Trouble isolating plasmid from Pseudomonas aeruginosa - (Nov/13/2013 )

Hello,

I may have asked this before but I haven't gotten an answer and it is something that is very important for my research.

I am attempting to obtain a culture of Pseudomonas aeruginosa (PA14) electroporated with a recombinant expression vector.

I have gotten a few colonies to arise on antibiotic plates which I assumed were successful transformants however when I tried to miniprep the culture the gel shows a smear. I also tried colony pcr and that also resulted in a smear.

I want to be able to confirm that these cells indeed do have the expression vector but I am not sure how to do this if minipreps and colony PCR don't work. Does anyone have any helpful suggestions or any thoughts on why the minipreps appear as a smear (which appear even after using a brand new kit)?

Any lead would be greatly appreciated.

Thank you,

Ted

Can you miniprep another plasmid (perhaps an E. coli plasmid) and run it on a gel? How does your marker lane look on the gel?

Yes, along with the PA14 miniprep I also miniprep E.coli containing the same plasmid. When run together on the gel, the PA14 lane was smeared but the E.coli lane showed discrete bands and so did the marker lane...

A labmate suggested I do a rough plasmid extraction via smearing the PA14 culture on an epp tube, boiling for 10 minutes, centrifuging then extracting the supernatant. I am going to try this for now since I don't seem to have any other option...

_{Tihong, do you have any luck in getting the plasmid from Pseudomonas aeruginosa? What method or kit did you use to do the preps? Could you show us your gel? How intense was the smear? I assume that you had RNase A in the cell suspension buffer and the smear wasn't RNA. Maybe the plasmid wasn't replicate well in PA, or maybe PA has strong nucleases, or maybe your alkaline lysis was't potent enough to lyse the PA? If boiling won't help, I would like to suggest AquaPlasmid, which now uses a novel non-alkaline lysis mechanism to prevent pDNA denaturation during cell lysis and increase pDNA yield.}