Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Analysis of rAAV Sample Morphology Using Negative Staining and High-Resolution Electron Microscopy

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Negative staining is a simple and rapid method for studying the morphology and ultrastructure of small particulate specimens (e.g., viruses, bacteria, cell fragments, and isolated macromolecules such as proteins and nucleic acids). Samples stained in this manner show great structural integrity because the stains used not only delineate the ultrastructure but also act as a fixative, thus protecting samples from irradiation damage by the electron beam. Negative staining also reduces the surface-tension forces at the airliquid interface, thus reducing shrinkage and specimen collapse. These effects are promoted by the heavy metal salts used in negative stains. Although negative staining is one of the oldest techniques for studying the ultrastructure of particulate samples at the level of electron microscopy, it has never been surpassed by any other technique for determining the surface structures, size, and shapes of specimens such as viruses, bacteria, and macromolecules, with resolution down to the 2-nm level (Hayat 2000). Because it is a simple and rapid method, negative staining combined with high-resolution transmission electron microscopy is a very effective method for determining the morphology and relative purity of rAAVs. Images taken at 40,000 to 50,000 diameters in magnification can be quickly scanned to differentiate empty versus fully packaged virions. For a research-grade laboratory preparation of rAAVs, a full-to-empty ratio of at least 20 may be acceptable; however, the higher the ratio of full to empty, the better the result for in vivo applications of rAAVs.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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