thermopol buffer  (New England Biolabs)


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    Structured Review

    New England Biolabs thermopol buffer
    Ten-base CRT sequencing with dU.V . ( A ) 377 DNA sequencer gel image of a 10-cycle CRT experiment. Therminator polymerase was bound to primer/template complex and subjected to multiple cycles of incorporation (5 min) with 3 μM dU.V in 1× <t>ThermoPol</t> buffer, plus UV cleavage (4 min) in 50 mM sodium azide. P, dye-labeled primer (blue star represents BODIPY-FL label); 1a, first incorporation; 1b, first cleavage; 2a–10a, subsequent cleavage and incorporation cycles on dye-labeled primer. ( B ) Histogram plot of quantified gel bands in (A). Cycle efficiency was determined as a product of incorporation efficiency (1a: 100%) and cleavage efficiency (1b: 100%). Dephasing signals were also quantified as ‘% n –2’ (incomplete incorporation for the current cycle), ‘% n –1’ (incomplete UV cleavage from the previous cycle, extension of the ‘% n –2’ from the previous cycle and/or 3′-exonuclease activity followed by incorporation in the current cycle) and ‘% n +1’ [natural (photochemically cleaved) nucleotide carryover from the previous cycle]. ( C ) Histogram plot including the total signal for each cycle, representing the sum of the correct signal plus all dephasing signals.
    Thermopol Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates"

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1293

    Ten-base CRT sequencing with dU.V . ( A ) 377 DNA sequencer gel image of a 10-cycle CRT experiment. Therminator polymerase was bound to primer/template complex and subjected to multiple cycles of incorporation (5 min) with 3 μM dU.V in 1× ThermoPol buffer, plus UV cleavage (4 min) in 50 mM sodium azide. P, dye-labeled primer (blue star represents BODIPY-FL label); 1a, first incorporation; 1b, first cleavage; 2a–10a, subsequent cleavage and incorporation cycles on dye-labeled primer. ( B ) Histogram plot of quantified gel bands in (A). Cycle efficiency was determined as a product of incorporation efficiency (1a: 100%) and cleavage efficiency (1b: 100%). Dephasing signals were also quantified as ‘% n –2’ (incomplete incorporation for the current cycle), ‘% n –1’ (incomplete UV cleavage from the previous cycle, extension of the ‘% n –2’ from the previous cycle and/or 3′-exonuclease activity followed by incorporation in the current cycle) and ‘% n +1’ [natural (photochemically cleaved) nucleotide carryover from the previous cycle]. ( C ) Histogram plot including the total signal for each cycle, representing the sum of the correct signal plus all dephasing signals.
    Figure Legend Snippet: Ten-base CRT sequencing with dU.V . ( A ) 377 DNA sequencer gel image of a 10-cycle CRT experiment. Therminator polymerase was bound to primer/template complex and subjected to multiple cycles of incorporation (5 min) with 3 μM dU.V in 1× ThermoPol buffer, plus UV cleavage (4 min) in 50 mM sodium azide. P, dye-labeled primer (blue star represents BODIPY-FL label); 1a, first incorporation; 1b, first cleavage; 2a–10a, subsequent cleavage and incorporation cycles on dye-labeled primer. ( B ) Histogram plot of quantified gel bands in (A). Cycle efficiency was determined as a product of incorporation efficiency (1a: 100%) and cleavage efficiency (1b: 100%). Dephasing signals were also quantified as ‘% n –2’ (incomplete incorporation for the current cycle), ‘% n –1’ (incomplete UV cleavage from the previous cycle, extension of the ‘% n –2’ from the previous cycle and/or 3′-exonuclease activity followed by incorporation in the current cycle) and ‘% n +1’ [natural (photochemically cleaved) nucleotide carryover from the previous cycle]. ( C ) Histogram plot including the total signal for each cycle, representing the sum of the correct signal plus all dephasing signals.

