Replicator™ RNAi Kit

Efficient system for producing large quantities of dsRNA

Description

The 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 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

Contents

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 (New England Biolabs), Inorganic Pyrophosphatase, 10x dsRNA Synthesis Buffer, 50 mM MnCl₂ solution, 10x NTP mix and 8 M LiCl solution.

Storage and stability

Recommended storage temperature -20 °C. Stable for one year from the packaging date.

Ordering information

Replicator™ RNAi Kit
F-610	40 reactions in 50 μl volume