Transcription of RNA from PCR Product

1. Assemble the following transcription reaction:

4 [A 5 X commercial T7 or SP6 RNA polymerase buffer 8 ¡A RTP mix (10 mM each CTP, ATP, GTP, and UTP)

2 ¿il mM dithiothreitol (DTT) 1 fA T7 RNA polymerase

(Note: The remainder of the PCR product may be stored frozen and used as template to produce control RNA transcripts for comparison with selected RNA transcripts, for selection by another protein, or for further amplification as needed.)

2. Bring to 20 pi total volume with DEPC-H20 in 0.5-ml tubes.

4. Add 1.5 pi RQ 1 DNase (Promega) at 1 U/p.1. Incubate at 37°C for 10 min.

5. Precipitate, wash, and dry the pellet as in described in Section II,A.

6. Resuspend RNA in 20 ¡A DEPC-treated H20.

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