A preparative method for isolating high-molecular-weight DNA from animal cells is described.This method is based on the use of proteinase K, a powerful proteolytic enzyme with a broad action spectrum, which is very active in the presence of sodium dodecylsulfate and ethylenediamine tetraacetate. The DNA preparation is free of RNA, protein and degrading enzymes.The number-average molecular weight of the native DNA is I90 x lo6, whereas it is 9(& x lo6 for single-stranded DNA, indicating that the DNA molecules do not contain single-stranded nicks.The native DNA molecules range in molecular weight from 40 x lo6 to more than 500 > lo6. Common preparative methods [9] yield mammalian DNA molecules with molecular weights of 5 -20 x lo6, whereasDNA molecules of higher molecular weight (up to 60 x 106) were obtained using a more gentle enzymatic procedure [lo]. Both DNA preparations contain single-stranded nicks caused by the action of nucleases during the time between the death of the organism and the isolation of the DNA. It is obvious that it is impossible to prepare DNA molecules with molecular weights of 1 x loll, due to the well-known degrading effects of mechanical shear during the isolation procedure, but it should be possible to isolate fragments of molecules with molecular weights of several hundred millions containing very few single-stranded nicks, provided the action of degrading enzymes could be totally prevented.

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Poly(glycerol monomethacrylate-block-2-hydroxypropyl methacrylate) [PGMA−PHPMA] diblock copolymer vesicles are prepared by RAFT aqueous dispersion polymerization at 70 °C and then used as precursors for chain extension experiments with a third comonomer. For self-blocking experiments conducted with water-soluble HPMA, good living character is obtained, as judged by the relatively low final polydispersity (M w /M n < 1.30). This suggests that the RAFT chain-end fidelity is high, at least over time scales of a few hours. Using water-immiscible monomers such as methyl methacrylate (MMA) or benzyl methacrylate (BzMA) also leads to efficient chain extension under "seeded" emulsion polymerization conditions. High conversions are obtained within a few hours at 70 °C, but polydispersities are somewhat higher for the resulting ABC triblock copolymer chains. TEM studies indicate that introduction of a second hydrophobic block within the vesicle membrane produces remarkable changes in morphology. A distinctive framboidal morphology is obtained in the case of BzMA, which is attributed to microphase separation between the PHPMA and PBzMA hydrophobic blocks. Moreover, there is a monotonic increase in the size of the observed globular features as higher degrees of polymerization for the PBzMA block are targeted. Similar but less distinctive morphological changes are also observed when using MMA as the third comonomer, which suggests that somewhat weaker microphase separation occurs in this case. If a bifunctional monomer such as ethylene glycol dimethacrylate (EGDMA) is used as the third comonomer, highly crosslinked vesicles are obtained that can withstand a surfactant challenge, unlike the precursor linear diblock copolymer vesicles. In this case it is noteworthy that much better results are obtained if the EGDMA is added last, rather than during the HPMA polymerization. The robust, reproducible nature of these aqueous formulations is expected to lead to new opportunities in the growing field of block copolymer vesicles, since they allow access to new particle morphologies in multigram quantities at relatively high solids.

Critical examination finds that the longest chains play a catalytic role in the formation of shish kebabs recruiting other chains into the formation of this morphology. The longest chains in an ensemble are stretched by shear flow to form the "shish" upon which the bulk of the material crystallizes as "kebabs". A universal parameter for the formation of shish kebab structures, the specific mechanical work, and a method by which it may be measured for any given ensemble of polymers is provided. In rotating parallel-plate flow a clear boundary is observed between oriented and unoriented material which is dependent on both the shear rate and the total strain. It has been found that the necessary conditions for the formation of oriented nuclei is that the shear rate should be larger than the inverse Rouse time of the longest chain in the ensemble and that mechanical work above a critical threshold is required. The experimental procedure required to make such measurements, and the precautions necessary to avoid artifacts such as elongated spherulites, elastic instabilities and photoelasticity, are examined in detail. The concept of the critical work being a control parameter has been previously demonstrated using model linear-linear hydrogenated polybutadiene blends and this concept is extended to industrial polymers, low-density polyethylene and polypropylene, using both small-angle X-ray scattering and polarized light imaging to measure orientation. The approach proposed is simple, is elegant, and can be easily implemented in the laboratory to study the fundamental processes of flowinduced crystallization and to test commercial materials before processing in real applications.

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Hydroxy-functionalized polymersomes (or block copolymer vesicles) were prepared via a facile one-pot RAFT aqueous dispersion polymerization protocol and evaluated as Pickering emulsifiers for the stabilization of emulsions of n-dodecane emulsion droplets in water. Linear polymersomes produced polydisperse oil droplets with diameters of ~50 μm regardless of the polymersome concentration in the aqueous phase. Introducing an oil-soluble polymeric diisocyanate cross-linker into the oil phase prior to homogenization led to block copolymer microcapsules, as expected. However, TEM inspection of these microcapsules after an alcohol challenge revealed no evidence for polymersomes, suggesting these delicate nanostructures do not survive the high-shear emulsification process. Thus the emulsion droplets are stabilized by individual diblock copolymer chains, rather than polymersomes. Cross-linked polymersomes (prepared by the addition of ethylene glycol dimethacrylate as a third comonomer) also formed stable n-dodecane-in-water Pickering emulsions, as judged by optical and fluorescence microscopy. However, in this case the droplet diameter varied from 50 to 250 μm depending on the aqueous polymersome concentration. Moreover, diisocyanate cross-linking at the oil/water interface led to the formation of well-defined colloidosomes, as judged by TEM studies. Thus polymersomes can indeed stabilize colloidosomes, provided that they are sufficiently cross-linked to survive emulsification.

Block copolymer vesicles are conveniently prepared directly in water at relatively high solids by polymerization-induced self-assembly using an aqueous dispersion polymerization formulation based on 2-hydroxypropyl methacrylate. However, dynamic light scattering studies clearly demonstrate that addition of small molecule surfactants to such linear copolymer vesicles disrupts the vesicular membrane. This causes rapid vesicle dissolution in the case of ionic surfactants, with nonionic surfactants proving somewhat less destructive. To address this problem, glycidyl methacrylate can be copolymerized with 2-hydroxypropyl methacrylate and the resulting epoxy-functional block copolymer vesicles are readily cross-linked in aqueous solution using cheap commercially available polymeric diamines. Such epoxy-amine chemistry confers exceptional surfactant tolerance on the cross-linked vesicles and also leads to a distinctive change in their morphology, as judged by transmission electron microscopy. Moreover, pendent unreacted amine groups confer cationic character on these cross-linked vesicles and offer further opportunities for functionalization.

The study of mouse models is crucial for the functional annotation of the human genome. The recent improvements in mouse genetics now moved the bottleneck in mouse functional genomics from the generation of mutant mice lines to the phenotypic analysis of these mice lines. Simple, validated, and reproducible phenotyping tests are a prerequisite to improving this phenotyping bottleneck. We analyzed here the impact of simple variations in animal handling and housing procedures, such as cage density, diet, gender, length of fasting, as well as site (retro-orbital vs. tail), timing, and anesthesia used during venipuncture, on biochemical, hematological, and metabolic/endocrine parameters in adult C57BL/6J mice. Our results, which show that minor changes in procedures can profoundly affect biological variables, underscore the importance of establishing uniform and validated animal procedures to improve reproducibility of mouse phenotypic data.