T7 mScript™ Standard mRNA Production System

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

The T7 mScript™ Standard mRNA Production System provides all enzymes and enzyme-relatedreagents for making 5'-capped, 3'-polyadenylated mRNA. This mScript Kit includes modules for (i) in vitrotranscription of linear double-stranded DNA templates using the mScript T7 Enzyme Solution, thecanonical nucleotides ATP, CTP, GTP and UTP, (ii) enzymatic capping of the RNA using ScriptCap™Capping Enzyme System (for making mRNA with a Cap 0 cap structure), (iii) ScriptCap™2'-O-Methyltransferase (for optionally making mRNA with a Cap 1 cap structure) and (iv) A-Plus™ Poly(A)Polymerase for adding a 3'-poly(A) tail. Post-transfection, capped and tailed mRNA has increased stability and translation efficiency in mosteukaryotic cell lines. The mScript System improves upon existing capping methods by ensuring virtually100% transcript capping, all caps in the proper orientation and the ability to produce large amounts ofcapped RNA at a reasonable cost. This mRNA is suitable for use in transfection and microinjectionexperiments as well as in vitro translation systems.

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. A-Plus Poly(A) Polymerase is provided in a 50% glycerol solution containing 50 mM Tris-HCl, pH 7.5,0.5 M NaCl, 1 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100. ScriptGuard RNase Inhibitor is provided in a 50% glycerol solution containing 50 mM Tris-HCl, pH7.5, 100 mM NaCl, 10 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100. All other enzymes are provided in a 50% glycerol solution containing 50 mM Tris-HCl, pH 7.5,100 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 0.1% Triton X-100.

Unit Definitions One T7 mScript Standard mRNA Production System reaction produces 60 µg of 5'-capped, 3'-poly-(A)-tailed mRNA. One unit of RNase-Free DNase I digests 1 µg of pUC19 DNA to oligodeoxynucleotides in 10 minutesat 37°C. One unit of ScriptCap Capping Enzyme releases 1 nmole of inorganic phosphate from GTP in 10minutes at 37°C under standard assay conditions. One unit of ScriptCap 2'-O-Methyltransferase methylates one picomole of a control Cap 0 RNA in1 hour at 37°C under standard assay conditions. One unit of A-Plus Poly(A) Polymerase converts 1 nmole of ATP into acid-insoluble material in 10minutes at 37°C under standard assay reaction conditions. 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 T7 mScript Standard mRNA Production System is functionally tested under standard reactionconditions using the T7 Control Template DNA and independent transcripts. The in vitro transcriptionmodule must produce at least 60 µg of RNA from 1 µg of the T7 Control Template DNA in 15 minutesat 37°C. A-Plus Poly(A) Polymerase is functionally tested in 1X A-Plus Poly(A) Tailing Buffer with1 mM ATP and a 1.4 kb transcript. The capping module enzymes are tested independently usingnon-T7 control transcript RNA to assay for completeness of reaction. Contaminating Activity Assays All components of the T7 mScript Standard mRNA Production System are free of detectable RNaseand DNase activity, except for the inherent activity of the RNase-Free DNase I component.