In this section
@article{Zeden:2023:10.1101/pdb.prot107946,
author = {Zeden, MS and Schuster, CF and Gründling, A},
doi = {10.1101/pdb.prot107946},
journal = {Cold Spring Harb Protoc},
title = {Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli.},
url = {http://dx.doi.org/10.1101/pdb.prot107946},
volume = {2023},
year = {2023}
}
TY - JOUR
AB - We present a protocol for the generation of a gene-deletion allelic-exchange plasmid and its recovery in Escherichia coli for the purpose of constructing an in-frame gene deletion in Staphylococcus aureus Here, we present detailed methodologies for (i) the primer design (using the S. aureus tagO gene as our specific example); (ii) PCR amplification of the required gene fragments; (iii) preparation of the cloning vector (using the S. aureus allelic-exchange vector pIMAY as an example); (iv) the Gibson assembly cloning method; (v) introduction of the plasmid into E. coli; (vi) confirmation of the plasmid insert in E. coli by colony PCR; and, finally, (vii) confirmation of the insert by sequencing. We also consider the long-term storage of the E. coli strains containing the desired plasmid.
AU - Zeden,MS
AU - Schuster,CF
AU - Gründling,A
DO - 10.1101/pdb.prot107946
PY - 2023///
TI - Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli.
T2 - Cold Spring Harb Protoc
UR - http://dx.doi.org/10.1101/pdb.prot107946
UR - https://www.ncbi.nlm.nih.gov/pubmed/37117030
VL - 2023
ER -