Problems isolating plasmids from E.coli JM110

Sergio sergioal at bbm1.ucm.es
Mon Mar 5 09:39:49 EST 2001


Have you checked whether the coli is actually harbouring the plasmid?

Try a colony miniprep to verify you have enough plasmid before performing the
miniprep, although this technique doesn't work very well with low copy plasmids.

You could also check the plasmid integrity before the restriction...

Hope this helps

Sergio

Trond Erik Vee Aune wrote:

> Hi
>
> I am having problems with isolating plasmids from the dam negative
> E.coli strain JM110. I have read that it has a endonuclease that cleaves
> double stranded DNA and that this is the reason for the problems. Still
> I manage to isolate some of my plasmids, but not the others. I have
> tried SV Wizard miniprep (column) and ordinary isopropanol-method. After
> isolation and digestion with restriction enzymes I can see no bands on
> the gel.
>
> All the plasmids are low-copy, but the ones I have trouble with have a
> copy-up mutation that makes the copy number about 10 times higher than
> the ones I have managed to isolate from.
>
> Any help would be appreciated!
>
> Trond Erik Vee Aune






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