IG® RPA Master Mix

Recombinase Polymerase Amplification (RPA) Technology:

             IG® Recombinase Polymerase Amplification Kit Version 2 (IG® RPA V2) amplifies DNA at a  single and constant temperature (25 – 42°C) using a recombinase (e.g. UvsX), primers, a single-stranded DNA binding protein (SSB), and a strand displacing DNA polymerase. T4 UvsX is used in combination with its accessory protein, UvsY. The recombinase interacts with the primers to form nucleoprotein filament. This complex can bind with homologous double-stranded DNA through a strand exchange^1-3. After the exchange, a single-stranded binding protein, T4 gp32, stabilizes the displaced strand. Finally, Bsu/Sau DNA polymerase extends the DNA from the primers, creating a new complete copy of the template, and amplification can continue like in Polymerase Chain Reaction (PCR).

Applications and Benefits:

• Highly selective and sensitive isothermal amplification technique.
• Alternative to PCR. No thermocycler or other heavy equipment needed.
• Speed and sensitivity. Excellent for rapid point-of-care and on-site testing.
• No DNA pretreatment required.
• Flexible endpoint detection compatibility (e.g. lateral flow, real-time fluorescence).

Technical Support:

Intact Genomics is committed to supporting the worldwide scientific research community by supplying the highest quality reagents. Each new lot of our products is tested to ensure it meets the quality standards and specifications designated for the product.

Please follow the instructions carefully and contact us if additional assistance is needed. We appreciate your business and your feedback regarding the performance of our products in your applications.

General Guidelines:

• The master mix may have a pearl-colored appearance, which is normal. It typically clears during RPA.
• Setting up the reaction on ice is recommended for consistent performance.
• Mixing is very important to ensure a homogeneous reaction. Mix the reagents well by pipetting before using. Mix by pipetting half of the total volume 10 times.
• Low copy DNA targets may require longer incubation times, warming to 37ºC, and/or higher template concentration.
• Primers are optimally 25-35 BP in length and do not form either homo– or hetero-dimers. Primers should be checked to minimize secondary structures, e.g., hairpins.

Protocol:

1. Add the DNA template and IG RPA Master Mix in a tube on ice; mix by pipetting.
2. Add the forward and reverse primers.
3. Add the IG RPA Reaction Starter to initiate the reaction. Prepare per reaction mix according to the following table in order:

4. Mix half of the total volume 10 times by pipetting, then pulse spin the reaction tube for 1 second.

5. Incubate the tube at 37°C for 5 – 30 minutes or at 25°C for 10 – 45 minutes.

6. (Optional) Purify the DNA via ethanol precipitation. Analyze the DNA by gel electrophoresis on a 1% or 2% agarose gel.

Q&A

Q: How long should the RPA incubate?

A: The IG® RPA Master Mix is a very sensitive assay and extending the time may enhance the signal for low DNA copy targets (under 1000 copies). The table below is a flexible guide to help with designing applications:

Q: How important is mixing?

A: The reaction mixture is more viscous than saline solutions. It is advised to mix in the DNA template well by pipetting 10 times , then mix 10 times again after the RPA reaction starter is added.

Q: Should the reagents be kept cold?

A: For best results, set up the RPA reactions on ice and then begin your reaction by moving the reaction vial to ambient temperature or an incubator. Once the starter is added, the reaction rate is temperature dependent and the reaction is fast at ambient temperature, but very slow on ice. Store any unused IG RPA Master Mix in a freezer at –20ºC.

Related Products:

1. T4 UvsX Recombinase (Cat.# 3562) , Glycerol-free T4 UvsX DNA Recombinase (Cat.# 3562GF)
2. T4 gp32 Protein (Cat.# 3515), Glycerol-free T4 gp32 Protein (Cat.# 3513GF)
3. T4 UvsY Protein (Cat.# 3572), Glycerol-free T4 UvsY Protein (Cat.# 3572GF)
4. Bsu DNA Polymerase (Cat.# 3585), Glycerol-free Bsu (Cat.# 3585GF)
5. Exonuclease IV (Nfo) (Cat.# 3425)
6. Exonuclease III (Cat.# 3415)

References:

1. Cromie GA, Connelly JC, Leach DR (2001) Recombination at double-strand breaks and DNA ends: conserved mechanisms from phage to humans. Mol Cell 8: 1163–1174
2. Michel B, Grompone G, Flores MJ, Bidnenko V (2004) Multiple pathways process stalled replication forks. Proc Natl Acad Sci U S A 101: 12783–12788
3. Liu J, Ehmsen KT, Heyer WD, Morrical SW (2011) Presynaptic filament dynamics in homologous recombination and DNA repair. Crit Rev Biochem Mol Biol 46: 240–270