Agrobacterium miniprep problem - (Aug/11/2008 )

Hi all,
Recently I did a Agrobacterium miniprep using Qiagen miniprep spin kit. I use the user developed protocol: make 15ml Agrobacterium culture, then spin down the Agro. to collect them in two seperate falcon tubes. Then add 250ul P1 buffer to transfer to two 1.5ml eppendorf tubes. add 250ul P2, 350ul N3, spin for 10mins, and ran the solution through column twice. Then when I ran a Agrose gel, I found smear from 500bp to larger than 24kb.The concentration is about 300ng/ul, which is very high, 260/280 is about 1.80. I did the same miniprep before and it worked. The concentration is about 30ng/ul. Anyone can tell me what was the problem of the smear?

This sounds like nuclease contamination to me.

Also, to improve all aspects of the prep, make sure you use very cold resuspension buffer and very very cold neutralization bufferto eliminate salt precipitation

You do realize that minipreps only isolate plasmid DNA??

Genomic DNA cannot be isolated with a mini-prep. You would have to use a hirt prep for that.

are you using a plasmid that is 24Kb??

Thank you all, I think I figure out the problem.
Previously, I make the Agro. culture, and the cells aggregate. So after spin down the cells ,I add 250 ul P1 buffer to the cells. When I transfer them to 1.5ml tube, I used a pestle to grind cells. That may release the gDNA to the buffer. So this time I did not do this, the smear is there but very weak. And my plasmid can be seen very clearly. The concentration is back to 50ng/ul---very normal.
Thanks