RCA of Circular Probe
Chia-Hsien Shih
Published: 2021-10-18 DOI: 10.17504/protocols.io.by5fpy3n
Abstract
This protocol is to verify that circular probe can conduct RCA reaction.
Steps
Preparation
1.
Dilute the 1µM Circular probe into 100nM
1.1.
Add 5µL of 1µM circular probe
1.2.
Add 45µL of RNase-free water
1.3.
Spin down after vortex
Protocol
2.
Add 17.4µL RNase-free water into a eppendorf
3.
Add 3µL of 10X phi29 polymerase reaction buffer
4.
Add 3µL of 100nM circular probe
5.
Add 3µL of 100nM miRNA
6.
Add 3µL of 2mM dNTPs
7.
Add 0.6µL of 10U/µl Phi29 polymerase
8.
Pipetting the reaction solution
9.
Incubate for 2h 0m 0s at 31Room temperature
10.
Add 1.5µL of 20X evagreen dye
11.
Add 1.5µL of 0.5M EDTA buffer to suspend the reaction
Measuring
12.
Load 20µL of reaction solution into 384-well plate
13.
Measure the fluorescence excitation and emission intensity
