title: Magnetic trap construction.
abstract: In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to >100 picoNewtons (pN). In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. This protocol describes a procedure for building and using a magnetic trap. It gives a method for constructing a microchamber suitable for magnetic tweezers studies, including antibody coating and passivation. It also describes a series of simple steps to achieve end-labeling of DNA anchoring fragments. One anchoring fragment is biotin-labeled and the other is labeled with digoxigenin. The anchoring fragments are then digested and ligated to a central DNA region containing the sequence of interest. The biotinylated DNA is adsorbed onto streptavidin-coated magnetic beads, and the DNA-bead mixture attaches specifically to the antidigoxigenin-coated surface of the microchamber.
authors.forename: Timothée
authors.initials: T
authors.lastname: Lionnet
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Allemand
authors.forename: Andrey
authors.initials: A
authors.lastname: Revyakin
authors.forename: Terence R
authors.initials: TR
authors.lastname: Strick
authors.forename: Omar A
authors.initials: OA
authors.lastname: Saleh
authors.forename: David
authors.initials: D
authors.lastname: Bensimon
authors.forename: Vincent
authors.initials: V
authors.lastname: Croquette
mesh_terms: D001710:Biotin / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004076:Digoxigenin / Q000378:metabolism; D008280:Magnetics; D008863:Microspheres; D036103:Nanotechnology / Q000379:methods
chemical_list: D001710:Biotin; D004247:DNA; D004076:Digoxigenin