thermopol buffer  (New England Biolabs)


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    Name:
    ThermoPol Reaction Buffer Pack
    Description:
    ThermoPol Reaction Buffer Pack 6 0 ml
    Catalog Number:
    b9004s
    Price:
    22
    Size:
    6 0 ml
    Category:
    Buffers
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    Structured Review

    New England Biolabs thermopol buffer
    ThermoPol Reaction Buffer Pack
    ThermoPol Reaction Buffer Pack 6 0 ml
    https://www.bioz.com/result/thermopol buffer/product/New England Biolabs
    Average 95 stars, based on 122 article reviews
    Price from $9.99 to $1999.99
    thermopol buffer - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) 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:

    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 "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:

    4) 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

    Related Articles

    Amplification:

    Article Title: Optimization of trans-Splicing for Huntington's Disease RNA Therapy
    Article Snippet: For amplifications outside the HTT exon 1 CAG repeat and the adjacent GC-rich region, Pfu enzyme (prepared in-house) with Thermopol buffer (New England Biolabs, B9004S) was used. .. For amplification across the exon 1 CAG repeat, Taq PCRx with 2 × enhancer solution (Invitrogen, 11495-017) was used, per manufacturer instructions.

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: The 25 µl reaction mixture contained 2.5 µl of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs, Hertfordshire, UK), 4 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 0.1 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), 1 µl each of 10 pmol/µl forward and reverse primer , 15.4 µl of sterile Milli-Q water, and 1 µl of DNA template. .. PCR amplification was carried out in an Eppendorf Master Cycler (22331, Eppendorf, Germany) using the following temperature program: initial denaturation (1 min, 94°C) followed by 35 cycles of denaturation (1 min, 94°C), annealing (1 min, 55°C), and elongation (1 min, 72°C) and then final extension (7 min, 72°C).

    Article Title: Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia
    Article Snippet: Amplicon size was 53 bp. .. For site 1565, the 10 μL PCR reaction contained 2 μL of 1 in 10 diluted template DNA, 0.4 μL each of the primers at 10 μM, 1 μL of the ThermoPol reaction buffer (NEB Inc., Ipswich, MA, USA; Cat. No. B9004S), 0.064 μL of dNTP’s at 25 mM (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-39029), 0.4 μL of MgCl2 (50 mM) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047), 0.25 μL of the LightCycler® 480 High Resolution Melting Master (Roche, Mannheim, Germany; Cat. No. 04909631001), 0.01 μL of IMMOLASE™ DNA polymerase (10 u/μL) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047) and 5.476 μL of ddH2 O (Honeywell, Burdick and Jackson; Muskegon, MI, USA; Cat. No. 365-4).

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: PCR amplification was carried out in an Eppendorf Master Cycler (22331, Eppendorf, Germany) using the following temperature program: initial denaturation (1 min, 94°C) followed by 35 cycles of denaturation (1 min, 94°C), annealing (1 min, 55°C), and elongation (1 min, 72°C) and then final extension (7 min, 72°C). .. The 50 µl PCR assay mix contained 32.8 µl of sterile Milli-Q water, 1 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 5 µL of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs), 3 µl each of 10 pmol/µl forward and reverse primer , 0.2 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), and 5 µl of DNA template.

    Article Title: Reasons for the Invasive Success of a Guppy (Poecilia reticulata) Population in Trinidad
    Article Snippet: .. For DNA amplification the following PCR protocol was used: 2.5 µl [25 ng/µl] template, 4.4 µl H2 O, 1 µl PCR buffer [10× Thermobuffer (NEB B9004S)], 1 µl dNTPs [2 mM], 0.1 µl Taq polymerase [5000 U/m (NEB M0267L)], 0.5 µl Fw-primer and 0.5 µl Rev-primer [both 10 pmol/µl]. .. All PCR amplifications run in Peltier Thermal Cyclers pTC-200 from Bio-Rad under the following conditions: 96°C for 3 min, 40 cycles of 96°C for 30 sec., 56°C for 30 sec., 68°C for 30 sec.; 68°C for 5 min.

    Article Title: Metabolomic Profiles of a Midge (Procladius villosimanus, Kieffer) Are Associated with Sediment Contamination in Urban Wetlands
    Article Snippet: Samples were then centrifuged for 5 min at 2700× g , supernatant diluted 1/50 and stored at −20 °C until PCR amplification. .. Each PCR reaction was 25 μL and contained the following components: 5 μL Chelex extracted DNA, 2.5 μL ThermoPol 10× buffer (New England Biolabs Ipswich, United States (NEB) Cat. No. B9004S), 2 min μL dNTP (25 mM), 0.2 μL Taq DNA polymerase (NEB Cat. No. M0273S), 1.25 μL each LCO1490 and HC02198 primers (10 μM each) and 12.8 μL of double deionized water.

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: After 30 min of incubation, unbound origami and extra staple strands were removed by washing three times with 60 μl of fresh TAE/Mg buffer (40 mM Tris acetate, 1 mM EDTA buffer supplemented with 12.5 mM magnesium acetate) and three times with 60 μl of 1× ThermoPol buffer (NEB, Cat. No. B9004S, 10× ; 1× contains 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100). .. This minimizes any false-positive PCR amplification by the recording primers.

    Article Title: Phenotypic and genotypic characteristics of Trueperellapyogenes isolated from ruminants
    Article Snippet: The pld -negative isolates were examined for 8 known and putative virulence genes, utilizing previously developed PCR protocols and primers., The PCR program was performed as follows : denaturation at 95°C for 4 min; 35 cycles of 94°C for 1 min, annealing for 30 s at different temperatures (60°C for nanH -, nanP -, plo -, cbpA -, and fimC -specific PCRs; and 57°C for fimA - and fimG -specific PCRs), , 72°C for 3 min; and a final extension at 72°C for 7 min. All amplifications were carried out in 50 µL of the following reaction mixture: 10 pmol of each primer (0.5 µL each), 10 mM of each dNTP (N0447S, New England BioLabs, Ipswich, MA), 1× Taq polymerase buffer (B9004S, New England BioLabs), 1 U of Taq DNA polymerase (M0273L, New England BioLabs), and ~50 ng of genomic DNA. .. A fragment of the 16S ribosomal (r)RNA gene was amplified utilizing the universal primers UNF (5’-GAGTTTGATCCTGGCTCAG-3’) and UNR (5’-GGACTACCAGGGTATCTAAT-3’) targeting bases 9–27 and 805–786 of the 16S rRNA gene of Escherichia coli , respectively.

