RNA Probe Synthesis

1. Digest linearized plasmid equivalent to 1 |g of insert DNA (see Note 11) with proteinase K (see Note 12) (0.05 |g/|L) for 30 min at 37°C

2. Phenol/chloroform-extract and ethanol-precipitate.

3. Redissolve the DNA in TE so that 1 |g of insert DNA is in 4 |L.

4. Mix together 4 |L of linearized plasmid (1 |g of insert DNA), 2 |L of 10X transcription buffer, 2 |L of 10X nucleotide mix (with digoxigenin-UTP or fluores-cein-UTP), and 20 U of RNase inhibitor. Add water to give a final reaction volume of 20 |L and 40 U of the appropriate T7, T3, or SP6 RNA polymerase.

5. Incubate the mixture for 2 h at 37°C.

6. Add 40 U of DNase I, and incubate at 37°C for 15 min to remove the plasmid DNA.

7. Stop the reaction by adding 2 |L of 200 mM EDTA, pH 8.0.

8. Precipitate the RNA with 2.5 |L 4 MLiCl, and 75 |L prechilled ethanol (see Note 13).

9. Spin down the pellet, and redissolve in 100 |L of RNase-free water containing 40 U of RNase inhibitor.

10. Check the probe by running 2.5 |L on an 0.8% (w/v) agarose, 1X TBE minigel. Wash the apparatus thoroughly before preparing the gel, and run the samples quickly to avoid problems with RNase (see Note 14).

11. The probe can be used without further treatment (see Note 15). It should be split into aliquots and stored at -20°C.

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