Standard mRNA Synthesis (E2060)
Standard mRNA Synthesis
- Thaw the necessary kit components, mix and pulse-spin in microfuge to
collect solutions to the bottoms of tubes.
Assemble the reaction at room temperature in the following order:
REAGENT AMOUNT FINAL CONCENTRATION Nuclease-free water to 20 µl 2X ARCA/NTP Mix 10 µl 1 mM GTP, 4 mM ARCA,
1.25 mM CTP, 1.25 mM
UTP, >1.25 mM ATP final
Template DNA X µl 1 µg DTT (0.1m)*
T7 RNA Polymerase Mix 2 µl Total reaction volume 20 µl
*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.
- Mix thoroughly and pulse-spin in a microfuge. Incubate at 37°C for
Do not heat the reaction. Do not purify the RNA. Proceed to DNase treatment and Poly(A) tailing steps. Or you can store the reaction at –20°C for a few days.
Reaction time depends on template amount, quality and RNA transcript length. For reactions with transcripts longer than 0.5 kb, 30 min incubation should give you the maximum yield.
For reactions with short RNA transcripts (< 0.5 kb), incubation time of 1 hour or longer is necessary to achieve good yield. It is safe to incubate the reaction for 16 hours (overnight).
For reaction times of 60 minutes or less, a water bath or heating block may be used; for reaction times longer than 60 minutes, please use a dry air incubator or PCR machine.
- DNase treatment to remove template DNA. Add 2 μl of DNase I, mix well and incubate at 37°C for 15 minutes.
DNase treatment is optional if the template does not interfere with downstream experiment. If left untreated, DNA template containing eukaryotic promoters may produce a background in mRNA transfection experiments.
- Save 1 μl for gel analysis if desired. Do not heat the reaction or purify the RNA. Proceed to tailing reaction.
- Poly(A) tailing. Set up the tailing reaction as below. The unpurified IVT reaction contains enough ATP, no extra ATP is necessary for the tailing reaction. Standard tailing reaction volume is 50 μl. Tail length is slightly longer in a 100 μl reaction volume for some transcripts.
REAGENT AMOUNT AMOUNT H2O to 20 µl 65 µl IVT reaction 20 µl 20 µl 10X Poly(A)
Polymerase Reaction Buffer
5 µl 10 µl
Poly(A) Polymerase 5 µl 5 µl Total reaction volume 50 µl 100 µl
- Mix thoroughly and pulse-spin in a microfuge. Incubate at 37°C for 30 minutes. Save 1 μl for analysis if necessary.
A 30 min tailing reaction will give 150 nt or longer poly(A) tail for majority of mRNAs. Due to the nature of the tailing reaction, tail length will vary depending on RNA sequence, structure, yield, length, etc. For very short RNA (< 300 nt), tailing time can be extended to 1 hour to achieve sufficient tailing.
The 3´ end of RNA must be exposed for efficient tailing. If the 3´ end is buried inside the RNA structure, it will not be available for tailing. Redesigning the mRNA 3´ end sequence may resolve the problem.
- Proceed with mRNA purification. For purification, we recommend the Monarch RNA Cleanup Kits (NEB #T2040 or #T2050).