krs1 at mrc-lmb.cam.ac.uk
Thu Jan 30 12:54:23 EST 1997
In article <krs1-2701971238510001 at nadgee.mrc-lmb.cam.ac.uk>,
krs1 at mrc-lmb.cam.ac.uk (Kirby Siemering) wrote:
A while ago I posted a question about hygromycin selection. Here is a
compilation of some of the answers I received. Thanks to all who helped me
> I would like to select transformants in vitro from vacuum infiltration
> using hygromycin. What concentration should I use and how good is the
> selection (I'm using pBIB-HYG in C24)?
From: dfzhou at vt.edu (Dafeng Zhou)
Hi, I have post the same question some times ago. Here is my impression of
the answers: the uppper limit of HYG is 30 microgram/ml, some people even
use as little as 15 ug/ml. Right now, I am using 30 ug/ml, looks pretty
good. The difference between Kanamycin and HYG is the kanamycin selection is
easy to seeb(non transformed plants bleached in about 2 week). However, the
HYG selection does have this advantage, the selection needs a little longer
(3 weeks) before you can tell the difference based on
selection(nontransformed no longer grow and no real leaves)
From: Dave Twell <twe at leicester.ac.uk>
I don't know your vector but we are using hyg selection in C24
transformants and find that 5
mg/l usually works if you are looking for a minimum, 10 is better and 15
still better but may
start to have an effect on resistant plants.
From: Nicole Bechtold <bechtold at oxygene.versailles.inra.fr>
I use 25 mg/l hygromycine and the selection is very clear and rapid.
The frequency of transformation is variable, some times you can select 5
transformants per plate of 300-500 seeds some times 1 or 0.
From: Grazyna Rempulska <gbujas at botinst.unizh.ch>
Usually I used the concentration of 20 mg of hygromycin B per L
medium for the selection of A. thaliana transformants after infiltration.
I performed my transformation with pGDW 3.1 vector (from Dr. C. Koncz) and
pGV3101 strain of A. tumefaciens. You have to wait few weeks
(3-4 weeks) after sowing and your selection becomes clear. On the begin
all plants will be green or light green, and after few weeks (seedlings in
the stage of two cotyledones and two well developed leaves) the selection
will be obvious: only transformed plants will survive on hygr. containing
medium and remain green.
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