Purfiying Unmethylated Plasmid - unmethylated plasmid (Mar/18/2009 )

Hi

I've been having some problems with plasmid DNA purified from DAM- Bugs. My Plasmid is ~40Kb (high copy number), the bugs are Stratagene JM110 DAM- Chemical comp and the prep kit I'm using is Qiagen spin columns.

I'm trying to do an XbaI partial digest and self-ligation. One XbaI site is methylation sensitive hence the DAM- bugs. I purify the DNA on the spin columns, including an extra step recomended by Qiagen for endonuclease rich bugs. After a 1 hour partial digest I run my DNA on a gel but I get a smear rather than clear bands. There is a very faint high molecluar weight band at one of the expected sizes which I gel extract and ligate overnight and get a few incorrect colonies. The uncut is also a bit smeary but not quite so bad.

The miniprep kit/buffers are OK as I did other preps at the some time and they were fine. The TAE buffer I used for the gel was fresh and the ladders were still nice and crisp. I've purified this plasmid using these kits before and it worked very well. The XbaI, buffer and dH2O used in the digest cut methylated DNA well and gives clear bands.

I'm starting to run out of ideas and this is the final ligation of my PhD so I really need it to work. Any suggestions on what might be going wrong and how I could get round it?

Thanks

The problem is the JM110 strain.

It is a EndA+ strain. The wild-type endA gene encodes an endonuclease, which nonspecifically degrades the DNA. Thus the quality of the plasmid DNA is rather poor as you have observed.

Could you use another dam- strain, which is also endA-? This option is the easiest and probably most reliable.

If not you have to inactivate the endA nuclease before it does too much damage. It means you have to work fast. Keep the lysed cell solution cold. And inactivate the endonuclease quickly, I use phenol-chloroform extraction.

You could also templiphi or PCR amplify your plasmid from a DAM+ strain, which would remove the methylation.

After you do your miniprep, heat inactivate the endo-ases by heating at 70 for 15 minutes before you start your digestion.