Synthesis of DIG or FITCLabeled Riboprobes

1. Warm transcription buffer, nucleotides, DTT, and DNA templates to room temperature.

2. Prepare the following reaction mixture in this order: 11 ||L DEPC-treated water, 2 |L 10X DIG, or FITC ribonucleotide mix, 2 |L 10X transcription buffer, and 2 |L 200 mM DTT. Mix by gently pipeting up and down. Next add 1 |L RNase inhibitor and 1 |L appropriate RNA polymerase, and mix in the same manner (see Note 9).

3. Add 2 |L (1 |g) of the linear DNA template, and mix gently by pipeting.

5. Check that the transcription reaction has been successful (Fig. 1G, lane 1). Analyze 1 |L of product on a 1% (w/v) agarose gel in 1X TBE containing ethidium bromide (0.1 |g/mL). If approx 10 |g of probe have been synthesized, then the DNA band and RNA band will have about the same intensity of fluorescence under UV transillumination (ethidium bromide intercalates poorly into RNA).

6. Meanwhile proceed with the purification of the riboprobe as follows. Add 2 |L RNase-free DNase-1, and incubate at 37°C for exactly 15 min.

7. Add 100 |L TE, 10 |L 4 M LiCl, 300 |L ethanol, and vortex.

8. Incubate at room temperature for 30 min, and spin in a microfuge at 10,000g for 15 min.

9. Remove the supernatant, immediately resuspend pellet in 100 |L TE and add 10 |L 4 M LiCl and 300 |L ethanol. Again, incubate at room temperature for 30 min, and spin in a microfuge at 10,000g for 15 min (see Note 10).

10. Carefully wash the pellet with 70% ethanol (made with DEPC-treated water).

11. Remove the supernatant and immediately dissolve the pellet in TE to give about 1 |g/10 |L as estimated from the gel (Subheading 3.4., step 5; usually 100 |L), add 2 |L RNasin, and store probe in 10 |L aliquots at -70°C.

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