Materials
| Name | Company | Catalog Number | Comments |
| Reagent/Equipment | Company | Catalog number | Comments |
| Nd:YVO4 laser | Spectra Physics | T40-Z-106C | |
| Acousto-optic deflector | IntraAction | DTD-274HA6 | |
| Microscope Objective | Olympus | PlanApo | 60X, NA 1.4 |
| Piezo stage | Mad City Labs | Nano-LP100 | XYZ stage |
| CCD camera | PixeLink | PL-A741 | |
| Photodetector | Electro-Optics Tech | ET-3020 | |
| Polystyrene Beads | Spherotech | SVP-08-10 | 800nm, streptavidin coated |
| Anti-digoxigenin | Roche | 11333089001 | From sheep |
| Primers | MWG operon | Custom oligos | One primer: biotin Other : digoxigenin |
| PCR reagents | New England Biolabs | TAQ polymerase, dNTPs | |
| Coverglass | Fisher Scientific | ||
| Other chemicals for buffer | Fisher Scientific | ||
Supplementary Materials |
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A. Hydrodynamic Friction C–fficient |
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For determining the hydrodynamic friction c–fficient of the microsphere near a surface one can use the following expansion5,10: |
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where the following shorthand has been introduced: |
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The friction c–fficient is defined in terms of the fluid viscosity η and the radius of the microsphere, with the microsphere's center located a distance η above the surface. The summation converges reasonably well when expanded to about ten terms. |
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B. Influence of Axial Position on Stiffness Calibration |
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The calibration of the trap stiffness involves a tradeoff between the accuracy of the calibration, which increases with increasing distance from the surface, and the actual axial position where the trap is used experimentally. In general, the trap is calibrated at around 800-1000 nm from the surface, which is higher than the actual experimental condition. |
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C. Modified Worm-Like Chain (WLC) Model |
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The force extension curves can be fit to a modified WLC model that accounts for volume exclusion effects at zero optical force as follows: |
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where Fopt is the optical force, xo is a fit parameter for the zero force extension,xopt is the extension under force, l is the contour length of the DNA, and l*p is a second fit parameter for an "effective" persistence length. Fwlc is given by the usual WLC model11 |
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where ε is the relative DNA extension.References
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- Tethered Particle Microscopy (TPM) Protocol. Meiners Lab. , University of Michigan. (2011).
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- Chen, Y. F., Milstein, J. N., Meiners, J. C. Protein-mediated DNA loop formation and breakdown in a fluctuating environment. Phys. Rev. Lett. 104, 258103-258103 (2010).
- Chen, Y. F., Milstein, J. N., Meiners, J. C. Femtonewton entropic forces can control the formation of protein-mediated DNA loops. Phys. Rev. Lett. 104, 048301-048301 (2010).
- Raghunathan, K., Milstein, J. N., Juliar, B., Blaty, J., Meiners, J. C. Sequence Dependent Effects on the Elasticity of Short DNA Molecules. , Forthcoming (2011).
