By William S. M. Wold, Ann E. Tollefson
Adenovirus equipment and Protocols, moment version, now in volumes, is a vital source for adenovirus (Ad) researchers starting within the box, and an inspirational start line for researchers seeking to department into new parts of advert research. as well as updating and increasing the 1st variation, the authors have additional new chapters that tackle leading edge components of emphasis in advert examine, together with advert vector building and use, real-time PCR, use of recent animal types, and techniques for quantification of advert virus or virus expression/interactions. all of the protocols offered in those volumes is written by means of trendsetting researchers.
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Centrifuge the suspension at 35,000 rpm (g) for 16 h in a Sorvall TV865 rotor (or equivalent). Two closely spaced virus bands should be visible in the center of the tube. 11. Fractionate the gradient into single-drop fractions through a hole made in the bottom of the tube. 12. Measure the radioactivity in each fraction by Cherenkov counting. Two more or less well-separated peaks should appear (Fig. 1). 24 Ketner and Boyer Fig 1. Defective Adenovirus Deletion Mutants 25 13. Pool the fractions that comprise the lighter peak (see Fig.
5. Store at –20°C. 23. 1 μg/μL Sheared salmon sperm DNA (29). Store at –20°C. 24. Mouse 3T6 cells (American Type Culture Collection). 25. 100X Glutamine: 29 g L-glutamine in 1 L of water. Filter-sterilize and aliquot. Store at –20°C long term or at 4°C while in use. 26. 100X Pen/strep: 10,000 U (Gibco 15140-122). Store at –20°C long term or at 4°C while in use. 27. 1X Dulbecco’s modified Eagle’s medium (DMEM) (Gibco 12100-046): make according to manufacturer’s directions. 2 with HCl. 2-μm filter and place one bottle at 37°C overnight to check for contamination.
Store at room temperature. 15. Optical grade CsCl (Pharmacia 17-0845-02). 16. 5-mL Microfuge tubes. 17. 0. Store at room temperature. 18. EcoRI (20 U/μL). 19. 1 M MgCl2, 10 mM dithiothreitol (DTT). Add DTT before using. Store at room temperature. 20. 100 mM DTT. Store at –20°C. 21. Klenow polymerase (5 U/μL). Store at –20°C. 22. 5. Store at –20°C. 23. 1 μg/μL Sheared salmon sperm DNA (29). Store at –20°C. 24. Mouse 3T6 cells (American Type Culture Collection). 25. 100X Glutamine: 29 g L-glutamine in 1 L of water.
Adenovirus Methods and Protocols: Adenoviruses, Ad Vectors, Quantitation, and Animal Models by William S. M. Wold, Ann E. Tollefson