Oct 17, 2021
Version 2
Background control of Gotcha RCA V.2
This protocol is a draft, published without a DOI.
- 1Chung Shan Medical University
- CSMU_Taiwan
Protocol Citation: Chia-Hsien Shih 2021. Background control of Gotcha RCA. protocols.io https://protocols.io/view/background-control-of-gotcha-rca-by5jpy4nVersion created by Chia-Hsien Shih
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 17, 2021
Protocol Integer ID: 54155
Abstract
This protocol aims to verify that GotCha design works as expected.
Preparation
Preparation
Add 5 µL of functional beads(Gotcha) into eppendorf
Centrifuge for 15000 rpm, 00:05:00 and remove supernatant. Make sure that eppendorf should put on DynaMag when removing supernatant.
5m
Protocol of Control group without miRNA
Protocol of Control group without miRNA
Add 3 µL of 10X phi29 polymerase reaction buffer into eppendorf with functional beads
Add 23.4 µL RNase-free water
Add 3 µL of 2mM dNTPs
Add 0.6 µL of 10U/ul phi29 polymerase
Pipetting to mix well
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
Protocol of Control group without phi29 polymerase
Protocol of Control group without phi29 polymerase
Add 3 µL of 10X phi29 polymerase reaction buffer into eppendorf with functional beads
Add 21 µL RNase-free water
Add 3 µL of 2mM dNTPs
Add 3 µL of 100nM miRNA
Pipetting to mix well
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