Oct 18, 2021

Public workspaceRCA of Gotcha

DOI
dx.doi.org/10.17504/protocols.io.by53py8n
  • 1Chung Shan Medical University
  • CSMU_Taiwan
Chia-Hsien Shih
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DOI: dx.doi.org/10.17504/protocols.io.by53py8n
Protocol Citation: Chia-Hsien Shih 2021. RCA of Gotcha. protocols.io https://dx.doi.org/10.17504/protocols.io.by53py8n
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 18, 2021
Last Modified: October 18, 2021
Protocol Integer ID: 54171
Abstract
This protocol is to test that GotCha is functional and can works as designed.
Preparation
Preparation
Add Amount5 µL of GotCha(functional beads) into eppendorf

Centrifuge for Centrifigation15000 rpm, 00:05:00 and remove supernatant. Make sure that eppendorf should put on DynaMag when removing supernatant.

5m
Protocol
Protocol
Add Amount3 µL of 10X phi29 polymerase reaction buffer into eppendorf with GotCha

Add Amount20.4 µL RNase-free water

Add Amount3 µL of 100nM miRNA

Add Amount3 µL of 2mM dNTPs

Add Amount0.6 µL of 10U/ul phi29 polymerase

Pipetting to mix well
Incubate for Duration02:00:00 at TemperatureRoom temperature

2h
Add Amount1.5 µL of 20X evagreen dye

Add Amount1.5 µL of 0.5M EDTA buffer to suspend the reaction

Measuring
Measuring
Load Amount20 µL of reaction solution into 384-well plate

Measure the fluorescence excitation and emission intensity