Jul 07, 2022

Public workspaceTransform pCas9

DOI
dx.doi.org/10.17504/protocols.io.eq2lynbopvx9/v1
  • 1Xi'an Jiaotong-Liverpool University
Qiaowa Gong
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DOI: dx.doi.org/10.17504/protocols.io.eq2lynbopvx9/v1
Protocol Citation: Qiaowa Gong 2022. Transform pCas9. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lynbopvx9/v1
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: July 07, 2022
Last Modified: July 07, 2022
Protocol Integer ID: 66195
Keywords: transformation, Crispr/Cas9, pCas9
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Abstract
this protocol is aimed to provide instructions of transforming pCas9, which is a key step of knock-out gene by Crispr/Cas9
Materials
competent MG1655, pCas9 plasmids, LB agar mediums (contain chloramphenicol), LB broth
Mix Amount50 µL of competent MG1655 with Amount10 µL of pCas9


Keep the reaction mix on ice forDuration00:30:00 minutes

30m
Then, transfer the mix to the water bath set at Temperature42 °C for Duration00:00:45


45s
Next, keep the mix on the ice for Duration00:02:00

2m
Add Amount940 µL LB broth and incubate the cells at Temperature37 °C with vigorous shaking (200 rpm) for Duration01:00:00

1h
Plate the cells on LB-agar plates containing chloramphenicol
Incubate at Temperature37 °C DurationOvernight