New England Biolabs, E2065S, HiScribe® T7 ARCA mRNA Kit

Now includes separate tube of DTT Related Categories RNA Capping,, RNA Synthesis In vitro Transcription (IVT),, Nucleotide Solutions for RNA Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) FAQ Q: HiScribe® T7 ARCA mRNA Kit (with tailing) What is the difference between the HiScribe T7 ARCA mRNA Kit (NEB #E2065) and the HiScribe T7 ARCA mRNA Kit (with Tailing)(NEB #E2060)? A: Both of the HiScribe T7 ARCA mRNA kits can be used to synthesize RNA by co-transcriptional capping with ARCA. The HiScribe T7 ARCA mRNA kits provide an easy solution for producing ARCA capped mRNAs. E2065 is intended for templates which have an encoded Poly(A) tail. For templates which do not have a encoded Poly(A) tails, #E2060 contains E. coli Poly(A) Polymerase for the addition of Poly(A) tails after transcription and capping. Q: I currently use MessageMAX™ T7 ARCA-capped Message Transcription Kit, which mRNA synthesis kit from NEB should I use? A: We recommend the HiScribe T7 ARCA mRNA kit (E2065) which will co-transcriptionally cap with ARCA but does not include Poly(A) polymerase. For use when enzymatic Poly(A) tailing is not needed, or the Poly(A) tail is encoded in the template. The HiScribe kit contains reagents and protocols for co-transcriptional ARCA synthesis and capping of 20 synthesis reactions. Q: I currently use mMessage mMachine® T7 Ultra Transcription Kit, which mRNA synthesis kit from NEB should I use? A: We recommend the HiScribe T7 ARCA mRNA kit (with tailing)(E2060) which can be used to co-transcriptionally cap with ARCA and subsequently add a poly(A) tail using E. coli Poly(A) Polymerase. The HiScribe kit contains reagents and protocols for complete co-transcriptional ARCA capping and tailing of synthesis reactions. Q: Can modified nucleotides be used with the HiScribe T7 ARCA mRNA kits? A: The HiScribe T7 ARCA mRNA kits are capable of incorporation of 5mCTP and Pseudo-UTP into mRNA. Up to 2.5mM total modified nucleotides can be added into the reaction without impacting the mRNA yield significantly. Other modified UTP and CTP may be used but the yield will vary depending on the property of the nucleotides. Modified GTP and ATP should not be used because they will interfere with capping and tailing efficiency. Q: What is the difference between the HiScribe T7 ARCA mRNA kits and the HiScribe T7 High Yield RNA Synthesis Kit (E2040) and HiScribe T7 Quick RNA Synthesis Kit (E2050)? A: Please see the selection chart to pick the appropriate HiScribe kit for your application. Q: Can I use the Monarch Spin RNA Cleanup Kits to cleanup my in vitro transcription (IVT) reaction? A: Yes, the Monarch Spin RNA Cleanup Kit (50 µg) (NEB #T2040) is suitable for cleaning up in vitro transcription reactions where the yield is not expected to exceed 50 µg. If the RNA yield of an in vitro transcription is expected to exceed 50 µg, we recommend the reaction be split among multiple Monarch Spin RNA Cleanup Columns (50 µg) or cleaned up using the Monarch Spin RNA Cleanup Kit (500 µg) (NEB #T2050), due to the larger RNA binding capacity. Q: How can I improve on a low yield of RNA from the transcription reaction? A: If the transcription reaction with your template generates full-length RNA but the yield is significantly lower than expected, it is possible that contaminants in the DNA template are inhibiting the RNA polymerase, or the DNA concentration may be incorrect. Alternatively, additional purification of DNA template may be required. Adding 5mM DTT (final) to the transcription reaction may also help improve RNA yield. Q: Are modified nucleotides included in the kit? A: Modified nucleotides are not included in the kit but can be purchased separately. NEB can provide N1-Methyl-Pseudouridine-5’-Triphosphate (NEB #N0431), 5-Methyl-Cytidine-5’-Triphosphate (NEB #N0432), Pseudouridine-5’-Triphosphate (NEB #N0433) and 5-Methoxy-Uridine-5’-Triphosphate (NEB #N0434). The kit manual includes a detailed protocol for using modified nucleotides in the transcription reaction. Q: Do I need to add DTT to the reaction? A: 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.