100 bp plus marker, lanes 1 to 14: Ethiopian T . evansi stocks MCAM/ET/2013/MU/01-02-04-05-06-07-08-09-10-11-13-14-15-17, lane 15: T . b . gambiense LiTat 1.3, lane 16: T . b . brucei AnTat 1.1 E lane 17: T . evansi type A (RoTat 1.2), lane 18: T . evansi type B (KETRI 2479), lane 19: T . equiperdum Dodola 940, lane 20: T . b . gambiense ABBA, lane N: negative control

    Article Snippet: For direct sequencing, PCR was performed in 50–100 μl volumes and amplicons were cleaned up and concentrated using a PCR cleanup kit (QIAquick PCR Purification Kit, Qiagen, Germany) and sent out for bidirectional direct sequencing at the Genetic Sequencing Facility (VIB, Belgium) using the described PCR primers.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker, Negative Control