Cosmid, P1, BAC, or YAC DNA prepared for molecular combing (see recipe) 1 x 10-6 M YOYO (Molecular Probes)

22 x 22-mm glass coverslips

AES-coated microscope slides for molecular combing (see Support Protocol 2)

1. Dilute DNA with water to ~10 pg/^l (equivalent to several thousand molecules per Ml).

2. Stain the DNA with an equal volume of 10-6 M YOYO.

3. Place 2 Ml of this DNA on a 22 x 22-mm coverslip.

The coverslip should not be AES-treated.

4. Place an AES-coated microscope slide on top of the coverslip.

The combing of the DNA and the concentration of the combed DNA can now be evaluated using a standard fluorescence microscope equipped with a filter set for FITC and a standard HBO 100 light source. The YOYO-stained DNA molecules are visible as green fibers.

Drying at 4°C is much slower than at 20°C, but yields a better quality of combing.

6. Proceed to the in situ hybridization procedure (unit 8.3).

If the DNA concentration is high enough, dilute 2 ul of YOYO-stained DNA with 8 ul AF solution (see recipe). This minimizes breakage of the DNA before further dilution with water.

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