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Smear after digestion of a plasmid from a Dam- strain


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#1 Schevchen

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Posted 05 November 2012 - 02:35 AM

Hej

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?

thanks!

#2 phage434

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Posted 05 November 2012 - 05:00 AM

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.




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