RCA of Gotcha
Chia-Hsien Shih
Published: 2021-10-18 DOI: 10.17504/protocols.io.by53py8n
Abstract
This protocol is to test that GotCha is functional and can works as designed.
Steps
Preparation
1.
Add 5µL of GotCha(functional beads) into eppendorf
2.
Centrifuge for 15000rpm and remove supernatant. Make sure that eppendorf should put on DynaMag when removing supernatant.
Protocol
3.
Add 3µL of 10X phi29 polymerase reaction buffer into eppendorf with GotCha
4.
Add 20.4µL RNase-free water
5.
Add 3µL of 100nM miRNA
6.
Add 3µL of 2mM dNTPs
7.
Add 0.6µL of 10U/ul phi29 polymerase
8.
Pipetting to mix well
9.
Incubate for 2h 0m 0s at Room 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
