Hybridization

All solutions made should be sterile and put into sterile containers, either autoclaved glass or sterile disposable plastic. Try to keep things as sterile as possible while working. Fingerprints contain RNase. Gloves should be worn while working to reduce the chance of fingerprint contamination of any item used. Slides can be handled with forceps. Make sure the forceps are clean. If in doubt, wash in detergent, soak in IN NaOH, rinse in water, dip in 95% (v/v) EtOH, and let dry.

1. 1N NaOH (can be used to inactivate RNase that is present on any surface that needs to be used).

2. Aluminum foil: Fresh off the roll, aluminum foil provides a clean RNase-free surface on which your slides can be placed.

3. Square polypropelene slide mailers (A. H. Thomas, Swedesboro, NJ): Treat with deionized water containing DEPC (0.1% [v/v]) for several hours or overnight and then autoclave.

4. Phosphate-buffered saline (PBS: 10 mM phosphate buffer: To 0.2M stock diluted 20X with DEPC treated water, add 0.9% [w/v] NaCl).

5. 0.25% Acetic anhydride (v/v) in triethanolamine buffer: Make stock solutions of 0.1 M triethanolamine adjusted to pH 8 with HC1, DEPC treat with 0.1% (v/v) for several hours, and autoclave. Immediately before use, add 0.25 mL acetic anhydride/100 mL.

6. Absolute ethanol.

7 95% (v/v) and 70% (v/v) Ethanol: Dilute absolute ethanol with DEPC water.

8. Chloroform.

9. Parafilm.

10. Moisture tight plastic container with a flat bottom: Make perspex or Plexiglas™ (5-8 mm thick) racks to fit the interior. Use small squares of plastic glued to the bottom surface to space the racks apart when stacked one above the other. With the slides placed flat on these racks, this system allows you to place a large number of slides in the same box. The interior is kept moist by placing a wet filter paper in the bottom under the first plastic rack.

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