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    Structured Review

    Qiagen 1x q solution
    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    1x Q Solution, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection"

    Article Title: Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-13-206

    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    Figure Legend Snippet: Increasing the assay specificity of the JAK2 V617F mutation-specific PCR. Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate. A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification. B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification. C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification. D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification

    Related Articles

    Centrifugation:

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    Article Snippet: .. The MinElute columns were subsequently placed into 2-mL Eppendorf tubes, washed with PE buffer (Qiagen), and dried by centrifugation at maximum speed. .. The DNA was finally eluted with 25 μL Tris-EDTA-Triton X-100 buffer, incubated for 2 min, and collected by centrifugation for 1 min at maximum speed.

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    Article Title: SmpB functions in various steps of trans-translation
    Article Snippet: The cells were harvested by centrifugation, resuspended in 30 ml of buffer A [50 mM HEPES (pH 7.5), 100 mM potassium chloride, 10% glycerol (w/v) and 1 mM dithiothreitol] and lysed by sonication. .. Fractions containing SmpB were dialyzed with buffer B [50 mM HEPES (pH 7.5), 200 mM potassium chloride, 10% glycerol and 1 mM dithiothreitol] and loaded onto Ni-NTA agarose (Qiagen).

    Article Title: Differences in the Fitness of Two Diverse Wild-Type Human Immunodeficiency Virus Type 1 Isolates Are Related to the Efficiency of Cell Binding and Entry
    Article Snippet: .. Cells on the filter were washed twice with 300 μl of wash buffer (50 mM HEPES, 5 mM MgCl2 , 1 mM CaCl2 , 500 mM NaCl) by centrifugation at 300 × g for 2 min and then lysed in the buffer AVL containing carrier RNA (Qiagen) ( ). .. RNA was extracted by using a Qiagen viral RNA minikit and was reversed transcribed by using M-MLV RT (Invitrogen) and the E105 primer ( ). cDNA was then amplified with an external set of primers (ED33 and ED5) and a nested set of primers (E80 and E125) (C2-V3 env region) for HTA analysis (see above).

    Amplification:

    Article Title: Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2 †
    Article Snippet: Paragraph title: PCR amplification. ... Three microliters of each PCR product was checked for homogeneity and size on an agarose gel; 12 μl of the products was purified with a QIAquick PCR Purification kit (QIAGEN, Westburg, Germany), according to the protocol but with an extra washing step with 500 μl PE buffer (QIAGEN), and then eluted with 30 μl elution buffer after 1 min of incubation.

    Article Title: Differences in the Fitness of Two Diverse Wild-Type Human Immunodeficiency Virus Type 1 Isolates Are Related to the Efficiency of Cell Binding and Entry
    Article Snippet: Cells on the filter were washed twice with 300 μl of wash buffer (50 mM HEPES, 5 mM MgCl2 , 1 mM CaCl2 , 500 mM NaCl) by centrifugation at 300 × g for 2 min and then lysed in the buffer AVL containing carrier RNA (Qiagen) ( ). .. RNA was extracted by using a Qiagen viral RNA minikit and was reversed transcribed by using M-MLV RT (Invitrogen) and the E105 primer ( ). cDNA was then amplified with an external set of primers (ED33 and ED5) and a nested set of primers (E80 and E125) (C2-V3 env region) for HTA analysis (see above).

    Autoradiography:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: Beads were washed three times with GST-binding buffer, and then bound proteins were resolved by SDS-PAGE and detected by autoradiography. .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    Construct:

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    Article Snippet: A binding apparatus was constructed by fitting an extension reservoir removed from a Zymo-Spin V column (Zymo Research) into a MinElute silica spin column (Qiagen) and placing them into a 50-mL Falcon tube. .. The MinElute columns were subsequently placed into 2-mL Eppendorf tubes, washed with PE buffer (Qiagen), and dried by centrifugation at maximum speed.

