T7-Scribe™ Standard RNA IVT Kit

Synthesize up to 150 µg of uridine-containing IVT RNA (U-RNA) from 1 µg of template in two hours.
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-Scribe™ Standard RNA IVT Kit is specially formulated to produce high 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 two hour, 20 μl reaction will yield up to 150 μg of RNA from 1 μg of the control template and can be easily scaled up to produce milligram amounts of U-RNA. These yields are made possible by the high-performance properties of the T7-Scribe™ enzyme. The T7-Scribe™ Standard RNA IVT Kit produces high yields of either long or short transcripts and can be readily modified to prepare fluorescent-, biotinylated- or digoxigenin-labeled RNA. ScriptGuard™ RNase Inhibitor is included in the kit to protect synthesized RNA from RNase degradation.

T7-Scribe™ 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 two hour, 20 μl reaction will yield up to 150 μg of RNA from 1 μg of the control template. However, lower amounts of DNA template can be used successfully in a T7-Scribe™ reaction. Table 1 summarizes the yields obtained when reducing the amount of control template DNA in a T7-Scribe™ reaction. Results may vary depending on the template used. Increasing the reaction time to four to six hours may increase the yield of RNA.

Table 1. RNA yields from T7-Scribe™ Standard RNA IVT Kit reactions using different DNA inputs. The standard protocol uses
1 µg of DNA input.

Control Template Amount	RNA Yield
1000 ng (1 µg)	130-160 µg
500 ng (0.5 µg)	110-130 µg
100 ng (0.1 µg)	30-50 µg
50 ng (0.05 µg)	15-25 µg
10 ng (0.01 µg)	10-20 µg
1 ng (0.001 µg)	3-8 µg

Materials Supplied:

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

T7-Scribe™ Standard RNA IVT Kit Contents (50 reactions)
Kit Component	Reagent Volume
T7-Scribe™ Enzyme Solution	100 μl
10X T7-Scribe™ Transcription Buffer	100 μl
100 mM GTP	75 μl
100 mM ATP	75 μl
100 mM UTP	75 μl
100 mM CTP	75 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	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)

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.