Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Smear after digestion of a plasmid from a Dam- strain - (Nov/05/2012 )


I purified a plasmid from a dam- strain (GM3189) and restricted it with SalI and PstI (fast enzyme digestion). If I load it on a gel I can only see smear. Using exactly the same enzymes, buffers etc. for another plasmid, but purified from XL1blue, i can see a clear band.
I checked my buffer and it seems to be the reason of the smear. But using it afterwards for other digestions it works fine.

So what do I need to change for this dam- strain?



This sounds like endonuclease contamination of your prepared DNA. Likely your GM3189 cells are endA+. For these strains, it is very important to purify the DNA extensively prior to digestion. I'd recommend a phenol-chloroform purification of your DNA. Best would be to switch to a dam- endA- strain of E. coli to avoid the problem.