    Article Title: In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
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    SYBR Green Assay:

    Article Title: In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
    Article Snippet: .. Each PCR mixture consisted of a total volume of 50 μl and contained 5 μl of the appropriate primers (5 μM) (NIFGEO225F/560R or RECGEO202F/670R), 5 μl of the RT-PCR product, 5 μl of 10× SYBR green PCR buffer (PE Biosystems, Foster City, Calif.), 6 μl of MgCl2 solution (25 mM; PE Biosystems), 4 μl of the deoxynucleoside triphosphate mix (2.5 mM; PE Biosystems), 10 μl of buffer Q solution (QIAGEN), 2 μl of bovine serum albumin (10 mg/ml; New England Biolabs, Beverly, Mass.), 0.5 U of AmpErase uracil- N -glycosylase (PE Biosystems), and 0.5 U of Taq polymerase (QIAGEN). .. The RT-PCR products used to construct the TaqMan standard curve were purified by the protocol for phenol extraction and ethanol precipitation of nucleic acids outlined in Current Protocols in Molecular Biology ( ).

    Incubation:

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    Article Title: Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2 †
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    Expressing:

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    Article Title: In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
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    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
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    Transformation Assay:

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    Article Snippet: Briefly, 8 L transformed bacterial culture was grown to a density of OD600 ~0.5 and induced with 1-mM IPTG for 4 h at 37°C. .. Cells were collected by centrifugation and resuspended in Buffer A (6-M Guanidine HCl, 0.1-M NaH2PO4, 0.01-M Tris pH 8.0, 5 mL/g cell pellet weight), and the clarified supernatant was incubated with Ni-NTA nickel resin beads (Qiagen) for 1 h at room temperature (RT).

    Competitive Binding Assay:

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    Article Snippet: Paragraph title: Whole virus-cell competitive binding assay. ... Cells on the filter were washed twice with 300 μl of wash buffer (50 mM HEPES, 5 mM MgCl2 , 1 mM CaCl2 , 500 mM NaCl) by centrifugation at 300 × g for 2 min and then lysed in the buffer AVL containing carrier RNA (Qiagen) ( ).

    Flow Cytometry:

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    Article Snippet: .. The flow-through was discarded and 750 µl Qiagen Buffer PE (PE) was added and incubated for 2 min before centrifuging at 12,000 rpm for 1 min. ..

    Article Title: SmpB functions in various steps of trans-translation
    Article Snippet: The S100 fraction prepared from the lysate was loaded on an SP Sepharose Fast Flow column (Amersham Pharmacia Biotech). .. Fractions containing SmpB were dialyzed with buffer B [50 mM HEPES (pH 7.5), 200 mM potassium chloride, 10% glycerol and 1 mM dithiothreitol] and loaded onto Ni-NTA agarose (Qiagen).

    Protease Inhibitor:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: In vitro translated proteins were incubated at 4 °C for 3 h with GST or GST fusion protein in 700 μl of GST-binding buffer containing 20 m m Tris-HCl (pH 8.0), 0.5% Nonidet P-40, 150 m m NaCl, 1 m m EDTA, 1 m m dithiothreitol, 10% glycerol, 1 m m phenylmethylsulfonyl fluoride, and a protease inhibitor mixture. .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    Cell Culture:

    Article Title: SmpB functions in various steps of trans-translation
    Article Snippet: Fractions containing SmpB were dialyzed with buffer B [50 mM HEPES (pH 7.5), 200 mM potassium chloride, 10% glycerol and 1 mM dithiothreitol] and loaded onto Ni-NTA agarose (Qiagen). .. The column was washed with buffer C [50 mM HEPES (pH 7.5), 200 mM potassium chloride, 10% glycerol, 20 mM imidazole and 1 mM dithiothreitol] and eluted with a linear gradient from 20 to 1000 mM imidazole in buffer C. Typically, 0.2 mg of SmpB with > 95% purity was yielded from a liter of cell culture.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
    Article Snippet: .. Each PCR mixture consisted of a total volume of 50 μl and contained 5 μl of the appropriate primers (5 μM) (NIFGEO225F/560R or RECGEO202F/670R), 5 μl of the RT-PCR product, 5 μl of 10× SYBR green PCR buffer (PE Biosystems, Foster City, Calif.), 6 μl of MgCl2 solution (25 mM; PE Biosystems), 4 μl of the deoxynucleoside triphosphate mix (2.5 mM; PE Biosystems), 10 μl of buffer Q solution (QIAGEN), 2 μl of bovine serum albumin (10 mg/ml; New England Biolabs, Beverly, Mass.), 0.5 U of AmpErase uracil- N -glycosylase (PE Biosystems), and 0.5 U of Taq polymerase (QIAGEN). .. The RT-PCR products used to construct the TaqMan standard curve were purified by the protocol for phenol extraction and ethanol precipitation of nucleic acids outlined in Current Protocols in Molecular Biology ( ).

