T7 mScript™ Standard mRNA Production System V2

All-in-one kit to produce uridine-containing, fully 5’ capped and 3’ poly(A) tailed mRNA (U-mRNA).
Combine the benefits of high-yield transcription with post-transcriptional capping and tailing.
Obtain virtually 100% 5’-capped transcripts.
Create variable 3’ poly(A) tail lengths, from 50 to >300 A’s.
Two kit sizes to produce 0.6 or 1.5 mg of U-mRNA.
ScriptGuard™ RNase Inhibitor included to protect synthesized mRNA from RNase degradation.

Product is for research use only (RUO)

Product Description:

The T7 mScript™ Standard mRNA Production System V2 provides all enzymes and reagents for making canonical uridine (U)-containing, 5'-capped and
3'-polyadenylated mRNA. The kit includes reagents for four workflow modules, (1) high-yield in vitro transcription of linear double-stranded DNA templates using the T7 mScript™ Enzyme Solution and the canonical nucleotides ATP, CTP, GTP and UTP, (2) enzymatic capping of the RNA using the ScriptCap™ Cap 1 Capping System (contains both ScriptCap™ Capping Enzyme and 2'-O-Methyltransferase) for making mRNA with a Cap 1 cap structure, (3) A-Plus™ Poly(A) Polymerase for adding a
3' poly(A) tail and (4) 5 M NH₄OAc as a convenient RNA/mRNA purification method. ScriptGuard™ RNase Inhibitor is included in the kit to protect synthesized mRNA from RNase degradation.

Post-transfection, capped and tailed mRNA has increased stability and translation efficiency in most eukaryotic cell lines. The mScript™ System improves upon co-transcriptional capping and tailing methods by ensuring virtually 100% transcript capping and enabling users to define their desired tail lengths, even greater than 300 A's. In this way, poly(A) tail lengths can be generated much longer than is possible using a template-encoded tail. mRNAs with longer poly(A) tails are expressed for longer periods of time in cells. mScript™ U-mRNA is suitable for use in transfection and microinjection experiments as well as in vitro translation systems.

For reduction of immunogenicity, combine mScript™ U-mRNA with the Min-Immune™ Gold dsRNA Removal Kit to produce virtually dsRNA-free (to <0.005% of sample [LLOQ]), ultra-low immunogenicity mRNA that is suitable for downstream applications such as cell and gene therapy research and mRNA vaccine development.

CELLSCRIPT™ also offers the INCOGNITO™ T7 mScript™ N1meΨ-mRNA Production System and INCOGNITO™ T7 mScript™ Ψ-mRNA Production System to produce low immunogenicity mRNA containing the modified nucleotides N1-methyl-pseudouridine and pseudouridine, respectively.

Materials Supplied:

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

T7 mScript™ Standard mRNA Production System V2 Kit Contents (Module 1 of 4)
Kit Module	Kit Component	Reagent Volume
Kit Module	Kit Component	C-MSC11610 10 Reactions	C-MSC100625 25 Reactions
In Vitro Transcription	T7 mScript™ Enzyme Solution	20 µl	50 µl
	10X T7 mScript™ Transcription Buffer II	20 µl	50 µl
	100 mM Dithiothreitol (DTT)	20 µl	50 µl
	NTP Solution 25 mM each GTP, ATP, UTP, CTP	80 µl	180 µl
	RNase-Free DNase I, 1 U/μl	10 µl	25 µl

T7 mScript™ Standard mRNA Production System V2 Kit Contents (Module 2 of 4)
Kit Module	Kit Component	Reagent Volume
Kit Module	Kit Component	C-MSC11610 10 Reactions	C-MSC100625 25 Reactions
Post-Transcriptional Capping	ScriptCap™ Capping Enzyme, 10 U/μl	40 µl	100 µl
	ScriptCap™ 2'-O-Methyltransferase, 100 U/μl	40 µl	100 µl
	10X ScriptCap™ Capping Buffer 0.5 M Tris-HCl (pH 8.0), 60 mM KCl and 12.5 mM MgCl₂	100 µl	250 µl
	20 mM S-adenosyl-methionine (SAM)	50 µl	125 µl
	20 mM GTP	50 µl	125 µl

