Call for help! Plasmid extraction problem - (Mar/06/2014 )

Hello everyone!
I’m having a plasmid extraction problem. I hope that you guys can give me some advices.
I have been doing plasmid extraction from two Klebsiella pneumonia isolates for 2 months and got nothing ! I used the S1-PFGE protocol to confirm the existence of plasmids in these 2 strains.
Strain A has 2 plasmids, about 55kb and 200kb. Strain B has 3 plasmids, about 40kb, 75~80kb and 185kb respectively.
At first I used the alkaline/SDS method to extract plasmids. But it didn’t work. It might because the alkaline lysis solution degenerate chromosome as well as the large plasmid. So I followed the SDS lysis method as described in Molecular Cloning: A Laboratory Manual and still got nothing. Then I used some extraction kits, still nothing.
I used these protocols to extract plasmid from an E.coli conjugant with a plasmid about 7kb, all of them worked well!

I read many research papers, it seems plasmid extraction is never a problem. I’m desperate to know your protocol about plasmid extraction especially when dealing with large plasmid.

You could try some commercial kits for BAC preparation, but a carefully done phenol chloroform or CTAB chlorform extraction would be as good or better. You will not likely be able to separate genomic DNA from your plasmids. To maintain the plasmid integrity, you need to be very gentle and use wide bore pipet tips, since the DNA is very physically fragile.

Another approach is to isolate the DNA from a gel, by cutting the band and either electroeluting into a dialysis bag, or treating with beta agarase. Depending on what you want to do with the DNA, your best bet may be to leave it in the gel and treat it in-situ.

Ordinary plasmid preparation kits rely on the dramatic difference in length and compactness of small plasmids to physically separate them from genomic DNA. It is not surprising you have no success in isolating these large plasmids with those kits.

thanks a lot ! i've ordered a BAC plasmid extraction kit, hope it will work.

by the way, our budget is a little tight, so i bought a cheap one. i wonder if there are some protocols for large plasmid extraction by hand.

You could also look at this paper:

http://precedings.nature.com/documents/1249/version/1

thaks a lot!