    Sonication:

    Article Title: Chromatin Immunoprecipitation for Human Monocyte Derived Macrophages
    Article Snippet: Paragraph title: 2.6 Chromatin Reverse Cross-linking and Efficiency of Sonication ... The flow-through was discarded and 750 µl Qiagen Buffer PE (PE) was added and incubated for 2 min before centrifuging at 12,000 rpm for 1 min.

    Article Title: SmpB functions in various steps of trans-translation
    Article Snippet: The cells were harvested by centrifugation, resuspended in 30 ml of buffer A [50 mM HEPES (pH 7.5), 100 mM potassium chloride, 10% glycerol (w/v) and 1 mM dithiothreitol] and lysed by sonication. .. Fractions containing SmpB were dialyzed with buffer B [50 mM HEPES (pH 7.5), 200 mM potassium chloride, 10% glycerol and 1 mM dithiothreitol] and loaded onto Ni-NTA agarose (Qiagen).

    Affinity Purification:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: Glutathione S-Transferase Pulldown Assay —GST and GST fusion proteins were expressed in Escherichia coli strain BL21 and affinity-purified using glutathione-Sepharose beads (Amersham Biosciences). .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    Recombinant:

    Article Title: Dominant negative Bmp5 mutation reveals key role of BMPs in skeletal response to mechanical stimulation
    Article Snippet: Recombinant protein containing murine BMP5 pro region (amino acids 1–310) fused with thioredoxin was expressed under the IPTG-inducible pET32 system in BL21 E. Coli bacterial cells as instructed by the manufacturer (Novagen). .. Cells were collected by centrifugation and resuspended in Buffer A (6-M Guanidine HCl, 0.1-M NaH2PO4, 0.01-M Tris pH 8.0, 5 mL/g cell pellet weight), and the clarified supernatant was incubated with Ni-NTA nickel resin beads (Qiagen) for 1 h at room temperature (RT).

    DNA Extraction:

    Article Title: Museum samples reveal rapid evolution by wild honey bees exposed to a novel parasite
    Article Snippet: Paragraph title: Genomic DNA extraction ... Beads with bound DNA were washed with 200 μl PE buffer (Qiagen) for 10 min at room temperature on a rotary mixer followed by bead separation on a magnetic stand.

    Article Title: Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain
    Article Snippet: Paragraph title: DNA Extraction. ... The MinElute columns were subsequently placed into 2-mL Eppendorf tubes, washed with PE buffer (Qiagen), and dried by centrifugation at maximum speed.

    Article Title: The Arrival of Siberian Ancestry Connecting the Eastern Baltic to Uralic Speakers Further East
    Article Snippet: Paragraph title: DNA extraction ... The DNA solution was concentrated to 250 μl (Amicon Ultra-15 30 kDa, Merck Millipore) and purified in large volume columns (High Pure Viral Nucleic Acid Large Volume Kit, Roche) using 2.5 ml of PB buffer, 1 ml of PE buffer and 50 μl of EB buffer (MinElute PCR Purification Kit, QIAGEN).

    Article Title: Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics
    Article Snippet: Paragraph title: Genomic DNA extraction from museum specimens ... Beads with bound DNA were washed with 200 µl PE buffer (Qiagen) for 10 minutes at room temperature on a rotary mixer followed by bead separation on a magnetic stand.

    Nucleic Acid Electrophoresis:

    Article Title: Selection of reference genes for expression studies with fish myogenic cell cultures
    Article Snippet: For samples where enough RNA was obtained (excludes day 2), the integrity of the RNA was confirmed by gel electrophoresis. .. Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect reverse transcription kit (Qiagen Inc., Chatsworth, CA, USA).

