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Some molecules (<5%) are fractured, possibly during the mixing of the DNA into the high-viscosity buffer, but a visual inspection of the light intensity and size can generally rule out these smaller molecules
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we made our flow cells by etching silicon wafers with KOH and anionically bonding Pyrex coverslips to the silicon to seal the top surface of the channels
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G but something slightly larger due to blooming in the camera. Once the chain is extended about two times the equilibrium size, the blooming is reduced and the measurements correspond closely to the actual extension. We stained the λ-DNA (New England Biolabs) with YOYO-1 (Molecular Probes) at a dye:base-pair ratio of 1:4 for >1 hour. The experiment was performed in a high viscosity (η = 41 centipoise) buffer consisting of 10 mM tris-HCl, 2 mM EDTA, 10 mM NaCl, 4% β-mercaptoethanol, ∼50 μg/ml glucose oxidase (Boehringer Mannheim), ∼10 μg/ml catalase (Boehringer Mannheim), ∼18% (w/w) glucose and ∼40% (w/w) sucrose. The viscosity of each solution was measured in a temperature-stabilized viscometer and adjusted as needed. The flow cell was mounted on a copper block and stabilized to 22.7° ± 0.2°C.
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G but something slightly larger due to blooming in the camera. Once the chain is extended about two times the equilibrium size, the blooming is reduced and the measurements correspond closely to the actual extension. We stained the λ-DNA (New England Biolabs) with YOYO-1 (Molecular Probes) at a dye:base-pair ratio of 1:4 for >1 hour. The experiment was performed in a high viscosity (η = 41 centipoise) buffer consisting of 10 mM tris-HCl, 2 mM EDTA, 10 mM NaCl, 4% β-mercaptoethanol, ∼50 μg/ml glucose oxidase (Boehringer Mannheim), ∼10 μg/ml catalase (Boehringer Mannheim), ∼18% (w/w) glucose and ∼40% (w/w) sucrose. The viscosity of each solution was measured in a temperature-stabilized viscometer and adjusted as needed. The flow cell was mounted on a copper block and stabilized to 22.7° ± 0.2°C.
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relax) to relax back to equilibrium before entering the elongational flow.
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1842267898
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onset still occurred. The polymers had the general appearance of a dumbbell configuration subject to a large fluctuations in extension and shape
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onset still occurred. The polymers had the general appearance of a dumbbell configuration subject to a large fluctuations in extension and shape.
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G, which yields τ = 6.2 s, and is the relaxation time we reported in an earlier
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The longer DNA molecules were concatemers of λ-DNA (up to 250 μm). We were unable to systematically investigate the dynamics of longer molecules, because we did not have an adequately monodisperse sample and could not independently measure the length of individual molecules
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The longer DNA molecules were concatemers of λ-DNA (up to 250 μm). We were unable to systematically investigate the dynamics of longer molecules, because we did not have an adequately monodisperse sample and could not independently measure the length of individual molecules.
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onset, and from internal configurations (that is, folds). In mixed flows, a large fraction of the molecules are partially extended because of shearing, and this effect may eliminate some of the internal constraints that led to the observed dynamics
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onset, and from internal configurations (that is, folds). In mixed flows, a large fraction of the molecules are partially extended because of shearing, and this effect may eliminate some of the internal constraints that led to the observed dynamics.
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We thank G. Fuller, D. Hoagland, R. Larson, R. Pecora, and B. Zimm for helpful discussions and J. Shott for generous aid in lithographic design. Supported in part by grants from the U.S. Air Force Office of Scientific Research, NSF, and the Human Frontiers Foundation and by an endowment established by Theodore and Frances Geballe. We acknowledge the generous assistance of J. Spudich, including support through NIH grant GM33289 to J. Spudich. D.E.S. was supported by a fellowship from the Program in Mathematics and Molecular Biology at the University of California, Berkeley, through NSF grant DMS 9406348. S.C. was supported in part by a Guggenheim fellowship during the course of this work.
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