    Techniques Used: Sequencing, Labeling, Activity Assay

    2) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    3) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    4) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    5) Product Images from "Helix loop-mediated isothermal amplification of nucleic acids"

    Article Title: Helix loop-mediated isothermal amplification of nucleic acids

    Journal: RSC Advances

    doi: 10.1039/c8ra01201f

    Characterization of HAMP results. (a) Gel electrophoresis for HAMP products with or without digestion by the restriction enzyme Hind III. Lane 1, digested HAMP products; lane 2, HAMP products prior to digestion; lane 3, negative control of HAMP; lane 4, digested HAMP products applying acceleration probes; lane 5, HAMP products applying acceleration probes prior to digestion; lane 6, negative control of HAMP reaction applying acceleration probes; lane M, molecular weight marker. (b) HAMP amplification plot by monitoring the fluorescence intensity. (c) Melting curve analysis on HAMP products.
    Figure Legend Snippet: Characterization of HAMP results. (a) Gel electrophoresis for HAMP products with or without digestion by the restriction enzyme Hind III. Lane 1, digested HAMP products; lane 2, HAMP products prior to digestion; lane 3, negative control of HAMP; lane 4, digested HAMP products applying acceleration probes; lane 5, HAMP products applying acceleration probes prior to digestion; lane 6, negative control of HAMP reaction applying acceleration probes; lane M, molecular weight marker. (b) HAMP amplification plot by monitoring the fluorescence intensity. (c) Melting curve analysis on HAMP products.

    Techniques Used: Nucleic Acid Electrophoresis, Negative Control, Molecular Weight, Marker, Amplification, Fluorescence

    Visual detection of HAMP and RT-HAMP for nucleic acids. The concentration of both plasmid DNA and mimic RNA was 10 6 copies in the amplification system. Amplification was performed at 63 °C for 60 min. Reaction 1 and 2, MERS-orf1b plasmid DNA; reaction 3 and 4, negative control of 1 and 2; reaction 5 and 6, MERS-orf1b mimic RNA; reaction 7 and 8, negative control of 5 and 6.
    Figure Legend Snippet: Visual detection of HAMP and RT-HAMP for nucleic acids. The concentration of both plasmid DNA and mimic RNA was 10 6 copies in the amplification system. Amplification was performed at 63 °C for 60 min. Reaction 1 and 2, MERS-orf1b plasmid DNA; reaction 3 and 4, negative control of 1 and 2; reaction 5 and 6, MERS-orf1b mimic RNA; reaction 7 and 8, negative control of 5 and 6.

    Techniques Used: Concentration Assay, Plasmid Preparation, Amplification, Negative Control

    6) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    7) Product Images from "Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection"

    Article Title: Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection

    Journal: Science Advances

    doi: 10.1126/sciadv.abj1281

    Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, ThermoPol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. RNase, ribonuclease; TiPP, thermostable inorganic pyrophosphatase; DTT, dithiothreitol; dNTPs, deoxynucleotide triphosphates. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA (NTC), 10, 15, 20, 200, 2000, 2 × 10 4 , or 2 × 10 5 copies/40 μl of reaction. Left: The average ( n = 3) FAM signal detecting SARS-CoV-2. *Only two of the three replicates of 10 copies per reaction amplified so the average was from duplicate reactions. Right: TEX 615 signal detecting the IAC. The IAC was detected in the NTC ( n = 3) and in the single 10 copies per reaction that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only two of the three replicates of 10 copies per reaction amplified. IAC signals were properly detected in NTC ( n = 3) and one of the three replicates of 10 copies per reaction undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies per reaction of SARS-CoV-2 RNA ( n = 40), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time to result of individual samples is plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies per reaction (corresponding to 0, 1, or 2.5 copies/μl) was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3). All data were measured every 13 s using FAM and TEX 615 channels by a real-time thermal cycler in 1-hour reactions at 63.3°C.
    Figure Legend Snippet: Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, ThermoPol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. RNase, ribonuclease; TiPP, thermostable inorganic pyrophosphatase; DTT, dithiothreitol; dNTPs, deoxynucleotide triphosphates. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA (NTC), 10, 15, 20, 200, 2000, 2 × 10 4 , or 2 × 10 5 copies/40 μl of reaction. Left: The average ( n = 3) FAM signal detecting SARS-CoV-2. *Only two of the three replicates of 10 copies per reaction amplified so the average was from duplicate reactions. Right: TEX 615 signal detecting the IAC. The IAC was detected in the NTC ( n = 3) and in the single 10 copies per reaction that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only two of the three replicates of 10 copies per reaction amplified. IAC signals were properly detected in NTC ( n = 3) and one of the three replicates of 10 copies per reaction undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies per reaction of SARS-CoV-2 RNA ( n = 40), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time to result of individual samples is plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies per reaction (corresponding to 0, 1, or 2.5 copies/μl) was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3). All data were measured every 13 s using FAM and TEX 615 channels by a real-time thermal cycler in 1-hour reactions at 63.3°C.