    Synthesized:

    Article Title: Optimization of trans-Splicing for Huntington's Disease RNA Therapy
    Article Snippet: RNA was resuspended in 10 mM Tris–HCl pH 8.2, 1 mM EDTA, and concentrations were measured using a Nanodrop (Thermo Fisher). cDNA was synthesized using 1 μg of RNA and random primers following the SuperScript III protocol (Invitrogen, 18080-044). .. For amplifications outside the HTT exon 1 CAG repeat and the adjacent GC-rich region, Pfu enzyme (prepared in-house) with Thermopol buffer (New England Biolabs, B9004S) was used.

    Electrophoresis:

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: The 50 µl PCR assay mix contained 32.8 µl of sterile Milli-Q water, 1 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 5 µL of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs), 3 µl each of 10 pmol/µl forward and reverse primer , 0.2 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), and 5 µl of DNA template. .. All PCR amplicons were visualized by electrophoresis on 1% (1.2% for ITS-PCR) agarose gels containing 2 µl GelRed stain (41003, Biotium, Fremont, CA, USA); wells were loaded with 10 µl of PCR product + 2 µl of loading dye (R0611, Thermo Fisher Scientific, Loughborough, UK).

    Article Title: Phenotypic and genotypic characteristics of Trueperellapyogenes isolated from ruminants
    Article Snippet: The pld -negative isolates were examined for 8 known and putative virulence genes, utilizing previously developed PCR protocols and primers., The PCR program was performed as follows : denaturation at 95°C for 4 min; 35 cycles of 94°C for 1 min, annealing for 30 s at different temperatures (60°C for nanH -, nanP -, plo -, cbpA -, and fimC -specific PCRs; and 57°C for fimA - and fimG -specific PCRs), , 72°C for 3 min; and a final extension at 72°C for 7 min. All amplifications were carried out in 50 µL of the following reaction mixture: 10 pmol of each primer (0.5 µL each), 10 mM of each dNTP (N0447S, New England BioLabs, Ipswich, MA), 1× Taq polymerase buffer (B9004S, New England BioLabs), 1 U of Taq DNA polymerase (M0273L, New England BioLabs), and ~50 ng of genomic DNA. .. The PCR products were separated by electrophoresis through a 2% agarose gel in Tris–acetate–EDTA buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA; Thermo Fisher Scientific, Lenexa, KS), stained with ethidium bromide (MilliporeSigma), and visualized using an Alpha Imager HP (Alpha-Innotech, San Leandro, CA).

    Incubation:

    Article Title: ALKBH10B Is an RNA N6-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition
    Article Snippet: .. The mixture was incubated at 37°C for an additional 5 h. For quantification of cap m6 Am , 100 ng poly(A) RNA was decapped with 20 units of RNA 5′pyrophosphohydrolase (RppH; M0356; New England Biolabs) in 1×Thermopol buffer (B9004S; New England Biolabs) for 3 h at 37°C, and then treated with Nuclease P1 and alkaline phosphatase as described above. ..

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: .. After 30 min of incubation, unbound origami and extra staple strands were removed by washing three times with 60 μl of fresh TAE/Mg buffer (40 mM Tris acetate, 1 mM EDTA buffer supplemented with 12.5 mM magnesium acetate) and three times with 60 μl of 1× ThermoPol buffer (NEB, Cat. No. B9004S, 10× ; 1× contains 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100). .. To the chamber, a 40 μl solution containing 0.8 units/μl of Bst polymerase (NEB, Cat. No. M0275S), 100 μM dNTP (NEB, Cat. No. N0447S), and the relevant primer mixes typically at 100 nM each, in 1× ThermoPol reaction buffer, was added and incubated for 1 h at room temperature.

    Mass Spectrometry:

    Article Title: ALKBH10B Is an RNA N6-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition
    Article Snippet: The mixture was incubated at 37°C for an additional 5 h. For quantification of cap m6 Am , 100 ng poly(A) RNA was decapped with 20 units of RNA 5′pyrophosphohydrolase (RppH; M0356; New England Biolabs) in 1×Thermopol buffer (B9004S; New England Biolabs) for 3 h at 37°C, and then treated with Nuclease P1 and alkaline phosphatase as described above. .. The nucleosides were separated by UPLC (Shimadzu) equipped with a ZORBAX SB-Aq column (Agilent) and detected by MS/MS using a Triple Quad 5500 (AB SCIEX) mass spectrometer in positive ion mode by multiple reaction monitoring.

    Article Title: Phenotypic and genotypic characteristics of Trueperellapyogenes isolated from ruminants
    Article Snippet: The pld -negative isolates were examined for 8 known and putative virulence genes, utilizing previously developed PCR protocols and primers., The PCR program was performed as follows : denaturation at 95°C for 4 min; 35 cycles of 94°C for 1 min, annealing for 30 s at different temperatures (60°C for nanH -, nanP -, plo -, cbpA -, and fimC -specific PCRs; and 57°C for fimA - and fimG -specific PCRs), , 72°C for 3 min; and a final extension at 72°C for 7 min. All amplifications were carried out in 50 µL of the following reaction mixture: 10 pmol of each primer (0.5 µL each), 10 mM of each dNTP (N0447S, New England BioLabs, Ipswich, MA), 1× Taq polymerase buffer (B9004S, New England BioLabs), 1 U of Taq DNA polymerase (M0273L, New England BioLabs), and ~50 ng of genomic DNA. .. The pld -negative isolates were examined for 8 known and putative virulence genes, utilizing previously developed PCR protocols and primers., The PCR program was performed as follows : denaturation at 95°C for 4 min; 35 cycles of 94°C for 1 min, annealing for 30 s at different temperatures (60°C for nanH -, nanP -, plo -, cbpA -, and fimC -specific PCRs; and 57°C for fimA - and fimG -specific PCRs), , 72°C for 3 min; and a final extension at 72°C for 7 min. All amplifications were carried out in 50 µL of the following reaction mixture: 10 pmol of each primer (0.5 µL each), 10 mM of each dNTP (N0447S, New England BioLabs, Ipswich, MA), 1× Taq polymerase buffer (B9004S, New England BioLabs), 1 U of Taq DNA polymerase (M0273L, New England BioLabs), and ~50 ng of genomic DNA.

    Genome Wide:

    Article Title: Reasons for the Invasive Success of a Guppy (Poecilia reticulata) Population in Trinidad
    Article Snippet: Here population specific markers already existed due to a genome-wide SNP study of guppy population history in Trinidad and Venezuela . .. For DNA amplification the following PCR protocol was used: 2.5 µl [25 ng/µl] template, 4.4 µl H2 O, 1 µl PCR buffer [10× Thermobuffer (NEB B9004S)], 1 µl dNTPs [2 mM], 0.1 µl Taq polymerase [5000 U/m (NEB M0267L)], 0.5 µl Fw-primer and 0.5 µl Rev-primer [both 10 pmol/µl].

