[Arabidopsis] Embryogenic culture

punamaya Maharjan via arab-gen%40net.bio.net (by puna459 from hotmail.com)
Mon Jun 29 06:28:20 EST 2009


  Dear Sir/ madam
 I would like to post my query in this page .=20

Thank you .
Puna

 "Query"



    Dear=20
arab-gen users=2C=20

I am posting a query related to
the problem of contamination in embryogenic cultures. If anybody has idea t=
o overcome it=2C I highly appreciate any kind of your comments.=20

I
followed the method described in the paper by Mordhorst et al.=2C 1998.  As=
 mentioned in the Mordhorst et al.=2C 1998=2C seeds were
surface sterilized for 10 sec in 70% ethanol followed by a 10- min incubati=
on
in commercial bleach (final concentration 2% sodium hypochlorite=2C contain=
ing
0.3% Tween 20)=2C washed 9 times with sterile water=2C dried on filter pape=
r=2C and
stored at room temperature before use. Then 30 seeds were incubated in 20 m=
l  MS-4 (pH 5.8)
induction medium [Murashige and Skoog salts containing 2% (w/v) Sucrose =2C=
 4.5 uM 2=2C4-D=2C
and 10 mm MES
[2-(N-morpholino)-ethanesulonic
acid]. After a cold treatment of 4 d at 4=B0C=2C cultures were kept on a ro=
tary
shaker (100 rpm) at 25=B0C in the light or darkness. For seed incubation=2C=
 I used
the 250 ml flasks instead of 190-ml Greiner plastic Containers (Alphen a/d =
Rijn=2C The
Netherlands) mentioned in Mordhorst et al.=2C 1998. I have tried with
different sterilization buffers like 1) 5 % Clorax=2C 1% SDS 2) 0.05 % Trit=
onX-100
followed by absolute ethanol washing.  Still I am getting the contamination=
 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=2C 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.=20

=20

 Puna=20




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