Backscattering from a tethered bead as a probe of DNA flexibility

Applied Physics Letters

Volumn 73, Issue 3, 1998, Pages 291-293

  Shivashankar, G V ^a   Stolovitzky, G ^a   Libchaber, A ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 0002678831     PISSN: 00036951     EISSN: None     Source Type: Journal    
DOI: 10.1063/1.121798     Document Type: Article

References (20)

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13. To calibrate the bead displacement one chooses an immobilized bead on the coverslip. The fixed bead is aligned with respect to the laser axis and the backscattered signal is projected onto a quadrant position detector. A triangle ramp (1 μm amplitude and 1 Hz frequency) is setup to move the sample stage and the differential output signal of the quadrant detector is recorded. The minimum step size of the stage is ∼10 nm. From this data the calibration of the bead displacement is obtained.
14. Tethered beads are prepared by attaching one end of λ DNA (Promega) to a coverslip and the other end to a 3 μm latex bead (Polysciences), as described in Ref. 10. Experiments are carried out at pH 7.4 and in 0.1 M phosphate buffered saline. Typically the ratio of the number of beads to DNA molecules ∼1:10, for DNA attachment to the coverslip.
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16. 2∼25mM, DTT∼2mM, Tris-Hcl ∼20 mM, and 100 μg/400 μl acetylated BSA. The final pH is 7 and the experiments are done at 37°C.
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20. A monomer of Rec A binds to about three bases of DNA. The kinetics of polymerization depends on the state of the DNA molecule, whether it is in the random coil state or in a stretched form. Typical time scales for the polymerization of Rec A on a single DNA polymer, under the same biochemical conditions and T=37°C, are: 60 min for random coil and 20 min for a molecule under an applied external force of 5 pN.