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    Qiagen qiaquick pcr purification kit
    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen <t>QIAquick</t> <t>PCR</t> Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).
    Qiaquick Pcr Purification Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 136535 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Journal: Scientific Reports

    Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

    doi: 10.1038/s41598-020-58586-3

    Figure Lengend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

    Article Snippet: Kit extractions We tested three different silica-column kits: Zymo ZR Viral DNA/RNA Kit (outdated protocol, D7021), Zymo Quick-DNA/RNA Kit (updated protocol, D7021), and the QIAquick PCR Purification Kit (28104, Qiagen).

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Purification

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

    Journal: Virology Journal

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

    doi: 10.1186/s12985-016-0504-8

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

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

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

    Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Comparison of chromatin fragmentation by ultrasound alone or in combination with benzonase digestion. (A) Comparison of sonication efficiency at the L and H power outputs over time. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for various times (as indicated) in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The “Quick” lanes contain samples sonicated for 20 min and reverse cross-linked for 1 h at +60°C. (B) Coomassie Blue staining of protein fractions generated during the sonication time course shown in panel A. Note the absence of high molecular weight proteins after 20 min of ultrasound treatment. (C) Titration of benzonase (0.2U to 90U) to fragment chromatin solubilized by 2 min L-power sonication. Following the digest, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. See Material and Methods section for detailed reaction conditions. (D) Combination of brief sonication (2 min at L-power) and benzonase digestion (0.7U to 90U) preserves the integrity of large proteins.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Generated, Molecular Weight, Titration

    Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Systematic optimization of sonication conditions. Part 2. (A) The effect of sample position on sonication efficiency at low power setting. 500 μL cell suspensions were loaded into positions L1, L7, R1, R7, all other positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Left panel: Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. Right panel: Afterwards, the samples were sonicated for an additional 9 min and reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 . p -values for selected T-tests are shown on the graph. (B) The effect of pulse time on sonication efficiency. 500 μL cell suspensions were loaded into position R1; vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 2 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), with variable pulse times (as indicated), no rotation and no ice. Following sonication, samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. The control (CTRL) sample is the cell suspension before sonication. (C) The effect of buffer composition on sonication efficiency. 500 μL cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 10 min in 1:4 ELB:H 2 O supplemented with varying concentrations of SDS and Triton X-100 (as indicated), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Following sonication samples were reverse cross-linked overnight. DNA was purified from the resulting mixture using the Qiagen PCR clean-up kit and analysed on 1.1% agarose gel. (D) The effect of water level and sample volume on sonication efficiency. Cell suspensions were loaded into position R1; positions R4, R7 and R11 were filled with tubes containing 500 μL of water; other R-positions were left vacant. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation and no ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. Analysis of variance p -values are shown on the graph for each sample volume.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.

    Journal: PLoS ONE

    Article Title: Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins

    doi: 10.1371/journal.pone.0148023

    Figure Lengend Snippet: Systematic optimization of sonication conditions. Part 1. (A) Schematic of the Bioruptor XL water bath with tube positions numbered from 1 to 12 in the left (L) and right (R) carousels; red arrows indicate the alignment marks for carousel assembly and positioning. (B) The effect of sample volume on sonication efficiency at low power setting. Cell suspensions of variable volume (100–700 μL, as indicated) were loaded into positions L3, L4, L5, L9, L10 and L11. Vacant L-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 8 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Remaining intact cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 1.0×10 −5 , between all samples 1.4×10 −6 . p -values for selected T-tests are shown on the graph. (C) Reproducibility of sample sonication across positions L1–L12. Sonication was carried out for 40 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 24 sec ON/24 sec OFF pulses, with rotation and no floating ice. Samples were reverse cross-linked overnight and the resulting DNA was purified using the Qiagen PCR clean-up kit followed by 1.1% agarose gel analysis. (D) The effect of sample position and power setting on sonication efficiency. 500 μL cell suspensions were loaded into positions R10-R4 and vacant R-positions were filled with tubes containing 500 μL of water. Sonication was carried out for 1 min in 1:4 ELB:H 2 O (0.1% SDS final concentration), 5 sec ON/5 sec OFF pulses, no rotation, no ice. Intact remaining cells were counted three times and the respective means calculated; error bars reflect the standard deviation. The control (CTRL) sample is the cell suspension before sonication. p -value for analysis of variance between sonicated samples is 3.6×10 −9 (L-power) and 8.2×10 −10 (H-power). p -values for selected T-tests are shown on the graph.

    Article Snippet: In the second procedure, referred to as the “quick” procedure, samples were incubated for 1 h at +60°C, and the resulting DNA was purified with a PCR purification kit (Qiagen, 28104).

    Techniques: Sonication, Concentration Assay, Size-exclusion Chromatography, Standard Deviation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis