Dear Elisa,

Such a smear might be caused by DNAse degradation. Maybe one of the added components is contamined by a DNAse (e.g. the used water or buffer). You might test the individual compoments with some DNA sample to exclude a contamination (preferably in the presence of Mg2+).

Best,

Jan

I think that your DNA prep is primarily genomic DNA and not plasmid. The size of the uncut DNA is much too large for most normal plasmids and the smear starts at a high MW as well. If you had pure plasmid but DNAse contamination, then the smear would begin downward from the size of the plasmid.

I really thanks both of you.

Maybe I am wrong, but if it was DNase degradation also the 500bp fragment would be degraded or not Jan-Hendrik Heilers ?

I also believe it can be genomic DNA, so where do you think I was wrong? Michael J. Benedik

Hard to determine what went wrong. Sometimes if you are too vigorous in agitation when doing a plasmid prep you release genomic DNA.

Thank you!!

Dear Elisa Milano and Michael J. Benedik ,

I agree with Michael that it is probably gDNA. But you can clearly see the 500 bp in lane 4, which shows that the plasmid seems to be present in decent amounts in your sample. However, in lane 2 you only see the DNA on top of the gel. In turn I thought, that you are maybe working with a quite large plasmid?

A DNAse contamination might not be very high, so degradation occurs slowly over time, hence your product might be still visible.

If you got new results of another prep I'm curious to hear whether you could solve the problem.

Best,

Jan

Thank you very much, I'll let you know when I will repeat the prep (I will also try to change buffers)...

Best,

Elisa