    Article Title: Transcriptional Regulation of the IGF Signaling Pathway by Amino Acids and Insulin-Like Growth Factors during Myogenesis in Atlantic Salmon
    Article Snippet: For samples where enough RNA was obtained (excludes day 2), the integrity of the RNA was confirmed by gel electrophoresis. .. Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect reverse transcription kit (Qiagen Inc., Chatsworth, CA, USA).

    In Vivo:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: In Vivo Ubiquitination Assay —HEK-293T cells were transfected with pcDNA3-His-c-Jun or pcDNA3-His-c-Fos, pcDNA3-HA-HBZ, and pcDNA3-FLAG-ubiquitin (Ub) in different combinations. .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    Transfection:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: In Vivo Ubiquitination Assay —HEK-293T cells were transfected with pcDNA3-His-c-Jun or pcDNA3-His-c-Fos, pcDNA3-HA-HBZ, and pcDNA3-FLAG-ubiquitin (Ub) in different combinations. .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    Purification:

    Article Title: In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
    Article Snippet: Each PCR mixture consisted of a total volume of 50 μl and contained 5 μl of the appropriate primers (5 μM) (NIFGEO225F/560R or RECGEO202F/670R), 5 μl of the RT-PCR product, 5 μl of 10× SYBR green PCR buffer (PE Biosystems, Foster City, Calif.), 6 μl of MgCl2 solution (25 mM; PE Biosystems), 4 μl of the deoxynucleoside triphosphate mix (2.5 mM; PE Biosystems), 10 μl of buffer Q solution (QIAGEN), 2 μl of bovine serum albumin (10 mg/ml; New England Biolabs, Beverly, Mass.), 0.5 U of AmpErase uracil- N -glycosylase (PE Biosystems), and 0.5 U of Taq polymerase (QIAGEN). .. The RT-PCR products used to construct the TaqMan standard curve were purified by the protocol for phenol extraction and ethanol precipitation of nucleic acids outlined in Current Protocols in Molecular Biology ( ).

    Article Title: Chromatin Immunoprecipitation for Human Monocyte Derived Macrophages
    Article Snippet: The PCR Purification Column (Qiagen, Hilden, Germany) was used to purify the DNA instead of the phenol:chloroform method recommended by Active Motif. .. The flow-through was discarded and 750 µl Qiagen Buffer PE (PE) was added and incubated for 2 min before centrifuging at 12,000 rpm for 1 min.

    Article Title: The Arrival of Siberian Ancestry Connecting the Eastern Baltic to Uralic Speakers Further East
    Article Snippet: .. The DNA solution was concentrated to 250 μl (Amicon Ultra-15 30 kDa, Merck Millipore) and purified in large volume columns (High Pure Viral Nucleic Acid Large Volume Kit, Roche) using 2.5 ml of PB buffer, 1 ml of PE buffer and 50 μl of EB buffer (MinElute PCR Purification Kit, QIAGEN). .. Sequencing libraries were built using NEBNext DNA Library Prep Master Mix Set for 454 (E6070, New England Biolabs) and Illumina-specific adaptors [ ] following established protocols [ – ].

    Article Title: SmpB functions in various steps of trans-translation
    Article Snippet: Paragraph title: Purification of His-tagged SmpB protein ... Fractions containing SmpB were dialyzed with buffer B [50 mM HEPES (pH 7.5), 200 mM potassium chloride, 10% glycerol and 1 mM dithiothreitol] and loaded onto Ni-NTA agarose (Qiagen).

    Article Title: Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2 †
    Article Snippet: .. Three microliters of each PCR product was checked for homogeneity and size on an agarose gel; 12 μl of the products was purified with a QIAquick PCR Purification kit (QIAGEN, Westburg, Germany), according to the protocol but with an extra washing step with 500 μl PE buffer (QIAGEN), and then eluted with 30 μl elution buffer after 1 min of incubation. .. Sometimes, when possible donor mobile-element copies were examined, multiple bands were visible on the agarose gel, and then each band was excised from the gel and extracted with the QIAquick gel extraction kit (QIAGEN) according to the protocol, but using the optional step of an extra wash with buffer QG (QIAGEN), an extra washing step with 700 μl buffer PE incubated for 2 min, and elution in 30 μl elution buffer after a 1-min incubation.

