A standard in vitro transcription reaction (5) incorporating digoxygenin-11-UTP is performed. SP6, T7, or T3 RNA polymerase may be used. Temperature sensitive reagents should be kept on ice, but the reaction should be mixed at room temperature to avoid precipitation of the DNA template. Reaction: 5X Transcription buffer 10.0 |L
Linearized DNA template (1 |g/|L) 2.5 |L 1 M DTT 0.5 |L
1. Incubate reaction at 37°C for 1-2 h.
2. Check synthesis by running 1 |L of the reaction on an agarose gel, and estimate the amount of RNA by comparing the intensities of the RNA and DNA template.
3. Add 2 |L of RNase-free DNase I, and incubate at 37°C for 10 min.
4. Purify the probe from unincorporated nucleotides by passing the reaction through a G50 spin column. These columns can be homemade. Alternatively, disposable cartridges can be purchased from IBI (R50 Spun Columns, cat. no. 06080).
5. Precipitate the flowthrough from the spin column.
6. Redissolve the purified pellet in H2O. Probes can be reduced in size by limited alkaline hydrolysis if necessary (see Note 1). Store probe stock at -20°C. Probes are stable indefinitely.
7. Most probes work well when used at approx 1 |g/mL in hybridization buffer.
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