Protocol for Standard RNA Synthesis (NEB #M0378)

Assemble the reaction at room temperature in the following order.

Components
Amount

Concentration
H2O
X µl

 
10X Reaction Buffer 2 µl
 
NTP X µl

0.5 mM each
Template DNA X µl

0.2–1 µg
RNase Inhibitor (optional)
0.5 µl 1 U/µl final
Fresh DTT (optional) X µl
5 mM final
T3 RNA Pol 2 µl
 
Total Volume
20 µl
 

Incubate at 37°C for 1 hour. For shorter (< 300 nt) transcripts incubate at 37°C for 2–16 hours.