Nov 20, 2021
Basic Rolling Circle Amplification (RCA) Protocol (Thermo phi29 polymerase)
- 1Universitas Gadjah Mada
- Laboratory of Virology, Faculty of Agriculture, Universitas Gadjah Mada
Document Citation: argawikandito 2021. Basic Rolling Circle Amplification (RCA) Protocol (Thermo phi29 polymerase). protocols.io https://dx.doi.org/10.17504/protocols.io.bz8np9ve
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: November 20, 2021
Last Modified: November 20, 2021
Document Integer ID: 55278
Abstract
-
Basic Rolling Circle Amplification (RCA) Protocol (Thermo phi29 polymerase)
Aim: RCA is useful method to amplify circular DNA/RNA. By principle, RCA conducted using phi29 polymerase to obtain copies of DNA/RNA target in concatemeric form. phi29 works on isothermal reaction at 36-38⁰C.
Materials :
gDNA
10U phi29 polymerase (Thermo Scientific)
10X reaction buffer (Thermo Scientific)
10mM dNTP mix (Thermo Scientific)
100µM n-hexamer primer (or specific primers to obtain specific target) (Thermo Scientific)
Nuclease-free water (Thermo Scientific)
Thermal cycler
Micropippetes
Tubes 0.2µL
Steps :
DNA Denaturation
- Prep denaturation mixture on 0.2µLtube :
| Reagent | Volume (µL) | |
| 10X reaction buffer | 0.5 | |
| 100µM n-hexamer primer | 1 | |
| Nuclease-free water | 2-3 | |
| gDNA | 0.5-1 | |
| Total | 5 | |
| NOTE : you can change 100µM n-hexamer primer with specific primer to obtain more specific result |
- Incubate 95⁰C for 3 minutes
- Put on ice immediately for 3-5 minutes
Rolling Circle Amplification
- Prep amplification mixture
| Reagent | Volume (µL) | |
| Denaturation mixture | 5 | |
| 10X reaction buffer | 1.5 | |
| 10mM dNTP mix | 2 | |
| 10U phi29 polymerase | 1 | |
| Nuclease-free water | 10.5 | |
| Total | 20 | |
| NOTE : you can also add 100µM n-hexamer primer to increase RCA yield |
- Incubate 36⁰C for 18 hours
- Inactivate reaction at 65⁰C for 10 minutes