Miniprep DNA digestion smear - (Mar/16/2008 )

I would never do a digestion in such low volume. Try doing it at 50 ul final volume, keeping the DNA and enzyme amounts the same. What is the concentration of the DNA? 1 ug is a good amount in a 50 ul reaction.
You didn't say what kind of miniprep you are using.

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I usually do digestion in this volume without problem. I try to clone my cDNA full length+linker: (2.3kb) in pINDEX vector(13kb) then intorduce into A.tumefaciens.
later i used the same vector and same conditions to make a knock down construct + vector without problem

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your cells should be fine, and I think your digest set up is fine too (I usually use about the same mix, only doubled). I suspect that you are introducing DNase somewhere - maybe your prep just isn't clean enough this time around for some reason. Can you see your DNA if you just run it out on the gel without adding anything to it? Does it go away if you add just buffer and incubate it for a while (no enzymes)? If yes, then it would suggest that you have some DNase in your prep that starts to chew up your DNA as soon as you give it the right conditions. You can try to phenol extract the DNA to purify it further or try another miniprep protocol.

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hello again
I made another miniprep DNA, and I get the same result (smear). I made phenol/chlorophorm extraction, then chlorophorm extraction and ethanol precipitation.
and after digestion I get smear!!!

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can you run out your raw miniprep DNA on a gel and show us a picture.

Have you tried changing your buffers?

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Hello everybody

Thank you all for your reply.
The solution of my problem was very simple and easy than expected. The smear was not due to nuclease, enzymes, buffers, water,... but ..... the hight voltage I used during electrophoresis wich causes smear!!! So I reduced voltage and I get a nice band in all my miniprepDNA samples.

Have a nice day

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