1. How much RNA is needed? A maximum of about 60-fold molar excess of driver RNA is required for a proper subtraction. If necessary, this can be reduced by limiting the short hybridizations to about 4X molar excess and the long hybridizations to 10fold excess. If one takes the trouble to calculate the amount of cDNA remaining after each subtractive hybridization, the total amount may be significantly reduced.
2. Mix poly (A)+ RNA or in vitro synthesized RNA (10-30 pg/reaction) with 50 pg of photoactivatable biotin acetate (Clontech). With the tube tops open and the lamp 6 in. from the sample, irradiate on ice for 15 min. Be sure to support the tube in a water bath rack, since the sunlamp rapidly melts the ice.
3. Add 1/10 vol 1 M Tris-HCl, pH 9.0, and extract repeatedly with TE-saturated 2-butanol to remove unreacted photobiotin (until the butanol phase is clear).
4. CHCl3 extract the RNA, ethanol-precipitate, rinse, and dry. Resuspend in DEPC-treated H2O and repeat the photobiotinylation. At this stage, the pellet should be reddish if the biotinylation has been successful.
5. Pool identical RNAs, and store as ethanol precipitates at -20°C.
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