• My NEB   • Print   • PDF   • Standard RNA Synthesis (E2050)
We strongly recommend wearing gloves and using nuclease-free
tubes and reagents to avoid RNase contamination. Reactions are
typically 20 μl but can be scaled up as needed. Reactions should
be assembled in nuclease-free microfuge tubes or PCR strip
tubes.     1. Thaw the necessary kit components, mix and pulse-
spin in microfuge to collect solutions to the bottoms of tubes.
Keep on ice.      2. Assemble the reaction at room temperature
in the following order:       Nuclease-free water  X µl
NTP Buffer Mix 10 µl (10 mM each NTP final)       Template DNA X
µl (1 µg)       T7 RNA Polymerase Mix 2 µl       Total reaction
volume 20 µl       3. Mix thoroughly and pulse-spin in a
microfuge. Incubate at 37°C for 2 hours.        Reaction time
depends on template amount, quality and RNA transcript length.
For reactions with transcripts longer than 0.3 kb, 2 hour
incubation should give you the maximum yield. For reaction times
of 60 minutes or less,