Replicator™ RNAi Kit

Efficient system for producing large quantities of dsRNA

Description

The Thermo Scientific Replicator™ RNAi Kit is a complete kit for producing milligram quantities of double-stranded RNA. Perfectly duplexed RNA produced with this kit can be further processed into siRNA which is the key component in RNAi-mediated gene silencing experiments. The suitable DNA template is created by PCR amplification. This step is carried out using the Thermo Scientific Phusion® High-Fidelity DNA Polymerase to ensure highest possible fidelity. In the first step of the dsRNA synthesis, T7 DNA-dependent RNA Polymerase transcribes single-stranded RNA copies from the DNA template molecule. Subsequently, the ssRNA molecules are directly converted to dsRNA by Phi6 RNA-dependent RNA polymerase in the same reaction mixture. One kit contains materials for producing up to 2.5 mg of pure double-stranded RNA in a 2 ml reaction volume. The high yield of double-stranded RNA obtained with the system is due to a unique combination of the complementary polymerase activities in carefully optimized reaction conditions.

Production of dsRNA with the Replicator™ RNAi Kit

Transcription T7 RNA Polymerase transcribes ssRNA from a DNA template.
Replication The ssRNA molecules are directly converted to dsRNA by Phi6 RNA Replicase.

Applications

Production of double-stranded RNA in milligram quantities for gene silencing experiments based on the RNAi mechanism
Production of a dsRNA marker fragment with a specific size

Advantages

No RNA hybridization step in the reaction protocol
RNA produced with this kit is perfectly duplexed
No smeared product resulting from RNA secondary structure formation or misannealing on agarose gels
Even large dsRNA fragments up to 20 kb, which are out of reach for competing systems, can be amplified

The Replicator™ RNAi Kit includes Phusion® High-Fidelity DNA Polymerase, 5x Phusion HF Buffer, dNTP mix, λ Control DNA, Control Primer Mix, Phi6 Replicase, T7 RNA Polymerase, Inorganic Pyrophosphatase, 10x dsRNA Synthesis Buffer, 50 mM MnCl₂ solution, 10x NTP mix and 8 M LiCl solution.