T7 mScript™ Standard mRNA Production System V2 Kit Contents (Module 3 of 4)
Kit Module	Kit Component	Reagent Volume
Kit Module	Kit Component	C-MSC11610 10 Reactions	C-MSC100625 25 Reactions
Poly(A) Tailing	A-Plus™ Poly(A) Polymerase, 4 U/µl	52 µl	130 µl
	10X A-Plus™ Poly(A) Tailing Buffer 0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl and 100 mM MgCl₂	125 µl	300 µl
	20 mM ATP	60 µl	150 µl

T7 mScript™ Standard mRNA Production System V2 Kit Contents (Module 4 of 4)
Kit Module	Kit Component	Reagent Volume
Kit Module	Kit Component	C-MSC11610 10 Reactions	C-MSC100625 25 Reactions
Common Usage	ScriptGuard™ RNase Inhibitor, 40 U/µl	35 µl	90 µl
	RNase-Free Water	3 x 1.4 ml	12 ml
	5 M Ammonium Acetate	3 x 1.4 ml	12 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)
Optional: TE saturated phenol/chloroform, 0.5-1 M 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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FAQs: For answers to Frequently Asked Questions about our products, visit the FAQ library.

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Troubleshooting Guide:

Synthesis of IVT RNA
Symptom	Solution
Low yields or less than full-length transcripts	Cleanup the templates to remove any RNase or other contaminants.
	Verify that ScriptGuard™ RNase Inhibitor was added to the reaction.
	Extend the incubation time. Do not extend the reaction time beyond 3 hours.
	Increase the template concentration.
	Increase the reaction temperature to 42°C.
Assembled reaction formed an insoluble precipitate	Repeat assembly of the reaction at >22°C.
White precipitate in reaction buffer	Incubate the reaction buffer at 37°C for 5 minutes then mix thoroughly to dissolve the precipitate.
White precipitate in reaction buffer	Do not store the kit at –70°C.

Synthesis of Capped RNA
Symptom	Solution
Low capping efficiency	Cleanup the templates to remove any RNase or other contaminants.
	Verify that ScriptGuard™ RNase Inhibitor was added to the reaction.
	SAM slowly degrades at room temperature and above. Keep SAM solutions on ice at all times.
	Increase the capping reaction incubation time. For example, up to 3 hours at 37°C.
	Some RNAs form stable structures (e.g., homodimers, hairpins) at the 5' end, limiting access by Capping Enzyme or 2'-O-Methyltransferase. Analyze the sequence and increase the RNA denaturation temperature to above the T_m (e.g., to 65°C for 20 min, 75°C for 10 min, 85°C for 5 min). If the 5' end is highly structured, it might be necessary to modify the 5' end sequence using molecular biology techniques. Often this can be accomplished by making a single point mutation within the first 5 bases of the DNA template for the RNA transcript (non-coding region). Contact CELLSCRIPT™ Technical Support for suggestions and recommendations.
White precipitate in reaction buffer	Incubate the reaction buffer at 37°C for 5 minutes then mix thoroughly to dissolve the precipitate.
White precipitate in reaction buffer	Do not store the kit at –70°C.

Synthesis of Poly(A)-Tailed RNA
Symptom	Solution
Poly(A) tails are longer than expected	Decrease the time of incubation of the reaction.
Poly(A) tails are longer than expected	Decrease the amount of A-Plus™ Poly(A) Polymerase used in the reaction.
Poly(A) tails are shorter than expected	Increase the time of incubation of the reaction.
Poly(A) tails are shorter than expected	Increase the amount of A-Plus™ Poly(A) Polymerase used in the reaction.
No poly(A) tails are observed	Enzyme is inactive. Store only at –20°C. Keep on ice when not in the freezer.
No poly(A) tails are observed	ATP is hydrolyzed. Do not expose to elevated temperatures.