The transcription protocol is as follows:
1 ug template DNA
4 ul 5X RIBOMAX buffer
2 ul NTPs*
1 ul RNAsin
1 ul Polymerase
1 ul pyrophosphatase
20 ul final volume
RIBOMAX Buffer (5X):
400 mM HEPES-KOH, pH7.5
60 mM MgCl2
10 mM Spermidine
200 mM DTT
*NTPs: For cold non-capped transcript, use 25 mM each ATP, CTP, GTP, UTP.
For labeled transcript use 30 mM each ATP, CTP, GTP, and add 1 ul of 1 mM UTP, then label with 100 uCi of 32-P UTP for a hyb probe, or 10 uCi UTP for a Rnase protection assay probe.
For infectious transcripts, use 30 mM each ATP, CTP, UTP, and 6 mM GTP. Add 6 ul CAP analog at 10 mM, from New England Biolabs.
Incubate at 37 C for 1.5 hours. For capped transcripts add 1 ul of 25 mM GTP after 1 hour.
Add 1 ul RQ1 RNase-free DNase (Promega)
Incubate a further 15 minutes.
Phenol:Chloroform extract.
Ethanol precipitate with NH4OAc.
The pyrophosphatase is from Sigma, catalog # I 1891. Resuspend the entire
vial in 1 ml of 50% glycerol, and store at -20 as any other enzyme.
For modified SP6 promoters (most of the newer CMV and sat RNA clones) use SP6 RNA polymerase fr