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.