    Modification:

    Article Title: ALKBH10B Is an RNA N6-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition
    Article Snippet: Paragraph title: Quantitative Analysis of RNA Modification by LC-MS/MS ... The mixture was incubated at 37°C for an additional 5 h. For quantification of cap m6 Am , 100 ng poly(A) RNA was decapped with 20 units of RNA 5′pyrophosphohydrolase (RppH; M0356; New England Biolabs) in 1×Thermopol buffer (B9004S; New England Biolabs) for 3 h at 37°C, and then treated with Nuclease P1 and alkaline phosphatase as described above.

    Countercurrent Chromatography:

    Article Title: Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia
    Article Snippet: The non-synonymous mutation occurs in the first codon position, changing serine (TCC) to proline (CCC) [ ]. .. For site 1565, the 10 μL PCR reaction contained 2 μL of 1 in 10 diluted template DNA, 0.4 μL each of the primers at 10 μM, 1 μL of the ThermoPol reaction buffer (NEB Inc., Ipswich, MA, USA; Cat. No. B9004S), 0.064 μL of dNTP’s at 25 mM (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-39029), 0.4 μL of MgCl2 (50 mM) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047), 0.25 μL of the LightCycler® 480 High Resolution Melting Master (Roche, Mannheim, Germany; Cat. No. 04909631001), 0.01 μL of IMMOLASE™ DNA polymerase (10 u/μL) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047) and 5.476 μL of ddH2 O (Honeywell, Burdick and Jackson; Muskegon, MI, USA; Cat. No. 365-4).

    Flow Cytometry:

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: For recording reactions on origami, flow chambers were created by attaching a freshly cleaved mica (Ted Pella) piece to a channel slide system (ibidi, Sticky-Slide VI0.4, Cat. No. 80608), yielding a 30 μl chamber volume. .. After 30 min of incubation, unbound origami and extra staple strands were removed by washing three times with 60 μl of fresh TAE/Mg buffer (40 mM Tris acetate, 1 mM EDTA buffer supplemented with 12.5 mM magnesium acetate) and three times with 60 μl of 1× ThermoPol buffer (NEB, Cat. No. B9004S, 10× ; 1× contains 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100).

    Ligation:

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: The three cap state selections use a series of enzymatic treatments to reduce specific populations of RNAs to 5’ monophosphate, making them capable of ligation to an RNA adapter by T4 RNA ligase (NEB, cat. M0204S). .. 5’ cap was removed with RNA 5’ pyrophosphohydrolase, RppH (NEB, cat. M0356S), using ThermoPol buffer (NEB, cat. B9004S).

    Cell Culture:

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: Paragraph title: Cell Culture and CoPRO library preparation ... 5’ cap was removed with RNA 5’ pyrophosphohydrolase, RppH (NEB, cat. M0356S), using ThermoPol buffer (NEB, cat. B9004S).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Optimization of trans-Splicing for Huntington's Disease RNA Therapy
    Article Snippet: Paragraph title: RNA isolation and RT-PCR ... For amplifications outside the HTT exon 1 CAG repeat and the adjacent GC-rich region, Pfu enzyme (prepared in-house) with Thermopol buffer (New England Biolabs, B9004S) was used.

    Tandem Mass Spectroscopy:

    Article Title: ALKBH10B Is an RNA N6-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition
    Article Snippet: The mixture was incubated at 37°C for an additional 5 h. For quantification of cap m6 Am , 100 ng poly(A) RNA was decapped with 20 units of RNA 5′pyrophosphohydrolase (RppH; M0356; New England Biolabs) in 1×Thermopol buffer (B9004S; New England Biolabs) for 3 h at 37°C, and then treated with Nuclease P1 and alkaline phosphatase as described above. .. The nucleosides were separated by UPLC (Shimadzu) equipped with a ZORBAX SB-Aq column (Agilent) and detected by MS/MS using a Triple Quad 5500 (AB SCIEX) mass spectrometer in positive ion mode by multiple reaction monitoring.

    Sequencing:

    Article Title: Metabolomic Profiles of a Midge (Procladius villosimanus, Kieffer) Are Associated with Sediment Contamination in Urban Wetlands
    Article Snippet: Procladius species were identified by amplifying and sequencing a 710 bp fragment of the COI gene using the universal primers LCO1490: 5′-ggtcaacaaatcataaagatattgg-3′ and HC02198: 5′-taaacttcagggtgaccaaaaaatca-3′ Folmer, et al. [ ]. .. Each PCR reaction was 25 μL and contained the following components: 5 μL Chelex extracted DNA, 2.5 μL ThermoPol 10× buffer (New England Biolabs Ipswich, United States (NEB) Cat. No. B9004S), 2 min μL dNTP (25 mM), 0.2 μL Taq DNA polymerase (NEB Cat. No. M0273S), 1.25 μL each LCO1490 and HC02198 primers (10 μM each) and 12.8 μL of double deionized water.

    Recombinant:

    Article Title: Base-Pair Resolution Genome-Wide Mapping Of Active RNA polymerases using Precision Nuclear Run-On (PRO-seq)
    Article Snippet: Paragraph title: Enzymes and recombinant protein reagents ... Alternatively, RNA 5′ Pyrophosphohydrolase, 5 units/μl (RppH) (NEB, cat. no. M0356S) can be used with ThermoPol Reaction buffer (NEB, cat. no. B9004S).

    Nucleic Acid Electrophoresis:

    Article Title: Metabolomic Profiles of a Midge (Procladius villosimanus, Kieffer) Are Associated with Sediment Contamination in Urban Wetlands
    Article Snippet: Each PCR reaction was 25 μL and contained the following components: 5 μL Chelex extracted DNA, 2.5 μL ThermoPol 10× buffer (New England Biolabs Ipswich, United States (NEB) Cat. No. B9004S), 2 min μL dNTP (25 mM), 0.2 μL Taq DNA polymerase (NEB Cat. No. M0273S), 1.25 μL each LCO1490 and HC02198 primers (10 μM each) and 12.8 μL of double deionized water. .. Amplification bands of correct size were confirmed with gel electrophoresis, using 2% agarose gels, stained with sybr safe (Thermo Fisher Scientific Cat No.: S33102).

