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Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp <t>DNA-like</t> polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM <t>MgCl</t> 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.
Mgcl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Article Title: Mechanical properties of DNA-like polymers

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt808

Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.
Figure Legend Snippet: Example measurement of mechanical properties. ( A ) Cyclization time course for 207-bp DNA-like polymer 5 (pJ1744). DNA ligase-catalyzed cyclization reaction was performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ATP, 10 mM dithiothreitol) and a final concentration of 100 U/ml T4 DNA ligase. Aliquots (10 µl) were removed at 1–15 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3). Gel lanes contains Invitrogen 100 bp DNA ladder (M), linear monomer without ligase (0) and increasing 1-min time points of the ligation reaction ( 1–15 ) showing the evolution of linear monomer ( M ), linear dimer ( D ), circular monomer ( C M ) and circular dimer ( C D ). Nearest molecular weight bands are indicated. ( B ) Cyclization kinetics analysis for 207-bp DNA-like polymer 5 (pJ1744). Fitting of data in (A) determines the J -factor, as previously described ( 30 ) (see also Supplementary Data S3 ). ( C ) WLC analysis for DNA-like polymer 5 . Fit of experimental J -factor data using the WLC model. ( D ). Monte Carlo estimation of uncertainty. Fit of simulated J -factor data based on (C) using the WLC model.

Techniques Used: DNA Ligation, Concentration Assay, Electrophoresis, Ligation, Molecular Weight

Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).
Figure Legend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

Techniques Used: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

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Methylation Sequencing:

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Synthesized:

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Overlay Assay:

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Size-exclusion Chromatography:

Article Title: Histone-Acetylated Control of Fibroblast Growth Factor Receptor 2 Intron 2 Polymorphisms and Isoform Splicing in Breast Cancer
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Concentration Assay:

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Incubation:

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Polymerase Chain Reaction:

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Binding Assay:

Article Title: Transgenic Overexpression of LARGE Induces ?-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle
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Article Title: The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice
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