Hi all,
I want to do electroporation experiments with plasmidic extract (natural plasmids) from Klesbiella pneumoniae strains that seem resistant to carbapenem antibiotics to prove that the plasmids are responsible for the resistance.I am confused about the choice of the strain I should make competent for the electroporation. What features should the cells have to be suitable for the electroporation? Should they be devoid of their own plasmids? should they be sensitive to the antibiotics used to select the transformants?I read some papers where the authors make sensitive Klebsiella pneumoniae strains competent. But all of my Klebsiella strains show plasmids and they are resistant..I have chemically competent cells in lab, but I am aware that chemically competent cells must not be used for electroporation. So, I was wondering what normally people do , how they select these cells to make competent and how you can be sure that these cells are ok for your electroporation (e.g how can you be sure that these cells express the genes carried by your plasmid)..Please, give me some suggestions/explanations. I am quite confused.
I haven't any expriencewith k.pneu , but let go ahead step by step.
If you want transfer a k.pneu plasmid to another strain (please correct me if not your aim) you can easily do that with chemical transfer such cacl2 and heat shock. But if this trafnsfer with two different species its difficult and you should check , copy no ,antibiotic resistant cassetcassets and so on...
I suggest you chemical transformation is good. Durring transformation optimum temp(37) and 18h shake is nessesorry
For step two if you just want to replicat your aim plasmid you can used DH5 alpha competent for that.
After your transformed bacteria in antibiotic added agar (such LB agar) was grown , you can extracted plasmid (mini,midi,maxipreb) and with restriction mapping appproved.
Your selection medium is should created based on your plasmid antibiotic casset with optimum concentration
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