Photobiotinylation of Purified Driver cDNA

Photobiotin: l mg/mL in water, aliquot and store at -20°C in lightproof tubes. UV source: 300 W ultra vitalux bulb (Osram, Sylvania, Danvers, MA). (Do not allow exposure of skin to UV.)

4. 7.5 M Ammonium acetate.

6. 1 M EPPS, pH 8.5 and 8.25, autoclave and filter-sterilize.

8. Extraction buffer: 50 mM EPPS, pH 8.5, 0.5 M NaCl, 2 mM EDTA.

9. Streptavidin: 4 |g/|L in 50 mM EPPS, pH 8.25, store at -20°C in 5-|L aliquots to avoid freeze-thaw problems.

10. TE-saturated phenol: chloroform (1:1). (Prepare fresh immediately prior to use do not store). Add 700 |L phenol/chloroform (Gibco-BRL, Gaithersburg, MD) to 700 |L TE, pH 8.0, vortex mixture, centrifuge, decant aqueous phase, and repeat 3x.

11. TE-saturated 2-butanol: 25 mL of 2-butanol are mixed with 25 mL TE, pH 8.0. The mixture is centrifuged briefly and the lower aqueous phase is removed. This is repeated. The 2-butanol is in the upper phase.

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