    Article Title: Differences in the Fitness of Two Diverse Wild-Type Human Immunodeficiency Virus Type 1 Isolates Are Related to the Efficiency of Cell Binding and Entry
    Article Snippet: Viruses B5, C5, NL4-3B5 gp120 , and NL4-3C5 gp120 were purified, added to the cells at an MOI of 0.0001 and were competed off the cells by the addition of NSI isolate B2 (92BR017) or the SI isolate D1 (92UG021) at fivefold MOI intervals from 0.0001 to 0.0625. .. Cells on the filter were washed twice with 300 μl of wash buffer (50 mM HEPES, 5 mM MgCl2 , 1 mM CaCl2 , 500 mM NaCl) by centrifugation at 300 × g for 2 min and then lysed in the buffer AVL containing carrier RNA (Qiagen) ( ).

    Polymerase Chain Reaction:

    Article Title: In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
    Article Snippet: .. Each PCR mixture consisted of a total volume of 50 μl and contained 5 μl of the appropriate primers (5 μM) (NIFGEO225F/560R or RECGEO202F/670R), 5 μl of the RT-PCR product, 5 μl of 10× SYBR green PCR buffer (PE Biosystems, Foster City, Calif.), 6 μl of MgCl2 solution (25 mM; PE Biosystems), 4 μl of the deoxynucleoside triphosphate mix (2.5 mM; PE Biosystems), 10 μl of buffer Q solution (QIAGEN), 2 μl of bovine serum albumin (10 mg/ml; New England Biolabs, Beverly, Mass.), 0.5 U of AmpErase uracil- N -glycosylase (PE Biosystems), and 0.5 U of Taq polymerase (QIAGEN). .. The RT-PCR products used to construct the TaqMan standard curve were purified by the protocol for phenol extraction and ethanol precipitation of nucleic acids outlined in Current Protocols in Molecular Biology ( ).

    Article Title: Chromatin Immunoprecipitation for Human Monocyte Derived Macrophages
    Article Snippet: The PCR Purification Column (Qiagen, Hilden, Germany) was used to purify the DNA instead of the phenol:chloroform method recommended by Active Motif. .. The flow-through was discarded and 750 µl Qiagen Buffer PE (PE) was added and incubated for 2 min before centrifuging at 12,000 rpm for 1 min.

    Article Title: The Arrival of Siberian Ancestry Connecting the Eastern Baltic to Uralic Speakers Further East
    Article Snippet: .. The DNA solution was concentrated to 250 μl (Amicon Ultra-15 30 kDa, Merck Millipore) and purified in large volume columns (High Pure Viral Nucleic Acid Large Volume Kit, Roche) using 2.5 ml of PB buffer, 1 ml of PE buffer and 50 μl of EB buffer (MinElute PCR Purification Kit, QIAGEN). .. Sequencing libraries were built using NEBNext DNA Library Prep Master Mix Set for 454 (E6070, New England Biolabs) and Illumina-specific adaptors [ ] following established protocols [ – ].

    Article Title: Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2 †
    Article Snippet: .. Three microliters of each PCR product was checked for homogeneity and size on an agarose gel; 12 μl of the products was purified with a QIAquick PCR Purification kit (QIAGEN, Westburg, Germany), according to the protocol but with an extra washing step with 500 μl PE buffer (QIAGEN), and then eluted with 30 μl elution buffer after 1 min of incubation. .. Sometimes, when possible donor mobile-element copies were examined, multiple bands were visible on the agarose gel, and then each band was excised from the gel and extracted with the QIAquick gel extraction kit (QIAGEN) according to the protocol, but using the optional step of an extra wash with buffer QG (QIAGEN), an extra washing step with 700 μl buffer PE incubated for 2 min, and elution in 30 μl elution buffer after a 1-min incubation.

    Selection:

    Article Title: Genome-scale Mapping of DNaseI Hypersensitivity
    Article Snippet: Paragraph title: Size selection of DNaseI hypersensitive sites ... 19 After samples have been loaded onto Minelute columns, they are allowed to incubate with Qiagen wash buffer (PE) for 5 minutes before being filtered through the column.

