I have to electroporate pMAD cloned with an insert of 7.4 kb in my S. aureus isolate. When I electroporated in RN4220 I had no problems but when I put it in my isolate I just recover few colonies and they have the empty plasmid. Has anyone had this problem?

  • $\begingroup$ How do you know they're empty? Are you doing blue-white screening or actually isolating the plasmid? $\endgroup$ – canadianer Apr 13 '17 at 14:19
  • $\begingroup$ I ask because if your isolate contains an endogenous beta-galactosidase gene, blue-white screening will not work. $\endgroup$ – canadianer Apr 13 '17 at 14:33
  • $\begingroup$ I have the primers to amplifie the region of the multiple cloning sites and it gives me the fragment of an empty plasmid. Also I cut the plasmid with the same enzymes of the cloning and it gives me no fragment of the insert size, only the empty plasmid. When I do the same thing with the plasmids from RN4220, I have the good fragments... $\endgroup$ – Marina Apr 13 '17 at 15:16
  • $\begingroup$ A qualified guess is that either your insert is somehow toxic to the cells or you have a contamination issue with the empty plasmids far outweighing your plasmid with the insert concentration wise. $\endgroup$ – Jeppe Nielsen Apr 26 '17 at 0:48

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