Hello,
I am Monica Bodogai, graduate student in Hungary.
It seems that i have a problem with a mammalian cloning vector from BD clontech. I someone use it, i would be very greatful to any kind of information.
I received the plasmid from an assistent and i am not sure if she didn't mix the strain. The EcoRV is a unique site on the plasmid. That's what it is written in the manual. But sometimes I obtain 3 bands instead of one: 5200bp (the normal one) plus one of 3000 and one of 2000 like as the enzyme cuts in 2 places. I tried to clean the clone, but after a while even the clone which seemed good does the same thing.
Does it happens to other people too or I should get the plasmid from someone else, or i should try with something else?

EcoRV can have star activity with a large excess of enzyme. These factors can also contribute to star activity.
prolonged incubation time or a large excess of enzyme with respect to DNA
high glycerol concentration in the reaction mixture or the presence of other organic solvents, such as ethanol or dimethyl sulfoxide
low ionic strength or high pH values in the reaction buffer
substitution of cofactor Mg2+ with other divalent cation (such as Mn2+ or Co2+).

All the enzymes what i am using are from Fermentas and in the catalog they write all the effects which you can get when you work with a certain enzyme. And for EcoRV this is not there, that's why i never thought of this.

Not all manufacturers list EcoRV as having start activity it is true, but there are examples in the literature of it having this activity when used in excess

Fixing star activity

Use as few units as possible to get a complete digestion. This avoids overdigestion and reduces the final glycerol concentration in the reaction.

Make sure the reaction is free of any organic solvents such as alcohols which might be present in the DNA preparation.

Raise the ionic strength of the reaction buffer to 100-150 mM (provided the enzyme is not inhibited by high salt).

Lower the pH of the reaction buffer to pH 7.0.

Use Mg++ as the divalent cation.

Alternative explainations may include things such as the strain of bacteria that you use can also effect the cutting patterns. If you do not purify the plasmids well enough in some cases there may well be trace nuclease activity. This generally applies when using endA+ strains like HB101 or JM series bacteria.
Here's a cute refernce to explain things

http://www.promega.com/pnotes/53/5015j6/5015j6_core.pdf

If you are using XL-1 blue or DH5alpha this is unlikely to be such a problem.

Alternatively if you really think you have a mixed sample of plasmid, find a different enzyme that is unique. When you cut with that you should see the appropriate sized linear band on your gel. Even if you have extra bands cut out the one that is the expected size, purify it then religate it and then transform into E. coli. Sequence a small section to confirm identity if the plasmid.

Good luck

The cloning worked and the cells are already transfected:) Thank you very much for all your replies.