In Vitro Transcription of Phage DNA

1. Digest 10 |g of phage DNA in a final volume of 100 |L with NotI or XhoI, depending on whether sense or antisense transcripts are desired.

2. Phenol-extract, ethanol precipitate, rinse, and dry the digested DNA. Resuspend at 1 mg/mL.

3. Prepare sense (T3 polymerase and XhoI digest) or antisense (T7 polymerase and NotI digest) RNA using the Ambion Megascript kit following the instructions with the kit. This kit reproducibly gives yields of 70-100 molecules of RNA/molecule of template. We typically use 2 |g of template and scale the reaction up 2X.

4. If desired, one can incorporate biotin-21-UTP during the transcription reaction for the production of driver RNA. Add biotin-21-UTP (Clontech) at 1/20 the final concentration of UTP in the transcription reaction.

5. Recover the RNA after transcription by adding an equal volume of 5 M LiCl and incubating at -20°C overnight.

6. Spin the precipitate for 20 min at 4°C, rinse well, and dry. Resuspend in DEPC-treated H2O and quantitate.

7. Add 0.5 volume 7.5 MNH4-acetate and 2.5 vol of ethanol, mix well, and store at -20°C. Calculate the new concentration of RNA, and determine the volume/pg of RNA.

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