INCOGNITO™ T7 5mC- & -RNA Transcription Kit

Store at -20°C in a freezer without a defrost cycle. Do not store at -70°C

The INCOGNITO™ T7 5mC- & -RNA Transcription Kit* is optimized for high-yield synthesis of 5-methylcytosine- pseudouridine-containing RNA (5mC -RNA) by in vitro transcription (IVT) of a DNA templatethat has a properly-oriented phage T7 RNA polymerase promoter using T7-Scribe™ RNA polymerase,the canonical nucleotides ATP & GTP, and the modified nucleotides 5-methylcytosine-5'-triphosphate(5mCTP) & pseudouridine-5'-triphosphate ( TP). Although yield varies with the DNA template and otherfactors, a standard 20 μl IVT reaction using 1 μg of a control template DNA yields approximately 100 μgof 5mC -RNA in 2 hours at 37°C. The 5mC -RNA can be enzymatically 5'-capped and 3'-poly(A)-tailedusing CELLSCRIPT's ScriptCap™ m7G Capping System, ScriptCap™ 2'-O-Methyltransferase andA-Plus™ Poly(A) Polymerase to generate 5mC -mRNA for expression in cells. It has been shown that 5mC -mRNAs are translated into protein at higher levels and induce lower innateimmune responses in human and other mammalian cells that express various RNA sensors compared tocorresponding unmodified mRNAs.

RNase-Free DNase I is provided in a 50% glycerol solution containing 50 mM Tris-HCl, pH 7.5,10 mM CaCl2, 10 mM MgCl2 and 0.1% Triton® X-100. ScriptGuard RNase Inhibitor is supplied in a50% glycerol solution containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTAand 0.1% Triton® X-100. All other enzymes are provided in a 50% glycerol solution containing 50 mMTris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100.

Unit Definition One unit of RNase-Free DNase I digests 1 µg of pUC19 DNA to oligodeoxynucleotides in 10 minutesat 37°C. One unit of ScriptGuard RNase Inhibitor results in 50% inhibition of 5 ng of RNase A. Activity ismeasured by the inhibition of hydrolysis of cyclic 2',3'-CMP by RNase A. Functional Testing The INCOGNITO T7 5mC- & Ψ-RNA Transcription Kit is functionally tested under standard reactionconditions using the T7 Control Template DNA. The kit must produce at least 90 µg of RNA from1 µg of the T7 Control Template DNA in 2 hours at 37°C. Contaminating Activity Assays All components of the INCOGNITO T7 5mC- & Ψ-RNA Transcription Kit are free of detectable RNaseand DNase activity, except for the inherent activity of the RNase-Free DNase I component.