Oct 16, 2023
Plasmid construction
- 1Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Protocol Citation: Minghao Chen, Xuefeng Ren 2023. Plasmid construction. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x3b5lqe/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: October 16, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 89372
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
Abstract
plasmid construction
Amplify the insert gene fragment by PCR with primers including 21 nt of overlapping sequence with the target gene.
Linearize the backbone with restriction enzymes (NEB).
Treat linearized backbone with quick CIP (NEB).
Run PCR products and linearized backbone in an agarose gel to confirm the size.
Purify the DNA from gel using a Gel extraction kit (Bio Basic).
Ligate the linearized backbone and the insert with the T4 ligase (NEB).
Transform the ligation products into home made competent cells.
Perform colony PCR to screen for colons that with inserted gene.
Sequence to verify that the inserted gene is correct.