    Lysis:

    Article Title: Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain
    Article Snippet: Following lysis, the sample was extracted with the following protocol: the supernatant was added to 15 mL binding buffer containing 5 M guanidine hydrochloride, 40% (vol/vol) isopropanol, 0.05% Tween-20, and 90 mM sodium acetate. .. The MinElute columns were subsequently placed into 2-mL Eppendorf tubes, washed with PE buffer (Qiagen), and dried by centrifugation at maximum speed.

    SDS Page:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: Beads were washed three times with GST-binding buffer, and then bound proteins were resolved by SDS-PAGE and detected by autoradiography. .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    Ubiquitin Assay:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: In Vivo Ubiquitination Assay —HEK-293T cells were transfected with pcDNA3-His-c-Jun or pcDNA3-His-c-Fos, pcDNA3-HA-HBZ, and pcDNA3-FLAG-ubiquitin (Ub) in different combinations. .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    RNA Extraction:

    Article Title: Selection of reference genes for expression studies with fish myogenic cell cultures
    Article Snippet: Paragraph title: RNA extraction and cDNA synthesis ... Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect reverse transcription kit (Qiagen Inc., Chatsworth, CA, USA).

    Article Title: Transcriptional Regulation of the IGF Signaling Pathway by Amino Acids and Insulin-Like Growth Factors during Myogenesis in Atlantic Salmon
    Article Snippet: Paragraph title: RNA extraction and cDNA synthesis ... Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect reverse transcription kit (Qiagen Inc., Chatsworth, CA, USA).

    Binding Assay:

    Article Title: Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain
    Article Snippet: The 25- to 30-mL solution containing binding buffer and the extraction supernatant was poured into the extension reservoir and centrifuged for 5 min at 269 × g . .. The MinElute columns were subsequently placed into 2-mL Eppendorf tubes, washed with PE buffer (Qiagen), and dried by centrifugation at maximum speed.

    Article Title: Genome-scale Mapping of DNaseI Hypersensitivity
    Article Snippet: [*Author; please expand on what you mean by ‘using a vacuum manifold’, e.g., where is it attached, and also please clarify how the syringe is used in applying and efficiently binding the samples to the Qiagen column. .. 19 After samples have been loaded onto Minelute columns, they are allowed to incubate with Qiagen wash buffer (PE) for 5 minutes before being filtered through the column.

    Article Title: Differences in the Fitness of Two Diverse Wild-Type Human Immunodeficiency Virus Type 1 Isolates Are Related to the Efficiency of Cell Binding and Entry
    Article Snippet: Viruses were added to cells in binding buffer (50 mM HEPES, 5 mM MgCl2 , 1 mM CaCl2 , 5% bovine serum albumin [BSA], 0.1% NaN3 ) on ice and then incubated for 1 h at 15°C ( ). .. Cells on the filter were washed twice with 300 μl of wash buffer (50 mM HEPES, 5 mM MgCl2 , 1 mM CaCl2 , 500 mM NaCl) by centrifugation at 300 × g for 2 min and then lysed in the buffer AVL containing carrier RNA (Qiagen) ( ).

    Agarose Gel Electrophoresis:

    Article Title: Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2 †
    Article Snippet: .. Three microliters of each PCR product was checked for homogeneity and size on an agarose gel; 12 μl of the products was purified with a QIAquick PCR Purification kit (QIAGEN, Westburg, Germany), according to the protocol but with an extra washing step with 500 μl PE buffer (QIAGEN), and then eluted with 30 μl elution buffer after 1 min of incubation. .. Sometimes, when possible donor mobile-element copies were examined, multiple bands were visible on the agarose gel, and then each band was excised from the gel and extracted with the QIAquick gel extraction kit (QIAGEN) according to the protocol, but using the optional step of an extra wash with buffer QG (QIAGEN), an extra washing step with 700 μl buffer PE incubated for 2 min, and elution in 30 μl elution buffer after a 1-min incubation.

