Table 2.

In Vitro Transcription Protocol

Component	Volume (µl)	Final Concentration
Nuclease-free H2O	43.3	n/a
10× Transcription Buffer	10.0	100 mM HEPES, pH 7.5
	-	20 mM MgCl2
	-	2.5 mM spermidine
100 mM DTT	10.0	10 mM
100 mM rNTP mixture	4.0	4 mM, each rNTP
0.1 U/µl (5′)ppp(5′)G	13.0	0.013 U/µl
20 U/µl RNasin	2.0	0.4 U/µl
PCR product DNA	13.0	*
SP6 RNA polymerase	3.5	**
0.5 U/µl Pyrophosphatase	1.2	0.006 U/µl
Total	100.0

^*variable, depending on efficiency of PCR reaction and purification

^**variable, depending on source of polymerase