We have compared the efficiency of incorporating biotin in to the driver by the use of biotinylated oligonucleotides and nucleotides both separately and in conjunction with each other to that of the efficiency of photobiotinylation, and have repeatedly found that photobiotinylation is the most efficient and reliable method of biotin incorporation. However, despite the reliability of photobiotinylation, it is recommended that the efficiency of biotin incorporation into the driver is tested functionally as described prior to its use in the subtractive hybridization reaction.
1. Boil 40 |L (50-100 |g) driver cDNA for 2 min, then snap-cool on ice prior to adding 40 ||L of photobiotin (1 mg/mL), and mix well. Place tube with the lid open 10 cm from the UV source, and irradiate for 5 min. Leaving the UV source on (to preserve the bulb), take out the sample, and mix by flicking the tube and replace under the UV for an additional 5 min.
2. Remove sample, add a further 40 | L of photobiotin, mix well, and replace under UV for a further 5 min. Then add an equal volume of 200 mM Tris-Cl, pH 9.0, to stop the reaction.
3. Free photobiotin may be removed by precipitating the DNA by adding 80 |L 7.5 M ammonium acetate and 560 ||L 100% ethanol. Chill on ice 15 min, and spin full speed in a refrigerated centrifuge for 15 min. Discard supernatant, and wash pellet 1X with 80% ethanol, dry orange/brown pellet, and resuspend the biotinylated DNA pellet in 50 ||L HE, and re-estimate concentration (see Note 4).
4. Efficient biotinylation may then be assessed by adding 50 | L of fresh extraction buffer and 2 ||L of 4 |g/|L streptavidin to 2 |g biotinylated driver and incubating for 2 min at room temperature. At this stage, remove 10 |L for gel analysis, prior to adding 50 ||L TE-saturated phenol/chloroform. Vortex the sample for 30 s and then spin at full speed for 3 min. Remove the top three quarters of the aqueous phase (~25-30 |L) avoiding the interphase, and transfer to a fresh tube. Compare 10 |L of this extracted aqueous phase to the 10 |L removed before subtraction by agarose gel electrophoresis.
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