    Fluorescence:

    Article Title: Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia
    Article Snippet: For site 1565, the 10 μL PCR reaction contained 2 μL of 1 in 10 diluted template DNA, 0.4 μL each of the primers at 10 μM, 1 μL of the ThermoPol reaction buffer (NEB Inc., Ipswich, MA, USA; Cat. No. B9004S), 0.064 μL of dNTP’s at 25 mM (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-39029), 0.4 μL of MgCl2 (50 mM) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047), 0.25 μL of the LightCycler® 480 High Resolution Melting Master (Roche, Mannheim, Germany; Cat. No. 04909631001), 0.01 μL of IMMOLASE™ DNA polymerase (10 u/μL) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047) and 5.476 μL of ddH2 O (Honeywell, Burdick and Jackson; Muskegon, MI, USA; Cat. No. 365-4). .. Thermo cycling steps were: 95 °C for 10 min, 20 cycles of 95 °C for 5 s, 65 °C (reduce 0.5 °C each cycle) for 15 s, 72 °C for 15 s, followed by an additional 20 cycles of 95 °C for 5 s, 55 °C for 15 s, and 72 °C for 15 s. Fluorescence information was captured at the end of each 72 °C step.

    Methylation:

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: 5’ cap was removed with RNA 5’ pyrophosphohydrolase, RppH (NEB, cat. M0356S), using ThermoPol buffer (NEB, cat. B9004S). .. It is not sensitive to the subsequent methylation step of capping .

    Mutagenesis:

    Article Title: Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia
    Article Snippet: The non-synonymous mutation occurs in the first codon position, changing serine (TCC) to proline (CCC) [ ]. .. For site 1565, the 10 μL PCR reaction contained 2 μL of 1 in 10 diluted template DNA, 0.4 μL each of the primers at 10 μM, 1 μL of the ThermoPol reaction buffer (NEB Inc., Ipswich, MA, USA; Cat. No. B9004S), 0.064 μL of dNTP’s at 25 mM (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-39029), 0.4 μL of MgCl2 (50 mM) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047), 0.25 μL of the LightCycler® 480 High Resolution Melting Master (Roche, Mannheim, Germany; Cat. No. 04909631001), 0.01 μL of IMMOLASE™ DNA polymerase (10 u/μL) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047) and 5.476 μL of ddH2 O (Honeywell, Burdick and Jackson; Muskegon, MI, USA; Cat. No. 365-4).

    Isolation:

    Article Title: Optimization of trans-Splicing for Huntington's Disease RNA Therapy
    Article Snippet: Paragraph title: RNA isolation and RT-PCR ... For amplifications outside the HTT exon 1 CAG repeat and the adjacent GC-rich region, Pfu enzyme (prepared in-house) with Thermopol buffer (New England Biolabs, B9004S) was used.

    Article Title: Efficient and Specific Detection of Salmonella in Food Samples Using a stn-Based Loop-Mediated Isothermal Amplification Method
    Article Snippet: LAMP Reaction and LAMP Product Detection The LAMP reaction was performed in a total volume of 25 μ L containing the following components (final concentration): 1.6 μ M each of FIP and BIP primers, 0.2 μ M each of F3 and B3 primers, 0.8 μ M each of LF and LB primers (in the same LAMP reaction), 1.6 mM of deoxyribonucleotide triphosphate mixture (dNTPs), 1 M betaine (Sigma, B2629, St. Louis, USA), 6 mM MgSO4 , 1x thermopol buffer (New England Biolabs, B9004S, Beverly, USA), 1 μ L (8 U) of B st DNA polymerase large fragment (New England Biolabs, M0275S, Beverly, USA), and 5 μ L of DNA template solution. .. Typhimurium ATCC 23566 cells or its isolated DNA (1 ng/reaction) was used as positive controls.

    Article Title: Reasons for the Invasive Success of a Guppy (Poecilia reticulata) Population in Trinidad
    Article Snippet: DNA was isolated using the DNeasy Blood & Tissue Kit from Qiagen. .. For DNA amplification the following PCR protocol was used: 2.5 µl [25 ng/µl] template, 4.4 µl H2 O, 1 µl PCR buffer [10× Thermobuffer (NEB B9004S)], 1 µl dNTPs [2 mM], 0.1 µl Taq polymerase [5000 U/m (NEB M0267L)], 0.5 µl Fw-primer and 0.5 µl Rev-primer [both 10 pmol/µl].

    Avidin-Biotin Assay:

    Article Title: Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy
    Article Snippet: .. 5-(3-aminoallyl) dUTP (Fermentas #R0091) Adenosine 5′-O-(3-thiotriphosphate) (ATPγS Calbiotech #119120) ATTO565 NHS-ester (ATTO-TEC GmbH #AD 565-31) Avidin agarose (Thermo Scientific #20219) Biotin-11-dGTP (PerkinElmer #NEL541) Bovine serum albumin (BSA, Sigma-Aldrich #A-9647) Deoxyribonucleoside triphosphates (dNTPs: Invitrogen dATP (#55082), TTP (#55085), dCTP (#55083) and dGTP (#55084) for PCR) Dithiothreitol (DTT Fisher Scientific #BP172-25) CAUTION causes eye and skin irritation; handle wearing goggles, lab coat and gloves Ethanol (EtOH Gold Shield Chemical Company #43196-117) CAUTION flammable; avoid open flames Ethylenediaminetetraacetic acid (EDTA, J. T. Baker #8993-01) Klenow Fragment DNA polymerase I (NEB #M0212S) Magnesium acetate (Mg(OAc)2 J. T. Baker #2424-01) Magnesium chloride (MgCl2 ) Methanol (MeOH Fisher Scientific #A412-4) CAUTION flammable; avoid open flames NEB buffer #2 (NEB buffer supplied with Klenow Fragment DNA polymerase I NEB #B7002S) Phage λ DNA (NEB #N3013S) Potassium hydroxide (KOH Sigma-Aldrich #221473-1KG) CAUTION corrosive; handle wearing goggles, lab coat and gloves Primers for polymerase chain reaction (PCR) to yield a 430 bp product identical to λ DNA between base pairs 23,788 and 24,217: forward primer 5′-biotin-ACTGTTCTTGCGGTTTGGAGG-3′ reverse primer 5′-CTATCGGAAGTTCACCAGCCAG-3′ (can be purchased from any high quality source such as Sigma, Integrated DNA Technologies, or Invitrogen) Sodium bicarbonate (NaHCO3 J. T. Baker #3506-05) Sodium chloride (NaCl) Sodium hydroxide (NaOH) Streptavidin-coated polystyrene beads, 1 μm (Bangs Laboratories #CP01N/10021) Sucrose (Sigma-Aldrich #S7903-5KG) ThermoPol buffer (NEB buffer supplied with VentR ® (exo− ) DNA polymerase NEB #B9004S) Tris acetate (TrisOAc TRIZMA base Sigma-Aldrich #T-1503 + Acetic acid EMD #AX0073-9) CAUTION Acetic acid is corrosive; handle wearing goggles, lab coat and gloves in ventilated hood Water, ultrapure Type 1 (Nanopure (Barnstead) or Milli-Q (Millipore)) VentR ® (exo− ) DNA Polymerase (NEB #M0257) YOYO-1 (Invitrogen #Y3601) .. Abrasive blasting cabinet (Harbor Freight #42202) Computer with design software such as CorelDraw (Corel Corporation) or Illustrator (Adobe Systems Incorporated) Cover glass (Corning No. 1, 24×60 mm #2955-246) Dremel® rotary tool with a diamond coated bit (Dremel #7134) Epoxy, 5 minute (Devcon #14210) Gastight syringes 1000 μl (×4) and 50 μl (×1) (Hamilton #1001 and # 1705) Glass Microscope slides (Fisher Scientific 25×75×1 mm #12-550-A3) Heat block or water bath for maintaining temperature for various reactions High-pressure mercury plasma arc-discharge lamp for curing the optical adhesive (Zeiss 100 watt HBO lamp).

