DNA restriction problems

Chen Linan chenln at sina.com
Sun Apr 16 11:29:16 EST 2000


Check your antibiotics and E.coli to determine if the former is inactived
and the later is contanminated. The plasmid may be the very little
one(without any relation with your expected plasmid except for the specific
antibiotics resistant sequence). I met the similar problem(little plasmid<2
kb) which wasted me two months to find the cause. Finally, the cause is the
inactived antibiotics(kanamycin) and small plasmid contamination. Hope it
give you a hint.

Chen Linan

Dept. of Anatomy
Beijing Medical Unversity



At 05:16 PM 2000/04/10 +0200, you wrote:
>Hello,
>
>I work with plant receptors that resemble bacterial two component
>receptors.
>I have problems with multiplying the plasmid containing this receptor
>sequence in E. coli.
>The plasmid runs very much too fast: it seems to be extremely
>supercoiled.
>It is not or hardly cut by any restriction enzyme.
>A very strange thing is that it seems to be cut better just after the
>miniprep, and almost not anymore after the DNA is stored in the freezer
>for a few days. DNA from midiprep inoculated with an o/n miniprep
>culture can not be cut either.
>
>Can this be due to heavy methylation ?(although EcoRI is not affected by
>dam/dcm methylation, but still does not cut the DNA) Maybe the heavy
>methylation makes the whole plasmid inaccessible for restriction
>enzymes??
>
>Who knows these kinds of problems or is able to give good advise on what
>to try next??
>
>Ivo Rieu
>Dept. Plant Cell Biology
>University of Nijmegen
>The Netherlands
>
>
>

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