RNA Transcription

Stable transcripts and high yields of protein are produced from cDNAs cloned into the vector pSP64T (15) or pCS2+ (16,17). The original version of pSP64T is slightly inconvenient, because although it has several sites for linearization, the only cloning site is BglII. Several laboratories have, however, produced more user-friendly versions of pSP64T, which include a multiple cloning site, and two such vectors are available from our colleague Masazumi Tada. Plasmids should be linearized before transcription with a restriction enzyme that generates a blunt end or a 5'-overhang.

1. Linearized plasmid at 1 mg/mL.

2. 10X Transcription buffer: 400 mM Tris (pH 7.5), 60 mM MgCl2, 20 mM spermidine HCl, 50 mM NaCl.

9. RNasin.

10. SP6 RNA polymerase.

11. RNase-free water.

13. Phenol/chloroform.

14. Ethanol.

15. 1% Agarose TBE gel (RNase-free). 3. Methods

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