Pretreatment of Microscope Slides and Preparation of DNA Fibers on Glass

1. The derivatization of glass substrates is among the most critical steps of the procedure. The slides should have the capacity to bind DNA molecules at one or both ends but should allow the molecules to stretch during the subsequent drying.

2. Solid substrates for QDFM are prepared in batches of 20 to 50 by derivatization of glass microscope slides (see Note 7), cover slips, or sheets of mica with APS, resulting in primary amino groups on the glass surface (51,53; see Note 8).

3. Clean glass slides mechanically by repeated rubbing with wet cheesecloth to remove dust and glass particles.

4. Rinse several times with water, immerse slides in boiling water for 10 min, and air-dry.

5. Immerse slides in 18 M sulfuric acid for at least 30 min to remove organic residues, followed by immersion in boiling water for 2 min.

6. Immerse precleaned dry slides in a solution of 0.1% APS in 95% ethanol for 10 min.

7. Remove slides from the silane solution, rinse several times with water, and immerse in water for 2 min.

8. Dehydrate by immersing in absolute ethanol and dry slides upright for 10 min at 65°C on a hot plate.

9. Store slides for 2 to 6 wk at 4°C in a sealed box under nitrogen prior to use.

3.2.1. Molecular Combing

1. In a typical experiment, 1 to 2 pL of clonal DNA are mixed with an equal amount of YOYO-1 (1 or 0.1 pM) and 8 pL water. Then 1 or 2 pL of this diluted DNA is applied to an untreated cover slip, which is then placed DNA side down on the APS-derivatized slide.

2. The DNA concentration can be estimated in the fluorescence microscope using a filter set for FITC and adjusted as needed (see Notes 9 and 10). As early as after 2 min of incubation at room temperature, the untreated cover slip can be removed slowly from one end, allowing the receding meniscus to stretch the bound DNA molecules ("fibers") in one direction (53,59) (see Note 11).

3. Alternatively, the slide or cover slip sandwich can be allowed to dry overnight at room temperature, after which the untreated cover slip is removed by lifting it on one side with a razor blade. Slides carrying DNA fibers are rinsed briefly with water, drained, allowed to dry at room temperature, and "aged" in ambient air at 20°C for 1 wk before hybridization. Extra slides are stored at 4°C.

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