CleanCap® AG 3' OMe CleanScript™ IVT Kit

CleanCap AG (3′ OMe) is a modified version of CleanCap AG to improve protein expression. CleanCap AG (3′ OMe) includes the 5′ N7-methyl-3′-O-methylguanosine commonly found in mRNA capped using anti-reverse cap analog (ARCA), whereas CleanCap AG has a naturally occurring 5′ N7-methylguanosine structure.

It is TriLink's proprietary in vitro transcription (IVT) method to produce high-quality, high-yield mRNAs from a broad range of sequences. It has been optimized to minimize dsRNA and improve in vivo protein expression from the resulting mRNAs. Please download the technote to learn more.

CleanScribe RNA Polymerase is a novel enzyme designed to substitute wild-type T7 RNA polymerase in IVT and reduce dsRNA formation by up to 85%.

The kit contains an IVT buffer for co-transcriptional capping with the supplied CleanCap® analog. The buffer is proprietary and formulated based on TriLink's CleanScript® IVT method to increase yield and lower dsRNA formation.

The kit provides all necessary reagents sufficient for either 20 x 20 µL reactions (K-7413-20) or 125 x 20 µL reactions (K-7413-125) to synthesize capped RNA.

No, the plasmid DNA must be fully linearized. Residual circular plasmid will produce unwanted extended RNA byproducts that can affect your results.

Yes, PCR products can be used directly, though using silica-membrane-purified PCR products typically gives better results. Please ensure it contains the correct T7 promoter sequence and the start site as described in the template design section. The final concentration should be 0.025 ug/uL for PCR templates (compared to 0.05 ug/uL for plasmid templates).

The template must contain the correct T7 promoter sequence followed by an initiating sequence of 5' AGG 3' or 5' AGA 3' for proper use with CleanCap AG (3' Ome). Please refer to the template design section in protocol.

The reaction conditions have been tested with templates up to 6 kb in length resulting in 4.5 kb transcript.

A typical CleanScript® IVT reaction following the kit protocol yields 160-200 ug of RNA per 20 μL of reaction using unmodified NTPs or N1-methylpseudouridine in place of UTP.

The standard protocol calls for a 3-hour incubation at 37°C, using either a thermal cycler with heated lid or a dry air incubator to prevent evaporation and condensation.

While DNase treatment is optional, it is recommended to remove template DNA. You can either use the one-pot DNase protocol in the product manual or purify the RNA and then follow a DNase treatment.

You can use lithium chloride precipitation, spin columns like QIAGEN RNeasy kits, or oligo(dT) if your RNA has a poly(A) tail. Oligo (dT) purification often results in higher integrity mRNA due to removal of incomplete transcripts. The choice often depends on your scale and downstream applications.

The control template is included to verify RNA transcription and aid with troubleshooting of technical issues that might arise. We strongly recommend including a control reaction with the provided template when initially setting up your IVT. Following our recommended protocol, the template should yield 160-200 ug of 1.9 kb RNA transcript in 3 hrs of IVT per 20 µL reaction. When using TriLink’s provided FLuc control template, for a 20-μL IVT reaction add 2 μL of the 0.5 μg/μL linearized plasmid (1 μg total) or add 2 μL of the 0.25 μg/μL PCR template (0.5 μg total).

No, the FLuc plasmid template is provided linearized.

For K-7413-20, 2.5 ug of Fluc control DNA template (PCR) is provided that is sufficient for 5 x 20 µL reactions. For K-7413-125, 25 ug of Fluc control DNA template (linearized plasmid) is provided that is sufficient for 5 x 100 uL reactions.