    Techniques Used: Amplification

    8) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    9) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    10) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    11) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    12) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    13) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    14) Product Images from "Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases"

    Article Title: Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz008

    The incorporation of hTTP (H) and the 5-substituted hUTPs 1a - f (lanes a–f) opposite a poly-dA template overhang in a DNA duplex using the evolved HNA polymerases T6G12_I521L and T6G12. The modified nucleotides (hTTP and 1a – f ) are used at a concentration of 125 μM. After optimization of the reaction conditions, the enzymes are used at a final concentration of 51 nM for T6G12_I521L and 82 nM T6G12. The reactions with the enzyme T6G12 contain 0.5 mM freshly prepared MnCl 2 . All reactions are carried out in 1× Thermopol buffer (NEB) supplemented with 1.5 mM MgSO 4 . The reactions are incubated at 50°C overnight. The positions where a 5-substituted hUTP has to be incorporated opposite the template oligonucleotide, are underlined in the sequence below the gel image. The lanes indicated with ‘P’ show the primer control (no enzyme and no nucleotides added). The position for the full-length material of HNA (signifying the incorporation of ten hT nucleotides) is indicated by ‘FL HNA’ on the side of the gel image.
    Figure Legend Snippet: The incorporation of hTTP (H) and the 5-substituted hUTPs 1a - f (lanes a–f) opposite a poly-dA template overhang in a DNA duplex using the evolved HNA polymerases T6G12_I521L and T6G12. The modified nucleotides (hTTP and 1a – f ) are used at a concentration of 125 μM. After optimization of the reaction conditions, the enzymes are used at a final concentration of 51 nM for T6G12_I521L and 82 nM T6G12. The reactions with the enzyme T6G12 contain 0.5 mM freshly prepared MnCl 2 . All reactions are carried out in 1× Thermopol buffer (NEB) supplemented with 1.5 mM MgSO 4 . The reactions are incubated at 50°C overnight. The positions where a 5-substituted hUTP has to be incorporated opposite the template oligonucleotide, are underlined in the sequence below the gel image. The lanes indicated with ‘P’ show the primer control (no enzyme and no nucleotides added). The position for the full-length material of HNA (signifying the incorporation of ten hT nucleotides) is indicated by ‘FL HNA’ on the side of the gel image.

    Techniques Used: Modification, Concentration Assay, Incubation, Sequencing

    The incorporation of the 5-substituted hUTPs together with hATP, hCTP and hGTP into the P1T2 duplex overhang using T6G12_I 521L after cycling for 1 min at 94°C, followed by 5 min at 50°C and 2 h 65°C, for 16 h in total, in Thermopol buffer 1× containing an additional 2 mM MgSO 4 . ‘P’ indicates the primer control. Lane H shows the hNTP control. Lanes a + hACG to f + hACG show the incorporation of the hA, hC and hG building blocks together with 1a – f respectively.
    Figure Legend Snippet: The incorporation of the 5-substituted hUTPs together with hATP, hCTP and hGTP into the P1T2 duplex overhang using T6G12_I 521L after cycling for 1 min at 94°C, followed by 5 min at 50°C and 2 h 65°C, for 16 h in total, in Thermopol buffer 1× containing an additional 2 mM MgSO 4 . ‘P’ indicates the primer control. Lane H shows the hNTP control. Lanes a + hACG to f + hACG show the incorporation of the hA, hC and hG building blocks together with 1a – f respectively.

