3 replies to this topic

[mnqcljsm]

Hi everyone.

Im cutting my insert out of my vector to ensure that my plasmid is what i
think it is. My insert is flanked by 2 different restriction sites. I'm a lazy
student so i set up a quick double digest using an NEB buffer in
which each enzyme is only 50% (as opposed to doing a sequential). On my
gel i have a nice fine band for my cut vector but one daddy of a smeared band where my insert should be, although the smear is concentrated into a recognisable band at the appropriate position. In short, should i be worried that my plasmid is dodgy or does this simply represent non-specific munching by one of my enzymes in a sub-optimal buffer.

Any ideas?

Jon

[loustau]

Hi,

I would think your plasmid is OK.  The smear could either be some star activity of one of the enzymes, or some contamination from a crude DNA prep.  

If you want to be sure, I suggest you either do a sequential digest with the appropriate buffers, or run a non-digested sample on the next lane - depending on how lazy you really are   .

Also, what enzymes/buffer/sizes are we talking about?

Good luck!

[mnqcljsm]

We're talking Not1/Pme1, both 50% in NEB2 + an 8Kb vector (3kb insert).
Thanks for the help,
Jon

Edited by mnqcljsm, 18 March 2005 - 02:55 AM.

[george@CASE]

I think it's star activity. One of the enzymes might not be too happy at low ionic conc.

Solution: Don't be lazy *peace* . Do it once and do it right. Saves you time in the long run.