T7-FlashScribe™ Transcription Kit V2

Rapid, high yield production of up to 180 µg of uridine-containing IVT RNA (U-RNA) from 1 µg of template in just 30 minutes.
Produce high yields of either long or short transcripts.
Easily scale up reactions to produce milligram amounts of U-RNA.
ScriptGuard™ RNase Inhibitor included to protect synthesized RNA from RNase degradation.

Product is for research use only (RUO)

Product Description:

The T7-FlashScribe™ Transcription Kit V2 is specially formulated to maximize yields of canonical uridine-containing RNA (U-RNA) from an in vitro transcription (IVT) reaction. Although yield varies with the DNA template and other factors, a standard 30 minute, 20 μl reaction will yield up to 180 μg of RNA from 1 μg of DNA template. These high yields are due to the superior performance properties of the T7-FlashScribe™ enzyme. The standard reaction can be scaled up to generate milligram amounts of either long or short RNA transcripts. ScriptGuard™ RNase Inhibitor is included in the kit to protect synthesized RNA from RNase degradation.

T7-FlashScribe™ IVT U-RNA can be further processed into U-mRNA (5'-end capped and 3'-end poly[A] tailed) through the use of CELLSCRIPT™'s ScriptCap™ Cap 1 Capping System (contains both ScriptCap™ Capping Enzyme and 2'-O-Methyltransferase) and A-Plus™ Poly(A) Polymerase Tailing Kit (available separately).

For reduction of immunogenicity, combine 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™ line of products to produce low immunogenicity mRNA containing the modified nucleotides N1-methyl-pseudouridine, pseudouridine and/or 5-methyl-cytidine.

Product Performance:

The standard 30 minute, 20 μl T7-FlashScribe™ reaction was optimized for transcription using 1 μg of linearized DNA template. However, higher or lower amounts of DNA template can be used successfully with protocol modifications. Table 1 summarizes IVT RNA yields from varying the input amount of control template DNA and incubation time in T7-FlashScribe™ reactions. Results may vary depending on the template used.

Table 1. RNA yields from T7-FlashScribe™ transcription reactions with varying DNA inputs and incubation times. The standard protocol uses 1 µg of DNA input and 30 minute incubation time.

Materials Supplied:

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

T7-FlashScribe™ Transcription Kit V2 Contents (50 reactions)
Kit Component	Reagent Volume
T7-FlashScribe™ Enzyme Solution	100 μl
10X T7-FlashScribe™ Transcription Buffer II	100 μl
100 mM GTP	90 μl
100 mM ATP	90 μl
100 mM UTP	90 μl
100 mM CTP	90 μl
100 mM Dithiothreitol (DTT)	100 μl
RNase-Free DNase I, 1 U/μl	50 μl
ScriptGuard™ RNase Inhibitor, 40 U/μl	25 μl
T7 Control Template DNA, 0.5 μg/μl	20 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)

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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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^oC.
	Check for stable secondary structure (T_m >37^oC) 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^oC.
White precipitate in reaction buffer	Incubate the reaction buffer at 37^oC for 5 minutes then mix thoroughly to dissolve the precipitate.
White precipitate in reaction buffer	Do not store the kit at –70^oC.