PNA Reaction

1. Prepare the PNA reaction mixture: aliquots of 5 pL of each PNA probe (chromosome 1- and chromosome 18-specific probes) are mixed into a microcentrifuge tube.

2. Denature the PNA probe mixture at 73°C for 6 min.

3. After draining the excess 2X SSC off the slide, and before the slide is completely dry, apply the PNA reaction mixture on the slide, and cover with a 22 x 32 cover slip.

No additional denaturation of the slide is required after the PRINS reaction because the chromosomal DNA remains denatured through the PRINS incubation.

4. Seal the slide with rubber cement (see Note 5).

5. Put the slide in a humidified hybridization chamber 60 min at 37°C.

6. At the end of the hybridization, carefully remove the cover slip from the slide using a scalpel blade.

7. Transfer the slide in a Coplin jar containing 1X PBS, 0.1% Tween-20, and wash the slide for 2 min at room temperature with gentle agitation.

8. Transfer the slide to 58°C prewarmed 1X PBS, 0.1% Tween-20 for 10 min with gentle agitation

9. Rinse the slide in 2X SSC, 0.1% Tween-20 for 1 min.

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