    Size-exclusion Chromatography:

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: The 50 µl PCR assay mix contained 32.8 µl of sterile Milli-Q water, 1 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 5 µL of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs), 3 µl each of 10 pmol/µl forward and reverse primer , 0.2 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), and 5 µl of DNA template. .. PCR amplification was carried out using the following temperature program: initial denaturation (5 min, 95°C) followed by 25 cycles of denaturation (45 sec, 94°C), annealing (1 min, at primer-specific temperature [ ]), and elongation (1 min, 72°C) and then final extension (10 min, 72°C).

    Article Title: Reasons for the Invasive Success of a Guppy (Poecilia reticulata) Population in Trinidad
    Article Snippet: For DNA amplification the following PCR protocol was used: 2.5 µl [25 ng/µl] template, 4.4 µl H2 O, 1 µl PCR buffer [10× Thermobuffer (NEB B9004S)], 1 µl dNTPs [2 mM], 0.1 µl Taq polymerase [5000 U/m (NEB M0267L)], 0.5 µl Fw-primer and 0.5 µl Rev-primer [both 10 pmol/µl]. .. All PCR amplifications run in Peltier Thermal Cyclers pTC-200 from Bio-Rad under the following conditions: 96°C for 3 min, 40 cycles of 96°C for 30 sec., 56°C for 30 sec., 68°C for 30 sec.; 68°C for 5 min.

    Polymerase Chain Reaction:

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: .. The 25 µl reaction mixture contained 2.5 µl of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs, Hertfordshire, UK), 4 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 0.1 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), 1 µl each of 10 pmol/µl forward and reverse primer , 15.4 µl of sterile Milli-Q water, and 1 µl of DNA template. .. PCR amplification was carried out in an Eppendorf Master Cycler (22331, Eppendorf, Germany) using the following temperature program: initial denaturation (1 min, 94°C) followed by 35 cycles of denaturation (1 min, 94°C), annealing (1 min, 55°C), and elongation (1 min, 72°C) and then final extension (7 min, 72°C).

    Article Title: Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia
    Article Snippet: .. For site 1565, the 10 μL PCR reaction contained 2 μL of 1 in 10 diluted template DNA, 0.4 μL each of the primers at 10 μM, 1 μL of the ThermoPol reaction buffer (NEB Inc., Ipswich, MA, USA; Cat. No. B9004S), 0.064 μL of dNTP’s at 25 mM (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-39029), 0.4 μL of MgCl2 (50 mM) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047), 0.25 μL of the LightCycler® 480 High Resolution Melting Master (Roche, Mannheim, Germany; Cat. No. 04909631001), 0.01 μL of IMMOLASE™ DNA polymerase (10 u/μL) (Bioline, Alexandria, NSW, Australia; Cat. No. BIO-21047) and 5.476 μL of ddH2 O (Honeywell, Burdick and Jackson; Muskegon, MI, USA; Cat. No. 365-4). .. PCR amplification was carried out using the Roche LightCycler® 480 system (384-well format).

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: .. The 50 µl PCR assay mix contained 32.8 µl of sterile Milli-Q water, 1 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 5 µL of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs), 3 µl each of 10 pmol/µl forward and reverse primer , 0.2 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), and 5 µl of DNA template. .. PCR amplification was carried out using the following temperature program: initial denaturation (5 min, 95°C) followed by 25 cycles of denaturation (45 sec, 94°C), annealing (1 min, at primer-specific temperature [ ]), and elongation (1 min, 72°C) and then final extension (10 min, 72°C).

    Article Title: Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy
    Article Snippet: .. 5-(3-aminoallyl) dUTP (Fermentas #R0091) Adenosine 5′-O-(3-thiotriphosphate) (ATPγS Calbiotech #119120) ATTO565 NHS-ester (ATTO-TEC GmbH #AD 565-31) Avidin agarose (Thermo Scientific #20219) Biotin-11-dGTP (PerkinElmer #NEL541) Bovine serum albumin (BSA, Sigma-Aldrich #A-9647) Deoxyribonucleoside triphosphates (dNTPs: Invitrogen dATP (#55082), TTP (#55085), dCTP (#55083) and dGTP (#55084) for PCR) Dithiothreitol (DTT Fisher Scientific #BP172-25) CAUTION causes eye and skin irritation; handle wearing goggles, lab coat and gloves Ethanol (EtOH Gold Shield Chemical Company #43196-117) CAUTION flammable; avoid open flames Ethylenediaminetetraacetic acid (EDTA, J. T. Baker #8993-01) Klenow Fragment DNA polymerase I (NEB #M0212S) Magnesium acetate (Mg(OAc)2 J. T. Baker #2424-01) Magnesium chloride (MgCl2 ) Methanol (MeOH Fisher Scientific #A412-4) CAUTION flammable; avoid open flames NEB buffer #2 (NEB buffer supplied with Klenow Fragment DNA polymerase I NEB #B7002S) Phage λ DNA (NEB #N3013S) Potassium hydroxide (KOH Sigma-Aldrich #221473-1KG) CAUTION corrosive; handle wearing goggles, lab coat and gloves Primers for polymerase chain reaction (PCR) to yield a 430 bp product identical to λ DNA between base pairs 23,788 and 24,217: forward primer 5′-biotin-ACTGTTCTTGCGGTTTGGAGG-3′ reverse primer 5′-CTATCGGAAGTTCACCAGCCAG-3′ (can be purchased from any high quality source such as Sigma, Integrated DNA Technologies, or Invitrogen) Sodium bicarbonate (NaHCO3 J. T. Baker #3506-05) Sodium chloride (NaCl) Sodium hydroxide (NaOH) Streptavidin-coated polystyrene beads, 1 μm (Bangs Laboratories #CP01N/10021) Sucrose (Sigma-Aldrich #S7903-5KG) ThermoPol buffer (NEB buffer supplied with VentR ® (exo− ) DNA polymerase NEB #B9004S) Tris acetate (TrisOAc TRIZMA base Sigma-Aldrich #T-1503 + Acetic acid EMD #AX0073-9) CAUTION Acetic acid is corrosive; handle wearing goggles, lab coat and gloves in ventilated hood Water, ultrapure Type 1 (Nanopure (Barnstead) or Milli-Q (Millipore)) VentR ® (exo− ) DNA Polymerase (NEB #M0257) YOYO-1 (Invitrogen #Y3601) .. Abrasive blasting cabinet (Harbor Freight #42202) Computer with design software such as CorelDraw (Corel Corporation) or Illustrator (Adobe Systems Incorporated) Cover glass (Corning No. 1, 24×60 mm #2955-246) Dremel® rotary tool with a diamond coated bit (Dremel #7134) Epoxy, 5 minute (Devcon #14210) Gastight syringes 1000 μl (×4) and 50 μl (×1) (Hamilton #1001 and # 1705) Glass Microscope slides (Fisher Scientific 25×75×1 mm #12-550-A3) Heat block or water bath for maintaining temperature for various reactions High-pressure mercury plasma arc-discharge lamp for curing the optical adhesive (Zeiss 100 watt HBO lamp).

