Preparation of Template for Riboprobe Synthesis

1. The DNA fragment should have been previously cloned into a plasmid vector, such that prokaryotic RNA polymerase promoters flank it. Suitable vectors include the pGem series (Promega, Madison, WI) and the pKS or pBluescript series (Stratagene, La Jolla, CA). In our experience, T7 and T3 RNA polymerases give the best overall performance on a wide range of templates, whereas SP6 polymerase has problems transcribing certain templates.

2. Linearize plasmid (usually 10-20 pg) with a suitable restriction endonuclease using manufacturer's buffer and conditions (see Note 3).

3. Check that digestion is complete by analyzing 0.25 pg on a 1% (w/v) agarose gel in 1X TBE containing ethidium bromide (0.1 pg/mL) alongside some uncut plas-mid. If the digestion is incomplete, add more enzyme, incubate for a further period, and reanalyze by gel electrophoresis.

4. Extract the remainder of the reaction mixture once with phenol, once with phenol:chloroform (1:1), and twice with chloroform.

5. Precipitate the plasmid DNA by the addition of 0.1 vol of 3 M sodium acetate (pH 7.0) and 2.2 vol of ethanol followed by incubation on ice for 15 min.

6. Collect the precipitate by centrifugation at 10,000g in a microfuge for 10 min, aspirate off the supernatant, and wash the pellet with 70% (v/v) ethanol.

7. After allowing the last traces of ethanol to evaporate, the pellet is resuspended in DEPC-treated water to give a final concentration of 0.5 pg/pL and stored at -20°C.

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