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