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. 2020 Sep 29;10:15969. doi: 10.1038/s41598-020-72724-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Selection of candidate genes in the HSP23^−/− background at non-permissive temperatures (A) Schematic drawing of the transgene selection procedure. Six over expression plasmids harbouring LdBPK_351030 to LdBPK_351080 were co-transfected into HSP23^−/− null mutants and selected either at LT with the selective antibiotic, or at HT, generating HSP23^−/− [Mix]-LT or HSP23^−/− [Mix]-HT cell populations. After selection, plasmids were isolated from the Leishmania populations by Phenol–Chloroform extraction. (B) Analytical PCR using gene-specific primers to test for the presence of candidate transgenes in the recovered plasmids. Transgene plasmid DNA was used as positive control (( +) ctrl). PCR products were analysed by agarose gel electrophoresis and ethidium bromide staining; the position of corresponding DNA size marker bands is shown on the right. The left panel shows the PCR products after LT selection, the right panel shows the results after HT selection.