schaeff at lmb.uni-muenchen.de
Thu Mar 14 07:24:10 EST 1996
Here are the answers to my inquiry about cosmid vectors suitable for
subcloning YACs other than pBIC20 and pLZO3.
I can provide pOCA18 or pOCA28 if you need them.
Inserts are very stable in pOCA. It was built for stability. See: Nucleic
Acids Research 16:10765 (1988) for an analysis of the stability of an
arabidopsis library in pOCA18.
Cloning capacity of pOCA18 15-20 kb.
pOCA18 & 28 carry kanamycin plant markers (NOS:NPTII gene). This marker works
well for my lab with arabidopsis, tomato, tob.
pOCA18 has the disadvantage that the bacterial marker is tetracycline
resistance. The A136 chromosomal background (most nopaline strains) give rise
to spontanious resistance at a frequency of 10-5 (see the NAR paper). This can
be worked around but I made pOCA28 to solve this problem. The marker in pOCA28
I don't like poca, because it is high copy, and that seems like a fundamentally
bad idea to me. Brian Staskawicz uses a vector made by Jonathan Jones
called pCLD04541, which is Tc in bacteria, and Km in plants. They have
made several libraries from YACs now (they make a library from the whole yeast
strain that carries the YAC), and so far have not found any gaps in their
libraries. I also made a library in this vector, and the person who screened
it said it was complete for the YAC.
The only trouble is that the Agrobacterium strain normally used, GV3101,
mutates to Tc resistance at high frequency. The story from Brian's lab is
that to select Agro carrying the cosmids, select with Tc at 2ug/ml, and the
small colonies are the right ones. Large colonies, or colonies that appear
at Tc concetrations higher than 2, are spontaneous mutants. This sounds nasty
to me, but apparently they get it to work just fine. I havn't tried the
Agro transformation part myself, yet.
The vector is proprietary, you have to get it from Jonathan, but it's no
problem. He also has somme derivatives for Basta, but as of six months ago,
these had not actually been used for Arabidpsis trasformation, so I was
hesitant to use them They may have the same problem with Tc, I don't know.
If you decide to use pCLD04541, it might be worthwhile chatting with
Doug Dahlbeck in Brian's lab, to make sure you have the right story about
the Tc resistance.
I think I have POCA28 somewhere, if you want it.
Jane (Glazebrook, MGH, Harvard)
We have used the pOCA vectors and cosmid library prepared by Neil Olszewski
to clone a selection of Arabidopsis genes. He can be reached at the
Dept. of Plant Biology, University of Minnesota, on FON 612 625 3129 or FAX
612 625 1738
Dept. of Biological Sciences
University of Warwick
* Dr. Tony Schaeffner
* Institut f. Biochemie
* Wuermtalstrasse 221
* D-81375 Muenchen, Germany
* Tel. (0)89-7401-7311
* Fax. (0)89-7401-7314
* email: schaeff at lmb.uni-muenchen.de
More information about the Arab-gen