[Arabidopsis] Embryogenic culture

Eve Wurtele via arab-gen%40net.bio.net (by evewurtele from gmail.com)
Mon Jun 29 13:00:27 EST 2009


it might help to reduce the number of seeds you are using in each flask.
one bad seed contaminates the whole flask.

also, have you tried controls in which you do the same protocol, but do not
add ANY seeds-  and then put on shaker.

this will help you rule out something to do with flasks, media, etc.

On Mon, Jun 29, 2009 at 6:28 AM, punamaya Maharjan <puna459 from hotmail.com>wro=
te:

>
>  Dear Sir/ madam
>  I would like to post my query in this page .
>
> Thank you .
> Puna
>
>  "Query"
>
>
>
>    Dear
> arab-gen users,
>
> I am posting a query related to
> the problem of contamination in embryogenic cultures. If anybody has idea
> to overcome it, I highly appreciate any kind of your comments.
>
> I
> followed the method described in the paper by Mordhorst et al., 1998.  As
> mentioned in the Mordhorst et al., 1998, seeds were
> surface sterilized for 10 sec in 70% ethanol followed by a 10- min
> incubation
> in commercial bleach (final concentration 2% sodium hypochlorite,
> containing
> 0.3% Tween 20), washed 9 times with sterile water, dried on filter paper,
> and
> stored at room temperature before use. Then 30 seeds were incubated in 20
> ml  MS-4 (pH 5.8)
> induction medium [Murashige and Skoog salts containing 2% (w/v) Sucrose ,
> 4.5 uM 2,4-D,
> and 10 mm MES
> [2-(N-morpholino)-ethanesulonic
> acid]. After a cold treatment of 4 d at 4=B0C, cultures were kept on a ro=
tary
> shaker (100 rpm) at 25=B0C in the light or darkness. For seed incubation,=
 I
> used
> the 250 ml flasks instead of 190-ml Greiner plastic Containers (Alphen a/=
d
> Rijn, The
> Netherlands) mentioned in Mordhorst et al., 1998. I have tried with
> different sterilization buffers like 1) 5 % Clorax, 1% SDS 2) 0.05 %
> TritonX-100
> followed by absolute ethanol washing.  Still I am getting the contaminati=
on
> problem
> just 1 week after transferring the flasks from cold room.
>
>  Does anyone have the idea to overcome the
> problem?  Does this experiment need
> specific 190-ml Greiner plastic Containers
> (Alphen a/d Rijn, The Netherlands). I tried to find this container also b=
ut
> I
> could not get the information about it. Would you please give me
> suggestion?  I thank you in advance for
> your reply.
>
>
>
>  Puna
>
>
>
>
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--=20
Eve Syrkin Wurtele, Professor
Bioinformatics and Computational Biology
2624D Howe Hall, VRAC, Iowa State University,
Ames IA 50011, USA
515-708-3232 (cell)
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e
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