    In Vitro:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: In vitro translated proteins were incubated at 4 °C for 3 h with GST or GST fusion protein in 700 μl of GST-binding buffer containing 20 m m Tris-HCl (pH 8.0), 0.5% Nonidet P-40, 150 m m NaCl, 1 m m EDTA, 1 m m dithiothreitol, 10% glycerol, 1 m m phenylmethylsulfonyl fluoride, and a protease inhibitor mixture. .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    Ethanol Precipitation:

    Article Title: Selection of reference genes for expression studies with fish myogenic cell cultures
    Article Snippet: RNA was concentrated by ethanol precipitation and quantified using a NanaoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect reverse transcription kit (Qiagen Inc., Chatsworth, CA, USA).

    Article Title: In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
    Article Snippet: Each PCR mixture consisted of a total volume of 50 μl and contained 5 μl of the appropriate primers (5 μM) (NIFGEO225F/560R or RECGEO202F/670R), 5 μl of the RT-PCR product, 5 μl of 10× SYBR green PCR buffer (PE Biosystems, Foster City, Calif.), 6 μl of MgCl2 solution (25 mM; PE Biosystems), 4 μl of the deoxynucleoside triphosphate mix (2.5 mM; PE Biosystems), 10 μl of buffer Q solution (QIAGEN), 2 μl of bovine serum albumin (10 mg/ml; New England Biolabs, Beverly, Mass.), 0.5 U of AmpErase uracil- N -glycosylase (PE Biosystems), and 0.5 U of Taq polymerase (QIAGEN). .. The RT-PCR products used to construct the TaqMan standard curve were purified by the protocol for phenol extraction and ethanol precipitation of nucleic acids outlined in Current Protocols in Molecular Biology ( ).

    Article Title: Transcriptional Regulation of the IGF Signaling Pathway by Amino Acids and Insulin-Like Growth Factors during Myogenesis in Atlantic Salmon
    Article Snippet: RNA was concentrated by ethanol precipitation and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect reverse transcription kit (Qiagen Inc., Chatsworth, CA, USA).

    Spectrophotometry:

    Article Title: Selection of reference genes for expression studies with fish myogenic cell cultures
    Article Snippet: RNA was concentrated by ethanol precipitation and quantified using a NanaoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect reverse transcription kit (Qiagen Inc., Chatsworth, CA, USA).

    Article Title: In Situ Expression of nifD in Geobacteraceae in Subsurface Sediments
    Article Snippet: Each PCR mixture consisted of a total volume of 50 μl and contained 5 μl of the appropriate primers (5 μM) (NIFGEO225F/560R or RECGEO202F/670R), 5 μl of the RT-PCR product, 5 μl of 10× SYBR green PCR buffer (PE Biosystems, Foster City, Calif.), 6 μl of MgCl2 solution (25 mM; PE Biosystems), 4 μl of the deoxynucleoside triphosphate mix (2.5 mM; PE Biosystems), 10 μl of buffer Q solution (QIAGEN), 2 μl of bovine serum albumin (10 mg/ml; New England Biolabs, Beverly, Mass.), 0.5 U of AmpErase uracil- N -glycosylase (PE Biosystems), and 0.5 U of Taq polymerase (QIAGEN). .. UV2401-PC dual-beam spectrophotometer at an absorbance of 260 nm.

    Article Title: Transcriptional Regulation of the IGF Signaling Pathway by Amino Acids and Insulin-Like Growth Factors during Myogenesis in Atlantic Salmon
    Article Snippet: RNA was concentrated by ethanol precipitation and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Residual genomic DNA was removed using the genomic DNA wipeout buffer included in the Quantitect reverse transcription kit (Qiagen Inc., Chatsworth, CA, USA).

    Produced:

    Article Title: Human T-cell Leukemia Virus Type 1 HBZ Protein Bypasses the Targeting Function of Ubiquitination *
    Article Snippet: For in vitro translation, [35 S]methionine (Amersham Biosciences)-labeled proteins were produced using the T n T quick-coupled transcription/translation system (Promega). .. After treatment with MG132 (20 μ m ) for 12 h, cells were lysed in buffer A (8 m urea, 10 m m imidazole, 0.5% Triton X-100, 100 m m Hepes (pH 7.5)) and incubated with 20 μl of Ni-NTA beads (Qiagen) for 2 h at room temperature.

