The Phi29 polymerase RCA (Rolling Ring Amplification) kit contains all the reagents needed to achieve sensitive and efficient rolling ring amplification (RCA) , an isothermal amplification method that continuously amplifies circular DNA by producing long repeat copies of circular sequences. The kit contains phi29 DNA polymerase, dNTPs and random primers for exonuclease resistance (containing phosphorothioate bonds), ensuring the versatility of amplified circular DNA sequences. The starting material can be purified circular DNA (single or double stranded) or use liquid medium culture, agar plate colonies and glycerol directly without additional DNA extraction. RCA products can be used directly in downstream applications such as DNA sequencing, cell free DNA enrichment, cell-free protein expression, and DNA biosensors.
1. Prepare the reaction without adding enzymes, as shown in the table below. Mix thoroughly by pipetting or vortexing, but it should be gentle. Centrifuge briefly to collect the solution to the bottom of the tube.
Component | Volume | Final Concentration |
5 X RCA Reaction Buffer | 4 μl | 1 X |
Exonuclease-Resistant Random Primers (500 µM) | 2 μl | 50 µM |
Deoxynucleotide (dNTP) Solution Mix (10 mM) | 2 μl | 1 mM |
Circular DNA Template | X µl | 0.01-50 ng |
RNase Free dH2O | Up to 18 μl | 1 X |
2. Incubate in a thermal circulator with the lid set at 100°C and incubate at 95°C for 3 minutes. Then allow the sample to cool to room temperature.
3. Place samples on ice and add 2 µl of phi29 DNA Polymerase to each sample. Mix thoroughly, and gently, by pipetting or vortexing. Centrifuge briefly to collect solutions to the bottom of tubes.
4. Incubate in a thermocycler with the lid set at ≥ 75°C, incubate at 30°C for 2 hours, followed by 10 minutes at 65°C to inactivate the DNA polymerase. The RCA products can be kept overnight at 4°C or for long term storage at -20°C
1. RCA amplification is the amplification of circular templates using the characteristics of Phi29 DNA polymerase chain displacement activity and continuous synthesis ability. Therefore, its optimal substrate is circular ssDNA or dsDNA templates (C-DNA). In addition, the linear template (L-DNA) with larger molecular weight can also be continuously synthesized and amplified, such as genomic DNA, in which the amplification ratio is weaker than that of CDNA.
2. The RCA products may be viscous due to the high yield of high molecular weight DNA. Double dilution with nuclease-free water is recommended before using any downstream applications..
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