Materials

1. Clean microscope slides (see Note 1).

2. Clean siliconized cover slips, 24-mm square (see Note 1).

3. Clean siliconized cover slips, 22 mm x 50 mm (see Note 1).

4. Compressed air can (e.g., Dust-Off®; Falcon Safety Products, Inc. Sommerville, NJ).

7. 1 : 2 : 3 Fixative: 1 vol Lactic acid, 2 vol distilled water, 3 vol glacial acetic acid.

8. Liquid nitrogen.

9. 2X SSC: 300 mM NaCl, 30 mM sodium citrate, pH 7.0.

10. 70 mM Sodium hydroxide, freshly prepared from pellets.

12. Coplin jars, or similar, for incubating slides.

13. Oligolabeling buffer: Prepare as described in refs. 7 and 8 using the following solutions:

a. Solution A: Add 9 |L of P-mercaptoethanol, 12.5 |L of 20 mM dATP, 12.5 |L of 20 mM dCTP, 12.5 |L of 20 mM dGTP to 0.47 mL of solution O.

c. Solution C: Random hexanucleotides (Pharmacia) at a concentration of 90 A260 units/mL.

d. Solution O: 1.25 M Tris-HCl, pH 8.0, 125 mM MgCl2.

Prepare oligolabeling buffer by mixing solutions A, B, and C in the proportions 2 : 5 : 3. Oligolabeling buffer and its constituents should be stored at -20°C.

15. 1 mM Biotin-16-dUTP (Roche). Store at -20°C (see Note 2).

16. 50X Denhardt's solution: 5 g Ficoll, 5 g polyvinyl pyrrolidone, 5 g bovine serum albumin (BSA), water to 500 mL. Filter and store at -20°C.

17. 2X Hybridization solution: 8X SSC, 2X Denhardt's solution, 20% dextran sulfate, 0.8% sonicated salmon sperm DNA. Store at -20°C.

18. Plastic box with tightly fitting lid, lined with moist tissue paper.

19. Detek-1 streptavidin-horseradish peroxidase detection kit (Enzo), or ExtrAvidin-horseradish peroxidase conjugate (Sigma-Aldrich). The Enzo dilution buffer is phosphate-buffered saline (PBS) supplemented with 1% BSA, 5 mM EDTA (see Note 2).

20. PBS: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per 1 L.

21. PBS-TX: PBS containing 0.1% Triton X-100.

22. DAB solution: 0.5 mg/mL Diaminobenzidine in PBS, supplemented with 0.01% H2O2. DAB is a potent mutagen. Care should be taken at all times when working with solutions containing DAB. Gloves should be worn and DAB should be dis pensed in the fume hood. DAB should be inactivated in 50% bleach before disposal (see Note 2).

23. 0.89% Giemsa's staining solution in methanol/glycerol (Gurrs/BDH). Use as a 1 : 20 dilution in 10 mM sodium phosphate buffer, pH 6.8.

24. DPX mountant (Fluka).

25. Diamond pencil.

26. Nail polish.

27. Polytene chromosome maps (see Note 3). 3. Methods

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