    Article Title: Reasons for the Invasive Success of a Guppy (Poecilia reticulata) Population in Trinidad
    Article Snippet: .. For DNA amplification the following PCR protocol was used: 2.5 µl [25 ng/µl] template, 4.4 µl H2 O, 1 µl PCR buffer [10× Thermobuffer (NEB B9004S)], 1 µl dNTPs [2 mM], 0.1 µl Taq polymerase [5000 U/m (NEB M0267L)], 0.5 µl Fw-primer and 0.5 µl Rev-primer [both 10 pmol/µl]. .. All PCR amplifications run in Peltier Thermal Cyclers pTC-200 from Bio-Rad under the following conditions: 96°C for 3 min, 40 cycles of 96°C for 30 sec., 56°C for 30 sec., 68°C for 30 sec.; 68°C for 5 min.

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: .. The 25 µl reaction mixture contained 2.5 µl of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs, Hertfordshire, UK), 4 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 0.1 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), 1 µl each of 10 pmol/µl forward and reverse primer ( ), 15.4 µl of sterile Milli-Q water, and 1 µl of DNA template. .. PCR amplification was carried out in an Eppendorf Master Cycler (22331, Eppendorf, Germany) using the following temperature program: initial denaturation (1 min, 94°C) followed by 35 cycles of denaturation (1 min, 94°C), annealing (1 min, 55°C), and elongation (1 min, 72°C) and then final extension (7 min, 72°C).

    Article Title: Metabolomic Profiles of a Midge (Procladius villosimanus, Kieffer) Are Associated with Sediment Contamination in Urban Wetlands
    Article Snippet: .. Each PCR reaction was 25 μL and contained the following components: 5 μL Chelex extracted DNA, 2.5 μL ThermoPol 10× buffer (New England Biolabs Ipswich, United States (NEB) Cat. No. B9004S), 2 min μL dNTP (25 mM), 0.2 μL Taq DNA polymerase (NEB Cat. No. M0273S), 1.25 μL each LCO1490 and HC02198 primers (10 μM each) and 12.8 μL of double deionized water. .. The qPCR program was: 3 min at 95 °C, followed by 35 cycles of 1 min at 95 °C, 1 min at 40 °C and 1 min 30 s at 72 °C, followed by a final extension step for 5 min at 72 °C.

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: After 30 min of incubation, unbound origami and extra staple strands were removed by washing three times with 60 μl of fresh TAE/Mg buffer (40 mM Tris acetate, 1 mM EDTA buffer supplemented with 12.5 mM magnesium acetate) and three times with 60 μl of 1× ThermoPol buffer (NEB, Cat. No. B9004S, 10× ; 1× contains 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100). .. This minimizes any false-positive PCR amplification by the recording primers.

    Article Title: Phenotypic and genotypic characteristics of Trueperellapyogenes isolated from ruminants
    Article Snippet: .. The pld -negative isolates were examined for 8 known and putative virulence genes, utilizing previously developed PCR protocols and primers., The PCR program was performed as follows : denaturation at 95°C for 4 min; 35 cycles of 94°C for 1 min, annealing for 30 s at different temperatures (60°C for nanH -, nanP -, plo -, cbpA -, and fimC -specific PCRs; and 57°C for fimA - and fimG -specific PCRs), , 72°C for 3 min; and a final extension at 72°C for 7 min. All amplifications were carried out in 50 µL of the following reaction mixture: 10 pmol of each primer (0.5 µL each), 10 mM of each dNTP (N0447S, New England BioLabs, Ipswich, MA), 1× Taq polymerase buffer (B9004S, New England BioLabs), 1 U of Taq DNA polymerase (M0273L, New England BioLabs), and ~50 ng of genomic DNA. .. The PCR products were separated by electrophoresis through a 2% agarose gel in Tris–acetate–EDTA buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA; Thermo Fisher Scientific, Lenexa, KS), stained with ethidium bromide (MilliporeSigma), and visualized using an Alpha Imager HP (Alpha-Innotech, San Leandro, CA).

    Real-time Polymerase Chain Reaction:

    Article Title: Metabolomic Profiles of a Midge (Procladius villosimanus, Kieffer) Are Associated with Sediment Contamination in Urban Wetlands
    Article Snippet: Each PCR reaction was 25 μL and contained the following components: 5 μL Chelex extracted DNA, 2.5 μL ThermoPol 10× buffer (New England Biolabs Ipswich, United States (NEB) Cat. No. B9004S), 2 min μL dNTP (25 mM), 0.2 μL Taq DNA polymerase (NEB Cat. No. M0273S), 1.25 μL each LCO1490 and HC02198 primers (10 μM each) and 12.8 μL of double deionized water. .. The qPCR program was: 3 min at 95 °C, followed by 35 cycles of 1 min at 95 °C, 1 min at 40 °C and 1 min 30 s at 72 °C, followed by a final extension step for 5 min at 72 °C.

    Selection:

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: Between adapter ligations, cap state selection reactions were performed. .. 5’ cap was removed with RNA 5’ pyrophosphohydrolase, RppH (NEB, cat. M0356S), using ThermoPol buffer (NEB, cat. B9004S).

