Agrobacterium Ti plasmid preparation

jaydub jaydub at mindless.com
Thu Dec 3 00:23:34 EST 1998


Nancy Terryn wrote:

> Hi netfriends,
>
> well I guess the title says it all. I am trying to get relative pure
> Agrobacterium Ti plasmid out of strain c58. I have tried the old Hirsh
> protocol from 1980, but with low succes. We have used pronase to get a
> better lysis of the cells, but it does not seem to help much. I also
> tried a protocol from MIT to purify large BAC clones, but that didn't
> work either. Any suggestions, references etc would be very wellcome...
> Thanks!
> --
> ==================================================================
> Nancy Terryn
> DEPARTMENT OF GENETICS                         Fax:32 (0)9 2645349
> UNIVERSITY OF GENT, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
> Vlaams Instituut voor Biotechnologie                           VIB
> mailto:nater at gengenp.rug.ac.be             http://spider.rug.ac.be
> ==================================================================

Hi Nancy:

I have two suggestions, one of which I hope will help.  I have digested
Agrobacterium A348 to isolate chromosomal DNA successfully by adding
Pronase E (previously self-digested at 37 C for 2 hours) to a pellet
suspension in TE, after washing once with 0.85-percent NaCl and once with
TES (10 mM Tris-Cl, 25 mM Na-EDTA, 150 mM NaCl, pH 8.0).

The second suggestion is to look at a protocol developed by C.I. Kado and
S.T. Liu in the Journal of Bacteriology, Vol. 145, page 1365-1373 (March
1981).  The article is titled, "Rapid Procedure for Detection and
Isolation of Large and Small Plasmids".  Their procedure looks promising,
and was used by I Gede Putu Wirawan et. al. in Journal of Bacteriology,
Vol. 175, page 3208-3212 (May 1993).

I hope one of these will help.

    Jon White









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