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Probelm with double digestion - (Feb/15/2013 )

Dear Friends,
I have plasmid DNA and i wanted to separate my vector and my insert. I tried EcoR1 and BamHI (From NEB) with NEB 2. And i tried 30 ul reaction and here below i am giving reaction volumes:

10x buffer 2ul
BSA: 0.2ul
Water: 7.3ul
BamHI:1 ul
pDNA: 8ul

O/N incubation. For quality of digestion i used 0.8% Agarose gel and i loaded my samples along with ladder. I got clear ladder and i got plain smear my samples.

Can anyone help me get out from this problme


pay attention to these points, try again and see what happens. most probably the result will be good (or easier troubleshooting possible)
1. at which temp did you digest o/n? 37°C is to much, you'll degrade your plasmid (BamHI has star-activity under certain conditions). 2 hours is enough.
2. how much plasmid did you use? in ng/uL, not only the volume.
3. why do you use buffer 2? BamHI comes with buffer 3, EcoRl has a unique. Use buffer 3.
4. did you precipitate your plasmid prior to digestion? smear may be a contamination.
5. how do the controls look like? single digest with each enzyme as well as the uncut?