Equilibrium binding experiments using fluorescence and absorption techniques have been performed throughout a wide concentration range (1 nM to 30 microM) of the dye Hoechst 33258 and several DNAs. The most stable complexes found with calf thymus DNA, poly[d(A-T)], d(CCGGAATTCCGG), and d(CGCGAATTCGCG) all have dissociation constants in the range (1-3) X 10(-9) M-1. Such complexes on calf thymus DNA occur with a frequency of about 1 binding site per 100 base pairs, and evidence is presented indicating a spectrum of sequence-dependent affinities with dissociation constants extending into the micromolar range. In addition to these sequence-specific binding sites on the DNA, the continuous-variation method of Job reveals distinct stoichiometries of dye-poly[d(A-T)] complexes corresponding to 1, 2, 3, 4, and 6 dyes per 5 A-T base pairs and even up to 1 and 2 (and possibly more) dyes per backbone phosphate. Models are suggested to account for these stoichiometries. With poly[d(G-C)] the stoichiometries are 1-2 dyes per 5 G-C pairs in addition to 1 and 2 dyes per backbone phosphate. Thermodynamic parameters for formation of the tightest binding complex between Hoechst 33258 and poly[d(A-T)] or d-(CCGGAATTCCGG) are determined. Hoechst 33258 binding to calf thymus DNA, chicken erythrocyte DNA, and poly[d(A-T)] exhibits an ionic strength dependence similar to that expected for a singly-charged positive ion. This ionic strength dependence remains unchanged in the presence of 25% ethanol, which decreases the affinity by 2 orders of magnitude. In addition, due to its strong binding, Hoechst 33258 easily displaces several intercalators from DNA.
An instrument is described in which two solutions can be homogeneously mixed within several microseconds. The liquids flow separately through two coaxial capillaries with conical tips and then simultaneously around a sphere (50–100 μ in diameter) which has been positioned close to the end of the outer tip. The liquids flow with velocities of ∼100 m/s through the small passages (∼5 μ wide) separating the sphere and the wall of the outer capillary and mix in the turbulent liquid flow behind the sphere. The mixed liquids are then ejected as a narrow liquid jet for observation. Design characteristics and construction techniques are presented along with a discussion of the properties of the turbulent flow field and estimates of the expected practically realizable mixing times. The experimentally determined speed of mixing indicates that we have nearly achieved the proposed lower limits of the mixing time.
Laser Micro Sintering was introduced to the international community of freeform fabrication engineers in 2003 and has since been employed for a variety of applications. It owes its unique features to certain effects of q-switched pulses that formerly had been considered detrimental in selective laser sintering. Besides sub-micrometer sized powders also materials with grain sizes of 1-10 micrometers can be sintered. Surface and morphology of the product are influenced by grain size and process environment. First results have been achieved with processing ceramic materials. A comprehensive overview of the process and the features is given supported by experimental evidence. Routes of further development are indicated.
Miniaturization is one of the main imperatives in high‐tech development and therefore a persistent challenge for mechanical engineering. Recently a freeform technique – Laser Micro Sintering – has been developed by which micro parts with an overall resolution of 30 μm can be produced from powder materials. The technique is a generative freeform fabrication method based on selective laser sintering; it can produce hollows and undercuts and does not afford shape‐specific tools. Functional micro bodies can be generated from metals and ceramics.
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