    Agarose Gel Electrophoresis:

    Article Title: Phenotypic and genotypic characteristics of Trueperellapyogenes isolated from ruminants
    Article Snippet: The pld -negative isolates were examined for 8 known and putative virulence genes, utilizing previously developed PCR protocols and primers., The PCR program was performed as follows : denaturation at 95°C for 4 min; 35 cycles of 94°C for 1 min, annealing for 30 s at different temperatures (60°C for nanH -, nanP -, plo -, cbpA -, and fimC -specific PCRs; and 57°C for fimA - and fimG -specific PCRs), , 72°C for 3 min; and a final extension at 72°C for 7 min. All amplifications were carried out in 50 µL of the following reaction mixture: 10 pmol of each primer (0.5 µL each), 10 mM of each dNTP (N0447S, New England BioLabs, Ipswich, MA), 1× Taq polymerase buffer (B9004S, New England BioLabs), 1 U of Taq DNA polymerase (M0273L, New England BioLabs), and ~50 ng of genomic DNA. .. The PCR products were separated by electrophoresis through a 2% agarose gel in Tris–acetate–EDTA buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA; Thermo Fisher Scientific, Lenexa, KS), stained with ethidium bromide (MilliporeSigma), and visualized using an Alpha Imager HP (Alpha-Innotech, San Leandro, CA).

    Ethanol Precipitation:

    Article Title: Single-molecule nascent RNA sequencing reveals regulatory domain architecture at promoters and enhancers
    Article Snippet: All steps were carried out following the manufacturer’s protocol, with phenol:chloroform extraction and ethanol precipitation between steps. .. 5’ cap was removed with RNA 5’ pyrophosphohydrolase, RppH (NEB, cat. M0356S), using ThermoPol buffer (NEB, cat. B9004S).

    Produced:

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: After 30 min of incubation, unbound origami and extra staple strands were removed by washing three times with 60 μl of fresh TAE/Mg buffer (40 mM Tris acetate, 1 mM EDTA buffer supplemented with 12.5 mM magnesium acetate) and three times with 60 μl of 1× ThermoPol buffer (NEB, Cat. No. B9004S, 10× ; 1× contains 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100). .. After the recording reaction, the supernatant solution containing produced records was collected and the polymerase was heat inactivated by incubating the solution at 80 °C for 20 min. For samples used in the geometry studies (three-point and hexagonal grid patterns) and the state change study, before quenching the reaction, extra recording primers contained in the product solution were removed by mixing the solution with Exonuclease I (NEB, Cat. No. M0293S) at 9:1 volume ratio and incubating for 20 min at 37 °C.

    Concentration Assay:

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: The MIC was identified as the lowest antibiotic concentration at which no growth (turbidity) was observed. .. The 25 µl reaction mixture contained 2.5 µl of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs, Hertfordshire, UK), 4 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 0.1 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), 1 µl each of 10 pmol/µl forward and reverse primer , 15.4 µl of sterile Milli-Q water, and 1 µl of DNA template.

    Article Title: Efficient and Specific Detection of Salmonella in Food Samples Using a stn-Based Loop-Mediated Isothermal Amplification Method
    Article Snippet: .. LAMP Reaction and LAMP Product Detection The LAMP reaction was performed in a total volume of 25 μ L containing the following components (final concentration): 1.6 μ M each of FIP and BIP primers, 0.2 μ M each of F3 and B3 primers, 0.8 μ M each of LF and LB primers (in the same LAMP reaction), 1.6 mM of deoxyribonucleotide triphosphate mixture (dNTPs), 1 M betaine (Sigma, B2629, St. Louis, USA), 6 mM MgSO4 , 1x thermopol buffer (New England Biolabs, B9004S, Beverly, USA), 1 μ L (8 U) of B st DNA polymerase large fragment (New England Biolabs, M0275S, Beverly, USA), and 5 μ L of DNA template solution. ..

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: The MIC was identified as the lowest antibiotic concentration at which no growth (turbidity) was observed. .. The 25 µl reaction mixture contained 2.5 µl of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs, Hertfordshire, UK), 4 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 0.1 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), 1 µl each of 10 pmol/µl forward and reverse primer ( ), 15.4 µl of sterile Milli-Q water, and 1 µl of DNA template.

    Article Title: A DNA nanoscope via auto-cycling proximity recording
    Article Snippet: After washing the chamber three times with 60 μl of TAE/Mg buffer by adding the buffer to one reservoir (inlet) and subsequently taking out the same volume from the other side (outlet), a 30 μl solution containing origami at 50 pM concentration was introduced to the chamber. (In the first washing round, ~30 μl of buffer occupies the chamber and only ~30 μl of extra buffer comes out.) .. After 30 min of incubation, unbound origami and extra staple strands were removed by washing three times with 60 μl of fresh TAE/Mg buffer (40 mM Tris acetate, 1 mM EDTA buffer supplemented with 12.5 mM magnesium acetate) and three times with 60 μl of 1× ThermoPol buffer (NEB, Cat. No. B9004S, 10× ; 1× contains 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton X-100).

    Staining:

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: Bifidobacterium isolates were phenotypically characterized by Gram staining and catalase reaction. .. The 25 µl reaction mixture contained 2.5 µl of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs, Hertfordshire, UK), 4 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 0.1 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), 1 µl each of 10 pmol/µl forward and reverse primer , 15.4 µl of sterile Milli-Q water, and 1 µl of DNA template.

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: The 50 µl PCR assay mix contained 32.8 µl of sterile Milli-Q water, 1 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 5 µL of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs), 3 µl each of 10 pmol/µl forward and reverse primer , 0.2 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), and 5 µl of DNA template. .. All PCR amplicons were visualized by electrophoresis on 1% (1.2% for ITS-PCR) agarose gels containing 2 µl GelRed stain (41003, Biotium, Fremont, CA, USA); wells were loaded with 10 µl of PCR product + 2 µl of loading dye (R0611, Thermo Fisher Scientific, Loughborough, UK).

    Article Title: Investigating the transmissibility of tet(W) in bifidobacteria exposed to acid and bile stress
    Article Snippet: Bifidobacterium isolates were phenotypically characterized by Gram staining and catalase reaction. .. The 25 µl reaction mixture contained 2.5 µl of 10x PCR buffer including 20 mM MgCl2 (B9004S, New England Biolabs, Hertfordshire, UK), 4 µl of 1.25 mM dNTP mix (N0447, New England Biolabs), 0.1 µl of 5U Taq DNA polymerase (M0267X, New England Biolabs), 1 µl each of 10 pmol/µl forward and reverse primer ( ), 15.4 µl of sterile Milli-Q water, and 1 µl of DNA template.

