Oct 18, 2021
RCA of Circular Probe
- 1Chung Shan Medical University
- CSMU_Taiwan
Protocol Citation: Chia-Hsien Shih 2021. RCA of Circular Probe. protocols.io https://dx.doi.org/10.17504/protocols.io.by5fpy3n
License: This is an open access protocol 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
Protocol status: Working
We use this protocol and it’s working
Created: October 17, 2021
Last Modified: October 18, 2021
Protocol Integer ID: 54151
Abstract
This protocol is to verify that circular probe can conduct RCA reaction.
Preparation
Preparation
Dilute the 1µM Circular probe into 100nM
Add 5 µL of 1µM circular probe
Add 45 µL of RNase-free water
Spin down after vortex
Protocol
Protocol
Add 17.4 µL RNase-free water into a eppendorf
Add 3 µL of 10X phi29 polymerase reaction buffer
Add 3 µL of 100nM circular probe
Add 3 µL of 100nM miRNA
Add 3 µL of 2mM dNTPs
Add 0.6 µL of 10U/µl Phi29 polymerase
Pipetting the reaction solution
Incubate for 02:00:00 at Room temperature
2h
Add 1.5 µL of 20X evagreen dye
Add 1.5 µL of 0.5M EDTA buffer to suspend the reaction
Measuring
Measuring
Load 20 µL of reaction solution into 384-well plate
Measure the fluorescence excitation and emission intensity