    Techniques Used:

    15) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    16) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    17) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    18) Product Images from "Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection"

    Article Title: Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection

    Journal: medRxiv

    doi: 10.1101/2021.08.12.21261875

    Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, Thermopol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA ( NTC ; no template control), 10, 15, 20, 200, 2000, 2×10 4 , or 2×10 5 copies/40µL reaction ( rxn ). The left panel shows the average ( n = 3 ) FAM signal detecting SARS-CoV-2. *Only 2/3 replicates of 10 copies/rxn amplified so the average was from duplicate reactions. The right panel shows TEX 615 signal detecting the internal amplification control (IAC). The IAC was detected in the NTC ( n = 3) and in the single 10 copies/reaction that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only 2/3 replicates of 10 copies/reaction amplified. IAC signals were properly detected in NTC ( n = 3 ) and 1 of the 3 replicates of 10 copies/reaction undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies/reaction of SARS-CoV-2 RNA ( n = 40 ), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time-to-result of individual samples are plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies/reaction (corresponding to 0, 1, or 2.5 copies/µL) was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3 ). All data was measured every 13s using FAM and TEX 615 channels, by a real-time thermal cycler in 1h reactions at 63.3°C, and ( C ).
    Figure Legend Snippet: Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, Thermopol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA ( NTC ; no template control), 10, 15, 20, 200, 2000, 2×10 4 , or 2×10 5 copies/40µL reaction ( rxn ). The left panel shows the average ( n = 3 ) FAM signal detecting SARS-CoV-2. *Only 2/3 replicates of 10 copies/rxn amplified so the average was from duplicate reactions. The right panel shows TEX 615 signal detecting the internal amplification control (IAC). The IAC was detected in the NTC ( n = 3) and in the single 10 copies/reaction that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only 2/3 replicates of 10 copies/reaction amplified. IAC signals were properly detected in NTC ( n = 3 ) and 1 of the 3 replicates of 10 copies/reaction undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies/reaction of SARS-CoV-2 RNA ( n = 40 ), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time-to-result of individual samples are plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies/reaction (corresponding to 0, 1, or 2.5 copies/µL) was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3 ). All data was measured every 13s using FAM and TEX 615 channels, by a real-time thermal cycler in 1h reactions at 63.3°C, and ( C ).

    Techniques Used: Amplification

    19) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    20) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    21) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    22) Product Images from "Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection"

    Article Title: Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection

    Journal: medRxiv

    doi: 10.1101/2021.08.12.21261875

    Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, Thermopol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA ( NTC ; no template control), 10, 15, 20, 200, 2000, 2x10 4 , or 2x10 5 copies/40µL reaction ( rxn ). The left panel shows the average ( n =3 ) FAM signal detecting SARS-CoV-2. *Only 2/3 replicates of 10 copies/rxn amplified so the average was from duplicate reactions. The right panel shows TEX 615 signal detecting the internal amplification control (IAC). The IAC was detected in the NTC ( n = 3) and in the single 10 copies/rxn that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only 2/3 replicates of 10 copies/rxn amplified. IAC signals were properly detected in NTC ( n = 3 ) and 1 of the 3 replicates of 10 copies/rxn undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies/rxn of SARS-CoV-2 RNA ( n = 40 ), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time-to-result of individual samples are plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies/rxn was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3 ). All data was measured every 13s using FAM and TEX 615 channels, by a real-time thermal cycler in 1h reactions at 63.3°C, and ( C ).
    Figure Legend Snippet: Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, Thermopol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA ( NTC ; no template control), 10, 15, 20, 200, 2000, 2x10 4 , or 2x10 5 copies/40µL reaction ( rxn ). The left panel shows the average ( n =3 ) FAM signal detecting SARS-CoV-2. *Only 2/3 replicates of 10 copies/rxn amplified so the average was from duplicate reactions. The right panel shows TEX 615 signal detecting the internal amplification control (IAC). The IAC was detected in the NTC ( n = 3) and in the single 10 copies/rxn that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only 2/3 replicates of 10 copies/rxn amplified. IAC signals were properly detected in NTC ( n = 3 ) and 1 of the 3 replicates of 10 copies/rxn undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies/rxn of SARS-CoV-2 RNA ( n = 40 ), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time-to-result of individual samples are plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies/rxn was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3 ). All data was measured every 13s using FAM and TEX 615 channels, by a real-time thermal cycler in 1h reactions at 63.3°C, and ( C ).

