RNA Synthesis with Modified Nucleotides (E2040)
Introduction
Dye or Biotin-NTP is not supplied. The recommended molar ratio of modified NTP (Biotin-, Fluorescein-, Digoxigenin-, or Aminoallyl-NTP) to standard NTP is 1:3 or 1:2. The following reaction set up assumes modified UTP is used.
Protocol
- Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to bottom of tubes. Keep on ice.
- Assemble the reaction at room temperature in the following order:
REAGENT AMOUNT FINAL CONCENTRATION Nuclease-free water X μl
10X Reaction Buffer 1.5 μl 0.75X final
ATP (100 mM) 1.5 μl 7.5 mM final GTP (100 mM) 1.5 μl 7.5 mM final CTP (100 mM) 1.5 μl 7.5 mM final UTP (100 mM) 1 μl 5 mM final Modified UTP (10 mM) 5 μl 2.5 mM final Template DNA X μl 1 μg DTT (0.1M)*
1 μl
5 mM
T7 RNA Polymerase Mix 1.5 μl
Total reaction volume 20 μl
- Mix thoroughly, pulse-spin and incubate at 37°C for 2 hours. For short (< 300 nt) transcripts incubate at 37°C for 4–16 hours.
Modified ribonucleotides reduce transcription efficiency; therefore lower transcription yields should be expected as compared to transcription using unmodified NTP. In general, Biotin-NTP and Aminoallyl-NTP have an insignificant effect on yields, while 50% or lower yields can be expected for transcription reactions containing Fluorescein-NTP or Cy-NTP. In addition, transcripts containing modified ribonucleotides have reduced electrophoresis mobility due to higher molecular weight.
- Optional: To remove template DNA, add 70 μl nuclease-free water to each 20 μl reaction, 10 μl of 10X DNase I Buffer, and 2 μl of RNase-free DNase I, mix and incubate at 37°C for 15 minutes.
- Proceed with purification of synthesized RNA or analysis of transcription productsgel electrophoresis (for purification, we recommend the Monarch RNA Cleanup Kits (NEB #T2040 or #T2050)).
*Addition of DTT (5mM final) to the reaction is optional but recommended.The RNA polymerase in the kit is sensitive to oxidation and could result in lower RNA yield over time due to repeated handling etc. Adding DTT to the reaction may help restore the kit performance in such cases. Adding DTT will not compromise the reaction in any situation.
With this set up, the kit contains sufficient materials for 65 reactions. Note the ratio of UTP/modified UTP can be adjusted to meet specific needs. The total amount of UTP can be lowered if higher RNA yield is not necessary. For example, in the above reaction, 3 mM UTP and 1.5 mM modified UTP can be used without affecting the labeling density of the transcript.