Download PDF by Abelson J.N., Simon M.I., Phillips I.M.: Antisense Technology: Applications Part B

By Abelson J.N., Simon M.I., Phillips I.M.

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18. Save the supernatant; transfer equal amounts (each 10 ml) into two 30-ml Corex (or plastic) tubes. Add 6 ml of 2-propanol to each tube and mix. 19. Incubate at room temperature for 10 min. 20. Spin at 10,000g at room temperature for 10 min. 21. 4 (10 mM Tris-HC1 with 1 mM EDTA). 22. 2 g of CsCI to each tube, mix, and dissolve. 24 ml of ethidium bromide (10 mg/ml) and mix. 23. 9-ml Optiseal centrifuge tube (Beckman, Fullerton, CA). 24. Spin in a NVT90 rotor (Beckman) at 78,000 rpm, 15° for 4 hr.

Tu, H. Yang, and M. K. Raizada, Am. J. Physiol. 274(2 Pt. 2), H719 (1998). 38j. M. Bergelson, J. A. Cunningham, G. Drognett, E. A. Kurt-Jones, A. Krithivas, J. S. Hong, M. S. Horwitz, R. L. Crowell, and R. W. Finberg, Science 275(5304), 1320 (1997). [3] ANTISENSE D N A DELIVERY FOR PROLONGED EFFECTS 39 episomal, that is, they do not integrate into the host DNA. They provide high levels of expression but the episomal DNA will invariably become inactive. , mice, this may take a long time compared with their life span, but in humans it limits the use of the virus as a vector.

5 ml. Tissue samples are incubated with [35S]TBPS for 3 hr at 25°. Nonspecific binding is estimated using picrotoxin (5 × 10-5 M). For specific binding of [3H]muscimol (10 nM), preferably frozen tissue pellets are resuspended in Tris-citrate at 2 mg/ml. Tissue samples are incubated with [3H]muscimol for 30 min at 0°. Nonspecific binding of [3H]muscimol is estimated using [3H]GABA (10 -4 M). 5 nM). Tissue samples (2 mg/ml) are incubated with [3H]QNB for 60 min at room temperature; nonspecific binding is measured by means of atropine (5 × 10 -6 M).

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Antisense Technology: Applications Part B by Abelson J.N., Simon M.I., Phillips I.M.


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