MessageMAX™ T7 ARCA-Capped Message Transcription Kit V2

Co-transcriptionally cap uridine-containing IVT RNA with Anti-Reverse Cap Analog (ARCA).
Optimized for maximum transcript capping and yield.
Produce up to 60 µg of ARCA-capped RNA from 1 µg of template in just 30 minutes.
ScriptGuard™ RNase Inhibitor included to protect synthesized RNA from RNase degradation.

Product is for research use only (RUO)

Product Description:

The MessageMAX™ T7 ARCA-Capped Message Transcription Kit V2 is optimized for high-yield synthesis of canonical uridine-containing Anti-Reverse Cap Analog (ARCA)-capped RNA from an in vitro transcription (IVT) reaction. A 30 minute, 20 μl reaction yields up to 60 μg of ARCA-capped RNA from 1 μg of the control template DNA. ARCA ensures correct 5′ end incorporation, resulting in RNA that translates more efficiently in vivo than standard cap analogs. ScriptGuard™ RNase Inhibitor is included in the kit to protect synthesized RNA from RNase degradation.

The ARCA / NTP PreMix contains all four ribonucleotides (canonical nucleotides GTP, ATP, UTP and CTP) and the ARCA. The PreMix ensures the optimal concentration of each NTP and ratio of ARCA to GTP (4:1), maximizing transcript capping (~80%) and yield.

MessageMAX™ ARCA-capped RNA can be further processed into mRNA for expression in cells through the use of post-transcriptional capping using CELLSCRIPT™'s ScriptCap™ 2'-O-Methyltransferase (for conversion to a Cap 1 structure) and post-transcriptional tailing using A-Plus™ Poly(A) Polymerase Tailing Kit (available separately).

For reduction of immunogenicity, combine ARCA-capped 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 ARCA 5mC- & Ψ-RNA Transcription Kit to produce low immunogenicity ARCA-capped RNA containing the modified nucleotides 5-methyl-cytidine and pseudouridine.

Materials Supplied:

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

MessageMAX™ T7 ARCA-Capped Message Transcription Kit V2 Contents (10 reactions)
Kit Component	Volume
MessageMAX™ T7 Enzyme Solution	20 μl
10X MessageMAX™ T7 Transcription Buffer II	20 μl
MessageMAX™ ARCA Cap/NTP PreMix (22.5 mM ATP, CTP & UTP; 4.5 mM GTP & 18 mM ARCA)	80 μl
20 mM GTP	20 μl
100 mM Dithiothreitol (DTT)	20 μl
RNase-Free DNase I, 1 U/μl	10 μl
ScriptGuard™ RNase Inhibitor, 40 U/µl	10 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)
Optional: TE saturated phenol/chloroform, 0.5-1 M EDTA

Terms & 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:

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 4 hours.
	Increase the template concentration.
	Increase the reaction temperature to 42°C.
	Check for stable secondary structure (T_m >37°C) in the DNA template which can cause T7 RNAP pausing or dissociation from the template.
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.