I am trying to extract plasmids from clinical samples of P. aeruginosa. I used the methods of Birboim and Kieser and none showed bands in electrophoresis. The E. coli controls work well and quantification of DNA is very high (believe it is genomic DNA).I cleansed with ethanol, isopropanol and phenol-chloroform (separately) and not getting results. Could anyone help me?
Hi Natalia, it seems that your problem goes back to transfection conditions only since the cells are efficient and the purification of DNA was able to detect the genomic DNA.. so I think you need to make sure your bacterial cells are accepting the plasmid DNA you need to propagate and being transfected well. You did not mention about your transfection protocol so I can not imagine which step could be the red point !!!
See this link, it might be helpful
https://www.lifetechnologies.com/jp/ja/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html
Hi Natalia,
Have a look on the link below. Alternatively you can use the QIAGEN Large-Construct kit.
Article Multidrug-resistant Pseudomonas aeruginosa strains harbourin...
May do PCR using a specific primer to the plasmid to check if it went into Pseudo. genome by DNA recombination.
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