Protocol for Standard RNA Synthesis with Hi-T7 RNA Polymerase (NEB #M0658)

Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottom of the tubes.

Assemble the reaction at room temperature in the following order.

  Volume Concentration
Nuclease-free H2O X µl  
10X Hi-T7 RNA Polymerase Reaction Buffer 2 µl 1X
NTPs X µl 0.5 mM each
Template DNA X µl 0.2 – 1 µg
RNase Inhibitor (optional) 0.5 µl 1 U/µl final
Hi-T7 RNA Polymerase (50 U/µl) 2 µl  
Total volume 20 µl  

Incubate at 50°C for 1 hour.