    Gel Extraction:

    Article Title: Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2 †
    Article Snippet: Three microliters of each PCR product was checked for homogeneity and size on an agarose gel; 12 μl of the products was purified with a QIAquick PCR Purification kit (QIAGEN, Westburg, Germany), according to the protocol but with an extra washing step with 500 μl PE buffer (QIAGEN), and then eluted with 30 μl elution buffer after 1 min of incubation. .. Sometimes, when possible donor mobile-element copies were examined, multiple bands were visible on the agarose gel, and then each band was excised from the gel and extracted with the QIAquick gel extraction kit (QIAGEN) according to the protocol, but using the optional step of an extra wash with buffer QG (QIAGEN), an extra washing step with 700 μl buffer PE incubated for 2 min, and elution in 30 μl elution buffer after a 1-min incubation.

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  • 99
    Qiagen 1x q solution
    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    1x Q Solution, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1x q solution/product/Qiagen
    Average 99 stars, based on 26 article reviews
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    1x q solution - by Bioz Stars, 2020-02
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    87
    Qiagen qiagen multiplex q solution
    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    Qiagen Multiplex Q Solution, supplied by Qiagen, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qiagen multiplex q solution/product/Qiagen
    Average 87 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    qiagen multiplex q solution - by Bioz Stars, 2020-02
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    84
    Qiagen 0 5x q solution
    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.
    0 5x Q Solution, supplied by Qiagen, used in various techniques. Bioz Stars score: 84/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/0 5x q solution/product/Qiagen
    Average 84 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    0 5x q solution - by Bioz Stars, 2020-02
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    Image Search Results


    Increasing the assay specificity of the  JAK2  V617F mutation-specific PCR.  Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate.  A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification.  B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification.  C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification.  D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.

    Journal: BMC Cancer

    Article Title: Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

    doi: 10.1186/1471-2407-13-206

    Figure Lengend Snippet: Increasing the assay specificity of the JAK2 V617F mutation-specific PCR. Samples tested: 100% mutant control DNA (MUT 100%) analysed in triplicate, 100% wild-type control DNA (WT 100%) analysed in 10 replicates, non-template control (NTC) analysed in triplicate. A . Mutant allele-specific PCR. The reactions contained two oligonucleotides: the mutant allele-specific forward primer and the reverse primer. The graph shows a significant number of false-positive amplifications. We observed a Cq value difference of 20 cycles between the MUT 100% and the first false-positive amplification. B . Mutant allele-specific PCR with the introduction of the wild-type specific 3′ dideoxy blocker. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele specific blocker and the reverse primer. The graph still shows the presence of a number of false positives. We observed a Cq value difference of 23 cycles between the MUT 100% and the first false-positive amplification. C . Mutant allele-specific PCR with the introduction of 1X Q-Solution. The reactions contained two oligonucleotides: the mutant allele-specific forward and the reverse primers. The graph shows a significant reduction of false-positive amplifications to a single false positive. We observed a Cq value difference of 16 cycles between the MUT 100% and the first false-positive amplification. D . Mutant allele-specific PCR with the introduction of both the 3′ dideoxy blocker and 1X Q-Solution. The reactions contained three oligonucleotides: the mutant allele-specific forward primer, the wild-type allele-specific blocker and the reverse primer. One false-positive amplification was observed, at a very late Cq value. We observed a Cq value of 23 cycles difference between the MUT 100% and the first false-positive amplification.

    Article Snippet: The JAK2 mutation-specific PCR reactions were performed in a 10 μl final volume comprising 1X PCR Buffer containing 1.5 mmol/L of MgCl2 , 0.5 mmol/L of extra MgCl2 (for a final concentration of 2 mmol/L of MgCl2 ), 200 μmol/L of each deoxynucleotide triphosphate, 200 nmol/L of the JAK2_Ex14_WT_Blocker_F oligo, 400 nmol/L of the JAK2_Ex14_Mut_F primer, 200 nmol/L of the JAK2_Ex14_R primer, 5 μmol/L of SYTO9, 1X Q-Solution (Qiagen), 0.25 units of HotStar Taq DNA polymerase and 2 μl of template (at a concentration of 16.5 ng/μl), for a total of about 10,000 JAK2 copies per reaction.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification