New England Biolabs, E2080L, HiScribe® T7 mRNA Kit with CleanCap® Reagent AG ...

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: Do I need to change my promoter sequence to use CleanCap® Reagent AG? A: The T7 RNA promoter sequence itself (5′ TAATACGACTCACTATA 3′) does not need to be changed as the kit still utilizes T7 RNA Polymerase. However, the two initiating bases (+1 and +2) that are just downstream of the T7 promoter need to be “AG”. (The +1 base following the standard T7 RNA Polymerase Promoter is always a “G”. However, the +2 base may be any base (N)). Original promoter with initiating base +1 (G) and +2 (N) 5′ TAATACGACTCACTATAGN 3′ New sequence required for use with CleanCap Reagent AG (with initiating bases AG) 5′ TAATACGACTCACTATAAG 3′ Q: How can I change the initiating sequence of my dsDNA template? A: If your sequence of interest is cloned into a plasmid we recommend using the Q5 Site-Directed Mutagenesis Kit (NEB #E0554S) to change the initiating +1 and +2 bases to AG. Before transcription, the plasmid will need to be linearized (using a restriction enzyme that leaves a blunt or 5′ end overhang) to create a double-stranded in vitro transcription template. See the product manual for DNA template preparation. Alternatively, PCR can be performed using a Forward Primer that is designed to include the T7 promoter (with a few bases upstream) and the G to A change in the initiating nucleotide as well as some downstream sequence. Design the Reverse Primer to anneal to the region where your sequence will be terminated. Q: Will the 5′ base of my RNA be an “A”? A: Yes. The +1 A will be the first base incorporated into the RNA transcribed using the HiScribe® T7 mRNA Kit with CleanCap® Reagent AG. The RNA will have a naturally occurring Cap1 structure. Q: Can an uncut plasmid be used as a template for the HiScribe® T7 mRNA Kit with CleanCap® Reagent AG? A: No. T7 RNA Polymerase is an extremely processive enzyme and will continue to transcribe around a circular template multiple times without disassociating. The plasmid can be linearized with restriction enzymes that leave a blunt or 5′ overhang downstream of the DNA of interest resulting in run-off transcription. See the product manual for DNA template generation. Q: Can modified nucleotides be used with the HiScribe® T7 mRNA Kit with CleanCap® Reagent AG? A: Yes. For complete substitution, leave out the unlabeled NTP provided with the kit and supplement with the same final concentration of a modified NTP (CTP or UTP) of your choice. Modified ATP should be avoided as it will interfere with CleanCap® Reagent AG incorporation. To note, some modified NTPs may impact final yield of the synthesis reaction and ratios of modified:unmodified NTPs may need to be optimized for partial substitutions. Please refer to the protocol in the product manual for use with modified NTPs. Q: How can I improve the yield of RNA when using the HiScribe® T7 mRNA Kit with CleanCap® Reagent AG? A: Make sure that the NTPs, CleanCap Reagent AG, 10X Buffer and double-stranded DNA template are thawed and kept at room temperature during reaction set up (keep the T7 RNA Polymerase Mix on ice). Make sure that these components (NTPs, CleanCap® Reagent AG, 10X Buffer and dsDNA template) are mixed well prior to use by gently vortexing for 2-3 seconds and spinning down in a microfuge to collect the liquid at the bottom of the tube Set the reaction up at room temperature in the order listed in the manual. Gently mix the reaction by pipetting up and down and briefly spin in a microfuge before moving the reaction at 37°C. In addition, make sure to use RNase-free tubes and filter tips, and always wear gloves when handling the reagents and reactions. Make sure that your DNA template is very clean and verify that the concentration is correct. Q: How can I purify the RNA synthesized using this kit? A: RNA >300 nucleotides can be purified using the Lithium Chloride (LiCl) solution provided with the kit following the directions in the manual. Alternatively, we recommend the Monarch® RNA Cleanup Kit, 500 µg (NEB #T2050) for RNA purification. The expected yield from a single reaction of unmodified RNA using this kit is >90 µg. Q: Can components from other HiScribe® kits, T7 RNA Polymerase (NEB #M0251) or RNAPol Reaction Buffer (NEB #B9012) be substituted in this kit? A: No. The formulation of the components in this kit have been optimized for in vitro transcription using 5 mM CTP, 5 mM GTP, 5 mM UTP, 6 mM ATP and 4 mM CleanCap® Reagent AG in a 20 µl reaction. Q: Do you recommend template-encoding the poly(A) tail or using E. coli Poly(A) Polymerase? A: Both methods will result in an mRNA with a poly(A) tail. However, using a template-encoded poly(A) tail will reduce the overall workflow time and result in a defined poly(A) tail length. If using Poly(A) Polymerase (NEB #M0276), we would recommend purifying the IVT reaction prior to the addition of the poly(A) tail. Using Poly(A) Polymerase will result in a range of tail lengths and an increased workflow time. Q: Can I use a different CleanCap® Reagent analog with this kit? A: We recommend using the CleanCap Reagent AG provided with this kit as the formulations have been optimized for use with this specific trinucleotide cap analog. CleanCap Reagent GG and Cleancap Reagent GG (3′ OMe) will not work with the formulations and NTPs ratios provided in this kit. TriLink does offer CleanCap Reagent AG (3′ OMe), but it has not specifically been tested with the formulations provided in this kit. 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.