Oct 17, 2021
Hybridization and Immobilization
- Chia-Hsien Shih1
- 1Chung Shan Medical University
- CSMU_Taiwan
Protocol Citation: Chia-Hsien Shih 2021. Hybridization and Immobilization. protocols.io https://dx.doi.org/10.17504/protocols.io.by5gpy3w
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 16, 2021
Last Modified: October 17, 2021
Protocol Integer ID: 54152
Keywords: immobilization probe, immobilization this protocol, immobilization, gotcha with circular probe, circular probe, magnetic bead, probe, hybridization
Abstract
This protocol is to assemble GotCha with circular probe, immobilization probe and magnetic beads.
Troubleshooting
Preparation
Dilute immobilization probe into 10µM
Protocol of Hybridization
Add 4.5 µL of 1µM circular probe
Add 9 µL of 10X 7.4 PBS buffer
Add 0.9 µL of 10µM immobilization probe
Add 75.6 µL RNase-free water
Vortex slightly and spin down
Heated to 90 °C for 00:00:30 then cool down to Room temperature and keep for 00:30:00
30m 30s
Protocol of Immobilization
36m
Take 22.5 µL of 10mg/ml magnetic beads into a eppendorf
Vortex then spin down in Centrifuge 15000 rpm, 00:02:00
2m
Carefully take the eppendorf onto the DynaMag and wait for 00:01:00 to collect the beads
1m
Wash beads with 1X PBS for three times. Make sure that eppendorf should put on DynaMag when removing supernatant.
Add 90 µL of hybridization solution into the eppendorf with dry beads
Incubate at Room temperature for 00:30:00 , then spindown in Centrifuge15000 rpm, 00:02:00
32m
Carefully take the eppendorf onto the DynaMag and wait for 00:01:00 to collect the beads
1m
Wash beads with 1X PBS for three times. Make sure that eppendorf should put on DynaMag when removing supernatant.
Preserve the functional beads in 90 µL 1X PBS