Agro competent stock cells

Moez Torki moez at POST.AGRI.PREF.IBARAKI.JP
Fri Sep 10 23:27:16 EST 1999


Hi again,

I prepared competent cells as http://ag.arizona.edu/~byeongha/agrotran.htm,
added DMSO 7% and stored them at -80 C. They seem to work fine, but LBA4404
are definitely more competent : 50 to 100 colonies/2 to 4 colonies for
LBA4404 versus C58 when transformed with 0,5 microg pBI. Even though, that
was sufficient for my purpose :-)

Moez


>moez torki wrote:
>
>> Hi all,
>>
>> I'm planning to transform Agro (C58 & LBA4404) with a binary vector. I
>> found a simple heat-schok protocol on
>> http://ag.arizona.edu/~byeongha/agrotran.htm (thanks to Lee Byeong-ha, :-).
>> The protocol recommend freezing the cells with liquid nitrogen, even they
>> are resuspended only in an aqueous sln (CaCl2 20 mM). I'm not realy
>> familiar with Agro strains, but when making E. Coli or pahges stocks to be
>> stored at -80 C, I always add Glycerol 15-20 % or DMSO 0,7 %.
>> Any hint ? Thanks.
>>
>> Moez Torki
>>
>> _________________________________________
>> Moez Torki, Ph.D.
>> Plant Biotechnology Institute,
>> Ibaraki Agricultural Center, 3165-1 Ago,
>> Iwama, Nishi-Ibaraki 319-0292, Japan
>>
>> +81-299-45-8330 (phone)
>> +81-299-45-8351 (fax)
>> torki at nocs.tsukuba-noc.affrc.go.jp
>> http://stafellow.jst.go.jp/life/today011/torki.html
>> _________________________________________
>> The World : once thought to be flat, then proved to be round. Now it's
>> quite definitely Web shaped....
>
>You are not freezing the cells to store them, you are freezing to make them
>competent. I do not recall that this procedure allows storage of the competent
>Agro and I generally use them right away. But if you would like something a
>lot more efficient and just as quick, electroporation is the way to transform
>Agro. You can simple scrape some off a plate (about 200 ul) and wash 5 times
>in cold sterile 10% glycerol and electroporate as you would for E. coli. You
>can also store the cells at -80 oC. Later,
>
>--
>  ------------------------------------------------------------------------
>
>Dr. Michael Cooley
>Waksman Institute
>Rutgers University
>190 Frelinghuysen Rd.
>Piscataway, NJ 08854
>
>732-445-3802
>FAX 732-445-5735
>cooley at waksman.rutgers.edu
>





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