Product Description:
The INCOGNITO™ T7-FlashScribe™ N1meΨ-RNA Transcription Kit is specially formulated to maximize yields of N1-methyl-pseudouridine-containing RNA (N1meΨ-RNA) from an in vitro transcription (IVT) reaction. Although yield varies with the DNA template and other factors, a standard one hour, 20 μl reaction will yield up to 160 μg of RNA from 1 μg of DNA template. These high yields are due to the superior performance properties of the T7-FlashScribe™ enzyme. The standard reaction can be scaled up to generate milligram amounts of RNA containing the canonical nucleotides ATP, CTP, GTP and the modified nucleotide N1-methyl-pseudouridine-5'-triphosphate (N1meΨTP).
Modified N1meΨ-mRNAs translate into higher protein levels and lower innate immune responses in human and other mammalian cells that express various RNA sensors as compared to unmodified mRNAs.
INCOGNITO™ T7 IVT N1meΨ-RNA can be further processed into low immunogenicity mRNA (5'-end capped and 3'-end poly[A] tailed) through the use of CELLSCRIPT™'s ScriptCap™ Cap 1 Capping System (contains both ScriptCap™ Capping Enzyme and 2'-O-Methyltransferase) and A-Plus™ Poly(A) Polymerase Tailing Kit (available separately).
For maximum reduction of immunogenicity, follow production of INCOGNITO™ N1meΨ-mRNA with dsRNA removal from your sample using the Min-Immune™ Gold dsRNA Removal Kit to produce virtually dsRNA-free, ultra-low immunogenicity mRNA that is suitable for downstream applications such as cell and gene therapy research and mRNA vaccine development.
Product Performance:
The standard 60 minute, 20 μl reaction was optimized for transcription using 1 µg of linearized DNA template. However, incubation times can be adjusted if desired. Table 1 summarizes transcription results with 1 µg of the control template DNA in a standard reaction with incubation times from 20-120 minutes. Results may vary depending on the template used.
Table 1. IVT RNA yields from INCOGNITO™ T7-FlashScribe™ N1meΨ-RNA Transcription Kit reactions incubated between 20-120 minutes. The standard protocol uses an incubation time of 60 minutes.
Incubation Time (minutes) |
20 |
30 |
60 |
90 |
120 |
RNA Yield (µg) |
78-80 |
100-109 |
120-175 |
120-172 |
121-199 |
Materials Supplied:
Important Store at -20°C in a freezer without a defrost cycle. Do not store at -70°C.
INCOGNITO™ T7-FlashScribe™ N1meΨ-RNA Transcription Kit Contents (25 reactions) |
Kit Component |
Reagent Volume |
T7-FlashScribe™ Enzyme Solution |
50 μl |
10X T7-FlashScribe™ Transcription Buffer II |
50 μl |
100 mM GTP |
45 μl |
100 mM ATP |
45 μl |
100 mM N1meΨTP |
45 μl |
100 mM CTP |
45 μl |
100 mM Dithiothreitol (DTT) |
50 μl |
RNase-Free DNase I, 1 U/μl |
25 μl |
ScriptGuard™ RNase Inhibitor, 40 U/μl |
15 μl |
T7 Control Template DNA, 0.5 μg/μl |
10 μl |
RNase-Free Water |
1.4 ml |
T7 Control Template DNA: Is a linearized 4.1 kb plasmid that contains a T7 promoter followed by a phage lambda dsDNA insert that encodes a 1,375 base runoff transcript. The Control Template DNA is provided at a concentration of 0.5 µg/µl in T10E1 Buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).
Materials Required, but not Supplied
- A DNA template for transcription of your RNA of interest
- Materials or kits for purification of the RNA product
- RNase-free TE Buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA)
Terms and Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.
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Troubleshooting Guide:
Uses and Label Licenses for Specific Products
INCOGNITO™ kits or other products or services comprising N1-methyl-pseudouridine, pseudouridine, 5-methyl-cytidine and/or other modified nucleotides for use in making RNA or mRNA (the “Products”) that are covered by technologies and IP disclosed in U.S. Patent Nos. 8,278,036 and 9,750,824 and PCT Publ. Nos. WO 2007/024708 and WO/2011/071931 and U.S. and international divisional, continuation, or other patents and patent applications derived therefrom that are owned by the University of Pennsylvania and licensed to CELLSCRIPT™ (“Products”) are sold for research use only.
By purchasing these Products from CELLSCRIPT™ or an authorized distributor of CELLSCRIPT™, Purchaser receives a limited non-exclusive, non-transferable right to use the Products purchased from CELLSCRIPT™ solely for its own internal laboratory research use (the “Product Use”). Such Product Use expressly excludes any Commercial Use comprising [A] clinical or other use in humans, [B] veterinary, livestock, agricultural or commercial use in animals, and/or [C] manufacture, distribution, importation, exportation, or sale of any products and/or services made using Products, it being understood that [A]–[C] are separately and/or collectively defined as a “Commercial Use” herein. By purchase of Products, Purchaser agrees not to make, import, use, transfer, offer for sale, or sell Products, components of Products, or derivatives of Products, including modified nucleoside-containing RNA or cells made by use of Products, or to provide services, information, or data obtained by the use thereof in exchange for money or other consideration. CELLSCRIPT™ provides no warranties (statutory or implied) concerning non-infringement of intellectual property rights of any other parties, and all such warranties are expressly disclaimed. Please contact CELLSCRIPT™ to inquire about a license for a Commercial Use.
The license remains in effect as long as the product is being used in accordance with the license terms. The license is terminated if the user breaches any of the license terms, such as using the product for commercial purposes without proper authorization.
