The plasmids pGEM-T Easy (Promega corporation Madison, WI, USA), pDE22 [24 (link)] and DNA vaccine vector pUMVC6 (Aldevron, Fargo, North Dakota, USA) were propagated in Escherichia coli strain TOP10 (ATCC, Manassas, VA, USA), and the plasmid pGESTH-1) [28 ] was propagated in E. coli strain BL-21 (Novagen, Madison, WI, USA), as described previously [29 (link), 30 (link)]. The shuttle vector pDE22 was used for the expression of pe35, esxa, esxb, rv2346c, rv2347c, rv3619c and rv3620c genes in M. smegmatis and M. vaccae, as described previously [24 (link), 31 (link)]. Genomic DNA isolated from M. tuberculosis H37Rv (obtained from the American Type Culture Collection, (Rockville, MD, USA) served as the source for the amplification and subsequent cloning of the genes, as previously described [28 , 30 (link)]. All DNA manipulations, restriction enzyme digestions and bacterial cell transformations with the plasmids were performed according to previously described procedures [28 –32 (link)].
Pumvc6
Manufactured by Aldevron
Sourced in United States
The PUMVC6 is a laboratory equipment product manufactured by Aldevron. It is a pump used for transferring and dispensing liquids in various lab applications. The core function of the PUMVC6 is to provide precise and controlled liquid handling capabilities for researchers and scientists. No further details on the intended use or specific applications of this product are available.
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2 protocols using pumvc6
1
Plasmid Propagation and Gene Expression
2
Cloning and Expression of M. tuberculosis rv3619c
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Genomic DNA isolated from M. tuberculosis H37Rv (obtained from the American Type Culture Collection, Rockville, MD, USA) served as the source for the amplification and subsequent cloning of the rv3619c gene, as previously described [9 (link)]. In brief, DNA corresponding to the rv3619c gene was amplified by PCR using genomic DNA isolated from M. tuberculosis and gene-specific primers (ThermoFisher Scientific, Ulm, Germany) (online suppl. Table S1 ; see www.karger.com/doi/10.1159/000525136 for all online suppl. material) and then ligated to appropriated cloning and expression vectors i.e., expression vector pGES-TH1 [9 (link)], shuttle vector pDE22 [12 (link)], and DNA plasmid vector pUMVC6 [12 (link)] (Aldevron, Fargo, ND, USA), as described previously [9 (link), 11 (link), 12 (link)] for expression in E. coli BL-21 (Novagen, Madison, WI, USA), M. smegmatis (ATCC 700084/mc(2)155, ATCC, Manassas, VA, USA), and DNA plasmid vector pUMVC6, respectively.
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