Component	Amount per reaction	Final concentration
Linear DNA template (Step 37A(iii))	Variable	2.5 ng/μl
HeLa nuclear extract 1× transcription buffer	(11–x) μl	—
MgCl2 (50 mM)	1.5 μl	3 mM
rNTP mixture (2.5 mM)	4 μl	0.4 mM
RNase inhibitor (40 U/μl)	0.25 μl	0.4 U/μl
HeLa nuclear extract	Variable (x μl)	0.32 U/μl
Nuclease-free water	Variable	—
Total volume	25 μl	—