Hybridization and Washing

1. To 1 mL of hybridization buffer, add 10 pL of 1M DTT, mix, and add 10 pL (-35 ng) of labeled probe and vortex. Ten microliters of this mix should count =100,000 cpm or above. If it is lower, the amount of DNA can be increased up to three times. Beyond this, there is too much background. Compare your probe labeling with the standard provided in the NEN kit.

2. To hybridize with a minimum of hybridization buffer, the slides need to be placed flat in a moist chamber. A Petri dish with moistened filter paper in the bottom will work for a few slides. Plastic refrigerator storage boxes work very well for larger numbers. Put moistened filter paper in the bottom, and place the slides flat. Pipet 50 pL onto each slide (this volume should be adjusted to the cover sections easily). A parafilm "cover slip" cut to fit over the sections can be used to spread the buffer over the sections and hold it there without excessive evaporation.There should be no air bubbles under the cover slip. Hybridize overnight at 37°C (see Note 1).

3. Wash sections in IX SSC. A typical schedule would be to wash at 55°C, with four changes of 20 min each, followed by two room temperature washes of 1 h each. The wash time and temperature will have to be adjusted for each probe. With an unknown probe, try several wash schedules aiming for a clear signal readily detected above background. The stringency of the wash can be varied low to high by increasing the temperature, lengthening the time, or reducing the salt concentration. Finally, dip the slides in distilled water, and let them dry.

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