Reversible, meniscus-free molecular combing of long-chain DNA.

We introduce a method for reversibly orienting long-chain DNA on solid hydrophilic substrates without a fluid meniscus. End-tethered lambda-DNA mushrooms are elongated by a hydrodynamic flow in the presence of trivalent cations, resulting in electrostatic adsorption of the extended DNA to the surface. By complexation of the cations the part of the DNA which is unspecifically bound to the surface desorbs quantitatively, and the mushroom conformation is restored. With the use of multiple deposition-combing steps, combined with a final desorption step, tethering densities higher than attainable with single deposition steps can be obtained.

Sodium chloride in separation medium enhances cell compatibility of free flow electrophoresis.

Free flow electrophoresis of cell suspensions in buffers containing sodium chloride was investigated using a modified procedure and the new apparatus Octopus PZE. The major methodical innovations are upward fluid flow, margin buffers flowing through the electrophoresis chamber at both sides of a central cell suspension buffer, adjacent to the electrode membranes, and a sample injection device which focuses the cells hydrodynamically to the middle of the chamber thickness. Mononuclear leukocytes, suspended in a buffer containing 35 mM NaCl, could be fractionated with the same accuracy as by conventional free flow electrophoresis, operated with a single NaCl-free chamber buffer. However, testing the vitality of separated cells with the help of the calcium indicator FURA2-AM clearly demonstrated the biological importance of the presence of a minimum amount of sodium chloride during cell electrophoresis. Only if at least 35 mM NaCl were present could an undisturbed cytosolic Ca2+ metabolism be maintained for the time of a free flow electrophoresis cell separation experiment.