    Techniques Used: Amplification

    23) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    24) Product Images from "Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection"

    Article Title: Harmony COVID-19: a ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection

    Journal: medRxiv

    doi: 10.1101/2021.08.12.21261875

    Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, Thermopol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA ( NTC ; no template control), 10, 15, 20, 200, 2000, 2×10 4 , or 2×10 5 copies/40µL reaction ( rxn ). The left panel shows the average ( n = 3 ) FAM signal detecting SARS-CoV-2. *Only 2/3 replicates of 10 copies/rxn amplified so the average was from duplicate reactions. The right panel shows TEX 615 signal detecting the internal amplification control (IAC). The IAC was detected in the NTC ( n = 3) and in the single 10 copies/reaction that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only 2/3 replicates of 10 copies/reaction amplified. IAC signals were properly detected in NTC ( n = 3 ) and 1 of the 3 replicates of 10 copies/reaction undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies/reaction of SARS-CoV-2 RNA ( n = 40 ), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time-to-result of individual samples are plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies/reaction (corresponding to 0, 1, or 2.5 copies/µL) was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3 ). All data was measured every 13s using FAM and TEX 615 channels, by a real-time thermal cycler in 1h reactions at 63.3°C, and ( C ).
    Figure Legend Snippet: Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, Thermopol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA ( NTC ; no template control), 10, 15, 20, 200, 2000, 2×10 4 , or 2×10 5 copies/40µL reaction ( rxn ). The left panel shows the average ( n = 3 ) FAM signal detecting SARS-CoV-2. *Only 2/3 replicates of 10 copies/rxn amplified so the average was from duplicate reactions. The right panel shows TEX 615 signal detecting the internal amplification control (IAC). The IAC was detected in the NTC ( n = 3) and in the single 10 copies/reaction that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only 2/3 replicates of 10 copies/reaction amplified. IAC signals were properly detected in NTC ( n = 3 ) and 1 of the 3 replicates of 10 copies/reaction undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies/reaction of SARS-CoV-2 RNA ( n = 40 ), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time-to-result of individual samples are plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies/reaction (corresponding to 0, 1, or 2.5 copies/µL) was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3 ). All data was measured every 13s using FAM and TEX 615 channels, by a real-time thermal cycler in 1h reactions at 63.3°C, and ( C ).

    Techniques Used: Amplification

    25) Product Images from "Controlled Microwave Heating Accelerates Rolling Circle Amplification"

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0136532

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Figure Legend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Techniques Used:

    26) Product Images from "Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection"

