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In vitro Transcription: INCOGNITO™ IVT and mRNA Production Systems

The new INCOGNITO™ kits

Evade cellular immune response to your RNA with the new INCOGNITO™ kits from Cellscript.

The new INCOGNITO™ kits * are designed for in vitro synthesis of RNA containing modified nucleosides such as pseudouridine and 5-methylcytidine. They assist in reducing immune response and enhancing protein translation. A range of these kits are available to end users depending on their specific research desires and downstream applications.

Cellscript’s INCOGNITO™ contain everything needed to make N1meΨ- or Ψ-containing Cap1 mRNA, including in vitro transcription reagents plus ScriptCap™ Capping Enzyme, ScriptCap™ 2’-O-Methyltransferase, and A-Plus™ Poly(A) Polymerase. - INCOGNITO™ T7-FlashScribe™

INCOGNITO™ T7 mScript™ mRNA Production Systems contain everything needed to make N1meΨ- or Ψ-containing Cap1 mRNA, including in vitro transcription reagents plus ScriptCap™ Capping Enzyme, ScriptCap™ 2’-O-Methyltransferase, and A-Plus™ Poly(A) Polymerase. - INCOGNITO™ T7-FlashScribe

The INCOGNITO™ SP6 Ψ-RNA Transcription Kit -optimized for high-yield synthesis of pseudouridine-containing RNA (Ψ-RNA) from an in vitro transcription (IVT) reaction.

The INCOGNITO™ T7 5mC- & Ψ-RNA Transcription Kit - optimized for high-yield synthesis of 5-methyl-cytidine- pseudouridine-containing RNA (5mCΨ-RNA) from an in vitro transcription (IVT) reaction.

Advantages

Optimized for high-yield synthesis of pseudouridine-containing IVT RNA (Ψ-RNA)
Produce up to 100 µg of Ψ-RNA from 1 µg of template ( depending on kit used).
Easily scale up reactions to produce milligram amounts of Ψ-RNA.
Lower immunogenicity in mammalian cells through incorporation of Ψ in synthesized mRNA.
Modified Ψ-mRNAs translate into higher protein levels
The standard reactions can be scaled up to produce milligram amounts of RNA canonical nucleotides ATP and GTP and modified pseudouridine-5'-triphosphate (ΨTP) and 5-methyl-cytidine-5'-triphosphate (5mCTP).

*Transcription Kits are for in vitro transcription of N1meΨ- or Ψ-containing RNA. - All kits above come with a label license to use them for internal research use under the University of Pennsylvania’s modified mRNA technologies patents.

INCOGNITO™ T7 mScript™ N1meΨ-mRNA Production System

CellScript

Catalogue No.	Description	Pack Size	Price	Qty	Change to: €
IMMY240625	INCOGNITO™ T7 mScript™ N1meΨ-mRNA Production System	25 rxns	POA	Quantity	Add to Order

Description

The INCOGNITO™ T7 mScript™ N1meΨ-mRNA Production System provides all enzymes and enzyme related reagents for making N1-methyl-pseudouridine (N1meΨ)-containing, 5'-capped, 3'-polyadenylated mRNA. The kit includes modules for (i) in vitro transcription of linear double-stranded DNA templates using the T7 mScript™ Enzyme Solution, the canonical nucleotides ATP, CTP, GTP and the modified nucleotide N1-methyl-pseudouridine-5'-triphosphate (N1meΨTP), (ii) 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, (iii) A-Plus™ Poly(A) Polymerase for adding a 3'-poly(A) tail. and (iv) 5 M NH₄OAc as a convenient RNA/mRNA purification method.

Post-transfection, capped and tailed mRNA has increased stability and translation efficiency in most eukaryotic cell lines. The mScript™ System improves upon existing capping methods by ensuring virtually 100% transcript capping, all caps in the proper orientation and the ability to produce large amounts of capped RNA at a reasonable cost. This mRNA is suitable for use in transfection and microinjection experiments as well as in vitro translation systems. It has been shown that N1meΨ-mRNAs are translated into protein at higher levels and induce lower innate immune responses in human and other mammalian cells that express various RNA sensors compared to corresponding unmodified mRNAs.^1-6

Uses and Label Licenses for Specific Products:

Purchaser receives a limited non-exclusive, non-transferable right to use the Products purchased from CELLSCRIPT™ solely for its own internal laboratory research use. See the Licensing tab for more information.

Materials Supplied

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

INCOGNITO™ T7 mScript™ N1meΨ-mRNA Production System Kit Contents 25 Reactions. (Module 1 of 4)
Kit Module	Kit Component	Reagent Volume
In Vitro Transcription	T7 mScript™ Enzyme Solution	50 µl
	10X T7 mScript™ Transcription Buffer II	50 µl
	100 mM Dithiothreitol (DTT)	50 µl
	N1meΨTP PreMix 25 mM each GTP, ATP, N1meΨTP, CTP	180 µl
	RNase-Free DNase I, 1 U/μl	25 µl

INCOGNITO™ T7 mScript™ N1meΨ-mRNA Production System Kit Contents 25 Reactions. (Module 2 of 4)
Kit Module	Kit Component	Reagent Volume
Post-Transcriptional Capping	ScriptCap™ Capping Enzyme, 10 U/μl	100 µl
	ScriptCap™ 2'-O-Methyltransferase, 100 U/μl	100 µl
	10X ScriptCap™ Capping Buffer 0.5 M Tris-HCl (pH 8.0), 60 mM KCl and 12.5 mM MgCl₂	250 µl
	20 mM S-adenosyl-methionine (SAM)	125 µl
	20 mM GTP	125 µl

INCOGNITO™ T7 mScript™ N1meΨ-mRNA Production System Kit Contents 25 Reactions. (Module 3 of 4)
Kit Module	Kit Component	Reagent Volume
Poly(A) - Tailing	A-Plus™ Poly(A) Polymerase, 4 U/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.	300 µl
	20 mM ATP	150 µl

INCOGNITO™ T7 mScript™ N1meΨ-mRNA Production System Kit Contents 25 Reactions. (Module 4 of 4)
Kit Module	Kit Component	Reagent Volume
Common Usage	ScriptGuard™ RNase Inhibitor, 40 U/µl	90 µl
	RNase-Free Water	12 ml
	5 M Ammonium Acetate	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.

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^oC.
Assembled reaction formed an insoluble precipitate	Repeat assembly of the reaction at >22^oC.
White precipitate in reaction buffer	Incubate the reaction buffer at 37^oC for 5 minutes then mix thoroughly to dissolve the precipitate.
	Do not store the kit at –70^oC.

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^oC.
	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^oC for 20 min, 75^oC for 10 min, 85^oC 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 Services for suggestions and recommendations.
White precipitate in reaction buffer	Incubate the reaction buffer at 37^oC for 5 minutes then mix thoroughly to dissolve the precipitate.
	Do not store the kit at –70^oC.

Synthesis of Poly(A)-Tailed RNA
Symptom	Solution
Poly(A) tails are longer than expected	Decrease the time of incubation of the reaction.
	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.
	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^oC. Keep on ice when not in the freezer.
	ATP is hydrolyzed. Do not expose to elevated temperatures.

FAQs: For answers to Frequently Asked Questions about our products, visit the FAQ library.