    Article Title: Metabolomic Profiles of a Midge (Procladius villosimanus, Kieffer) Are Associated with Sediment Contamination in Urban Wetlands
    Article Snippet: Each PCR reaction was 25 μL and contained the following components: 5 μL Chelex extracted DNA, 2.5 μL ThermoPol 10× buffer (New England Biolabs Ipswich, United States (NEB) Cat. No. B9004S), 2 min μL dNTP (25 mM), 0.2 μL Taq DNA polymerase (NEB Cat. No. M0273S), 1.25 μL each LCO1490 and HC02198 primers (10 μM each) and 12.8 μL of double deionized water. .. Amplification bands of correct size were confirmed with gel electrophoresis, using 2% agarose gels, stained with sybr safe (Thermo Fisher Scientific Cat No.: S33102).

    Article Title: Phenotypic and genotypic characteristics of Trueperellapyogenes isolated from ruminants
    Article Snippet: The pld -negative isolates were examined for 8 known and putative virulence genes, utilizing previously developed PCR protocols and primers., The PCR program was performed as follows : denaturation at 95°C for 4 min; 35 cycles of 94°C for 1 min, annealing for 30 s at different temperatures (60°C for nanH -, nanP -, plo -, cbpA -, and fimC -specific PCRs; and 57°C for fimA - and fimG -specific PCRs), , 72°C for 3 min; and a final extension at 72°C for 7 min. All amplifications were carried out in 50 µL of the following reaction mixture: 10 pmol of each primer (0.5 µL each), 10 mM of each dNTP (N0447S, New England BioLabs, Ipswich, MA), 1× Taq polymerase buffer (B9004S, New England BioLabs), 1 U of Taq DNA polymerase (M0273L, New England BioLabs), and ~50 ng of genomic DNA. .. The PCR products were separated by electrophoresis through a 2% agarose gel in Tris–acetate–EDTA buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA; Thermo Fisher Scientific, Lenexa, KS), stained with ethidium bromide (MilliporeSigma), and visualized using an Alpha Imager HP (Alpha-Innotech, San Leandro, CA).

    Fluorescence In Situ Hybridization:

    Article Title: Reasons for the Invasive Success of a Guppy (Poecilia reticulata) Population in Trinidad
    Article Snippet: In June and July 2010, all fish samples were taken to the Max Planck Institute for Developmental Biology in Tuebingen, Germany, for genetic analysis. .. For DNA amplification the following PCR protocol was used: 2.5 µl [25 ng/µl] template, 4.4 µl H2 O, 1 µl PCR buffer [10× Thermobuffer (NEB B9004S)], 1 µl dNTPs [2 mM], 0.1 µl Taq polymerase [5000 U/m (NEB M0267L)], 0.5 µl Fw-primer and 0.5 µl Rev-primer [both 10 pmol/µl].

    Hood:

    Article Title: Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy
    Article Snippet: .. 5-(3-aminoallyl) dUTP (Fermentas #R0091) Adenosine 5′-O-(3-thiotriphosphate) (ATPγS Calbiotech #119120) ATTO565 NHS-ester (ATTO-TEC GmbH #AD 565-31) Avidin agarose (Thermo Scientific #20219) Biotin-11-dGTP (PerkinElmer #NEL541) Bovine serum albumin (BSA, Sigma-Aldrich #A-9647) Deoxyribonucleoside triphosphates (dNTPs: Invitrogen dATP (#55082), TTP (#55085), dCTP (#55083) and dGTP (#55084) for PCR) Dithiothreitol (DTT Fisher Scientific #BP172-25) CAUTION causes eye and skin irritation; handle wearing goggles, lab coat and gloves Ethanol (EtOH Gold Shield Chemical Company #43196-117) CAUTION flammable; avoid open flames Ethylenediaminetetraacetic acid (EDTA, J. T. Baker #8993-01) Klenow Fragment DNA polymerase I (NEB #M0212S) Magnesium acetate (Mg(OAc)2 J. T. Baker #2424-01) Magnesium chloride (MgCl2 ) Methanol (MeOH Fisher Scientific #A412-4) CAUTION flammable; avoid open flames NEB buffer #2 (NEB buffer supplied with Klenow Fragment DNA polymerase I NEB #B7002S) Phage λ DNA (NEB #N3013S) Potassium hydroxide (KOH Sigma-Aldrich #221473-1KG) CAUTION corrosive; handle wearing goggles, lab coat and gloves Primers for polymerase chain reaction (PCR) to yield a 430 bp product identical to λ DNA between base pairs 23,788 and 24,217: forward primer 5′-biotin-ACTGTTCTTGCGGTTTGGAGG-3′ reverse primer 5′-CTATCGGAAGTTCACCAGCCAG-3′ (can be purchased from any high quality source such as Sigma, Integrated DNA Technologies, or Invitrogen) Sodium bicarbonate (NaHCO3 J. T. Baker #3506-05) Sodium chloride (NaCl) Sodium hydroxide (NaOH) Streptavidin-coated polystyrene beads, 1 μm (Bangs Laboratories #CP01N/10021) Sucrose (Sigma-Aldrich #S7903-5KG) ThermoPol buffer (NEB buffer supplied with VentR ® (exo− ) DNA polymerase NEB #B9004S) Tris acetate (TrisOAc TRIZMA base Sigma-Aldrich #T-1503 + Acetic acid EMD #AX0073-9) CAUTION Acetic acid is corrosive; handle wearing goggles, lab coat and gloves in ventilated hood Water, ultrapure Type 1 (Nanopure (Barnstead) or Milli-Q (Millipore)) VentR ® (exo− ) DNA Polymerase (NEB #M0257) YOYO-1 (Invitrogen #Y3601) .. Abrasive blasting cabinet (Harbor Freight #42202) Computer with design software such as CorelDraw (Corel Corporation) or Illustrator (Adobe Systems Incorporated) Cover glass (Corning No. 1, 24×60 mm #2955-246) Dremel® rotary tool with a diamond coated bit (Dremel #7134) Epoxy, 5 minute (Devcon #14210) Gastight syringes 1000 μl (×4) and 50 μl (×1) (Hamilton #1001 and # 1705) Glass Microscope slides (Fisher Scientific 25×75×1 mm #12-550-A3) Heat block or water bath for maintaining temperature for various reactions High-pressure mercury plasma arc-discharge lamp for curing the optical adhesive (Zeiss 100 watt HBO lamp).

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    New England Biolabs thermopol buffer
    The temperatures of <t>ThermoPol</t> Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Thermopol Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: The RCA components of the Bst DNA polymerase-LF reaction mixture were used for this purpose, because only the ThermoPol buffer of this polymerase is a nonproprietary reagent.

    Techniques:

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkz008

    Figure Lengend 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.

    Article Snippet: ThermoPol® buffer 10× and MgSO4 (100 mM) were purchased from NEB.

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkz008

    Figure Lengend 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.

    Article Snippet: ThermoPol® buffer 10× and MgSO4 (100 mM) were purchased from NEB.

    Techniques:

    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