    Article Title: Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection

    Journal: Science Advances

    doi: 10.1126/sciadv.abj1281

    Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, ThermoPol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. RNase, ribonuclease; TiPP, thermostable inorganic pyrophosphatase; DTT, dithiothreitol; dNTPs, deoxynucleotide triphosphates. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA (NTC), 10, 15, 20, 200, 2000, 2 × 10 4 , or 2 × 10 5 copies/40 μl of reaction. Left: The average ( n = 3) FAM signal detecting SARS-CoV-2. *Only two of the three replicates of 10 copies per reaction amplified so the average was from duplicate reactions. Right: TEX 615 signal detecting the IAC. The IAC was detected in the NTC ( n = 3) and in the single 10 copies per reaction that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only two of the three replicates of 10 copies per reaction amplified. IAC signals were properly detected in NTC ( n = 3) and one of the three replicates of 10 copies per reaction undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies per reaction of SARS-CoV-2 RNA ( n = 40), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time to result of individual samples is plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies per reaction (corresponding to 0, 1, or 2.5 copies/μl) was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3). All data were measured every 13 s using FAM and TEX 615 channels by a real-time thermal cycler in 1-hour reactions at 63.3°C.
    Figure Legend Snippet: Lyophilized RT-LAMP and its analytical sensitivity. ( A ) To set up the assay, a sample in elution/rehydration buffer containing magnesium, ThermoPol buffer, and detergents is added to rehydrate the lyophilized RT-LAMP reagents in a single step. RNase, ribonuclease; TiPP, thermostable inorganic pyrophosphatase; DTT, dithiothreitol; dNTPs, deoxynucleotide triphosphates. ( B ) Real-time amplification signal of RT-LAMP reactions containing SARS-CoV-2 RNA (NTC), 10, 15, 20, 200, 2000, 2 × 10 4 , or 2 × 10 5 copies/40 μl of reaction. Left: The average ( n = 3) FAM signal detecting SARS-CoV-2. *Only two of the three replicates of 10 copies per reaction amplified so the average was from duplicate reactions. Right: TEX 615 signal detecting the IAC. The IAC was detected in the NTC ( n = 3) and in the single 10 copies per reaction that did not detect SARS-CoV-2. ( C ) Time to detection of SARS-CoV-2 and IAC targets reported by the real-time thermal cycler. Individual data points are plotted. Note that only two of the three replicates of 10 copies per reaction amplified. IAC signals were properly detected in NTC ( n = 3) and one of the three replicates of 10 copies per reaction undetected for SARS-CoV-2. ( D ) Evaluation of analytical sensitivity at 20 copies per reaction of SARS-CoV-2 RNA ( n = 40), performed in two different runs ( n = 20 each) with two different serially diluted RNA samples. Time to result of individual samples is plotted along with medians (middle lines of the boxes), interquartile ranges (edges of the boxes), ranges (the ends of whiskers), and outliers (data points outside the whiskers). ( E ) Impact of nasal matrix. SARS-CoV-2 RNA at 0, 40, or 100 copies per reaction (corresponding to 0, 1, or 2.5 copies/μl) was amplified in lyophilized RT-LAMP in the presence of simulated nasal matrix ( n = 3). All data were measured every 13 s using FAM and TEX 615 channels by a real-time thermal cycler in 1-hour reactions at 63.3°C.

    Techniques Used: Amplification

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    New England Biolabs thermopol buffer
    Ten-base CRT sequencing with dU.V . ( A ) 377 DNA sequencer gel image of a 10-cycle CRT experiment. Therminator polymerase was bound to primer/template complex and subjected to multiple cycles of incorporation (5 min) with 3 μM dU.V in 1× <t>ThermoPol</t> buffer, plus UV cleavage (4 min) in 50 mM sodium azide. P, dye-labeled primer (blue star represents BODIPY-FL label); 1a, first incorporation; 1b, first cleavage; 2a–10a, subsequent cleavage and incorporation cycles on dye-labeled primer. ( B ) Histogram plot of quantified gel bands in (A). Cycle efficiency was determined as a product of incorporation efficiency (1a: 100%) and cleavage efficiency (1b: 100%). Dephasing signals were also quantified as ‘% n –2’ (incomplete incorporation for the current cycle), ‘% n –1’ (incomplete UV cleavage from the previous cycle, extension of the ‘% n –2’ from the previous cycle and/or 3′-exonuclease activity followed by incorporation in the current cycle) and ‘% n +1’ [natural (photochemically cleaved) nucleotide carryover from the previous cycle]. ( C ) Histogram plot including the total signal for each cycle, representing the sum of the correct signal plus all dephasing signals.
    Thermopol Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ten-base CRT sequencing with dU.V . ( A ) 377 DNA sequencer gel image of a 10-cycle CRT experiment. Therminator polymerase was bound to primer/template complex and subjected to multiple cycles of incorporation (5 min) with 3 μM dU.V in 1× ThermoPol buffer, plus UV cleavage (4 min) in 50 mM sodium azide. P, dye-labeled primer (blue star represents BODIPY-FL label); 1a, first incorporation; 1b, first cleavage; 2a–10a, subsequent cleavage and incorporation cycles on dye-labeled primer. ( B ) Histogram plot of quantified gel bands in (A). Cycle efficiency was determined as a product of incorporation efficiency (1a: 100%) and cleavage efficiency (1b: 100%). Dephasing signals were also quantified as ‘% n –2’ (incomplete incorporation for the current cycle), ‘% n –1’ (incomplete UV cleavage from the previous cycle, extension of the ‘% n –2’ from the previous cycle and/or 3′-exonuclease activity followed by incorporation in the current cycle) and ‘% n +1’ [natural (photochemically cleaved) nucleotide carryover from the previous cycle]. ( C ) Histogram plot including the total signal for each cycle, representing the sum of the correct signal plus all dephasing signals.

