Pretreatment and Denaturation of Chromosomes

I will describe two protocols used to denature polytene chromosomes prior to hybridization. The first uses alkali treatment and the second uses heat to denature the chromosomes. Although the latter is a quicker method, some probes can give a rather dispersed signal compared with chromosomes prepared using alkali denaturation.

3.2.1. Alkali Denaturation

1. Incubate the slides in 2X SSC at 65°C for 30 min. This step is intended to help preserve the morphology of the chromosomes.

2. Transfer the slides to 2X SSC at room temperature for 10 min, then denature the chromosomes by incubating the slides in 70 mM NaOH for 2 min. The 70 mM NaOH must be freshly prepared, using NaOH pellets rather than a concentrated stock solution.

3. Rinse the slides in 2X SSC.

4. Dehydrate through ethanol as described in Subheading 3.1., step 5 and air-dry. The slides should be used the same day.

3.2.2. Heat Denaturation

1. Place the slides directly in gently boiling 5 mM Tris-HCl, pH 7.5 for 2 min.

2. Place the slides in cold 70% ethanol for 5 min. Transfer the preparation through two 5-min changes of 96% ethanol and air-dry. Denatured slides should be used the same day.

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