    Journal: Nucleic Acids Research

    Article Title: Improved nucleotide selectivity and termination of 3?-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates

    doi: 10.1093/nar/gkq1293

    Figure Lengend Snippet: Ten-base CRT sequencing with dU.V . ( A ) 377 DNA sequencer gel image of a 10-cycle CRT experiment. Therminator polymerase was bound to primer/template complex and subjected to multiple cycles of incorporation (5 min) with 3 μM dU.V in 1× ThermoPol buffer, plus UV cleavage (4 min) in 50 mM sodium azide. P, dye-labeled primer (blue star represents BODIPY-FL label); 1a, first incorporation; 1b, first cleavage; 2a–10a, subsequent cleavage and incorporation cycles on dye-labeled primer. ( B ) Histogram plot of quantified gel bands in (A). Cycle efficiency was determined as a product of incorporation efficiency (1a: 100%) and cleavage efficiency (1b: 100%). Dephasing signals were also quantified as ‘% n –2’ (incomplete incorporation for the current cycle), ‘% n –1’ (incomplete UV cleavage from the previous cycle, extension of the ‘% n –2’ from the previous cycle and/or 3′-exonuclease activity followed by incorporation in the current cycle) and ‘% n +1’ [natural (photochemically cleaved) nucleotide carryover from the previous cycle]. ( C ) Histogram plot including the total signal for each cycle, representing the sum of the correct signal plus all dephasing signals.

    Article Snippet: Approximately 50 ng of genomic DNA was amplified with 0.4 µM of HNF1a_2.1 (F/R) or HNF1a_10.1 (F/R) primer pairs and one unit of Vent(exo− ) polymerase in 1× ThermoPol buffer [20 mM Tris–HCl, pH 8.8; 10 mM (NH4 )2 SO4 ; 10 mM KCl; 2 mM MgSO4 ; 0.1% Triton X-100; New England BioLabs], 1 M betaine ( , ) and 200 μM each of dATP, dCTP, dGTP and either TTP or HOMedUTP.

    Techniques: Sequencing, Labeling, Activity Assay

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Journal: PLoS ONE

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    doi: 10.1371/journal.pone.0136532

    Figure Lengend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Article Snippet: RCA mixtures each containing one component in 4-fold excess of its standard concentration in ThermoPol Buffer (NEB) were prepared as follows: 1 μL of each component (3 M Tris–HCl [pH 8.8], 1.5 M KCl, 1.5 M (NH4 )2 SO4 , and 0.3 M MgSO4 ) was added to an RCA mixture containing a dNTP mixture (2.5 mM each), two primers (10 pmol), circular template–primer complex (50 ng), Bst -LF, and ThermoPol Buffer (20 mM Tris–HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , and 0.1% Triton X-100).

    Techniques: