Dear Chuanmei Zhu
Washington University, St.Louis, MO,USA. 63130
the genomic DNA of arabidopsis has the introns,UTRS,etc thats why you may
not be able to amplify the expected amplicon, so its better to amplify from
CDNA, if you have plant tissue prepare RNA , from that prepare CDNA using
superscript or effecient MMLV(RTenzyme). so that you may amplify exact
amplicon then try to clone to cloning vector and subsequently sequencing an=
binary vector or interaction studies etc . hope this may give you some idea=
On 6/30/09, arab-gen-request from oat.bio.indiana.edu <
arab-gen-request from oat.bio.indiana.edu> wrote:
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>>> Today's Topics:
>> 1. M.Sc. Position available: ABC transporters in plant secondary
> metabolism (David Bird)
> 2. Re: Embryogenic culture (Eve Wurtele)
> 3. Could anyone help me on Arabidopsis genomic DNA cloning?
>> Message: 1
> Date: Mon, 29 Jun 2009 11:31:11 -0600
> From: David Bird <dbird from mtroyal.ca>
> Subject: [Arabidopsis] M.Sc. Position available: ABC transporters in
> plant secondary metabolism
> To: arab-gen from magpie.bio.indiana.edu> Message-ID:
>OF873D8FB6.FB20BA9E-ON872575E4.00602464-872575E4.00603C51 from mtroyal.ca>
> Content-Type: text/plain; charset=3D"US-ASCII"
>> M.Sc. Position available: ABC transporters in plant secondary metabolism.
>> A graduate student is sought for a(n) MSc/PhD project under the joint
> supervision of David Bird and Peter Facchini in the Department of
> Biological Sciences at the University of Calgary. The goal of this
> research program is to identify and characterize ABC transporters involve=
> in the transport of cuticular lipids and alkaloids in the model plants
> Arabidopsis thaliana and Papaver somniferum, respectively. The goal of th=
> Masters project is to identify candidate transporters using comparative
> genomics, bioinformatics, and gene silencing analysis. To be considered,
> the candidate should hold a B.Sc. in Biology, with an emphasis on
> molecular biology or biochemistry. The ideal candidate will hold an
> excellent academic record meeting the requirements for external
> scholarships and will have laboratory experience in biochemistry or
> molecular biology. Interested candidates should contact David Bird
> (dabird from ucalgary.ca) and provide a CV, academic transcripts, and the name=
> of two references.
>> This communication is intended for the use of the recipient to which it i=
> addressed, and may
> contain confidential, personal, and or privileged information. Please
> contact the sender
> immediately if you are not the intended recipient of this communication,
> and do not copy,
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> error, or subsequent
> reply, should be deleted or destroyed.
>> Message: 2
> Date: Mon, 29 Jun 2009 13:00:27 -0500
> From: Eve Wurtele <evewurtele from gmail.com>
> Subject: Re: [Arabidopsis] Embryogenic culture
> To: punamaya Maharjan <puna459 from hotmail.com>
> Cc: arab-gen from magpie.bio.indiana.edu> Message-ID:
> <7fdaa81b0906291100s4f453a7bg95a5aeb73d22574d from mail.gmail.com>
> Content-Type: text/plain; charset=3Dwindows-1252
>> 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 n=
> 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> >wrote:
> > 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 id=
> > to overcome it, I highly appreciate any kind of your comments.
> > I
> > followed the method described in the paper by Mordhorst et al., 1998. =
> > 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 pape=
> > and
> > stored at room temperature before use. Then 30 seeds were incubated in =
> > 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
> > shaker (100 rpm) at 25=B0C in the light or darkness. For seed incubatio=
> > used
> > the 250 ml flasks instead of 190-ml Greiner plastic Containers (Alphen
> > 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
> > 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
> > I
> > could not get the information about it. Would you please give me
> > suggestion? I thank you in advance for
> > your reply.
> > Puna
> > _________________________________________________________________
> > More than messages=96check out the rest of the Windows Live=99.
> > Arab-gen<
> > Arab-gen from net.bio.net> > http://www.bio.net/biomail/listinfo/arab-gen> >
> Eve Syrkin Wurtele, Professor
> Bioinformatics and Computational Biology
> 2624D Howe Hall, VRAC, Iowa State University,
> Ames IA 50011, USA
> 515-708-3232 (cell)
>https://www.metnetdb.org>https://www.metablast.org>> A neutron goes into a bar and asks the bartender, "How much for a beer?"
> bartender replies, "For you, no charge."
>> Message: 3
> Date: Mon, 29 Jun 2009 19:21:15 -0500
> From: =3D?GB2312?B?88O0q8PA?=3D <zhuchuanmei04 from gmail.com>
> Subject: [Arabidopsis] Could anyone help me on Arabidopsis genomic DNA
> To: arab-gen from magpie.bio.indiana.edu> Message-ID:
> <d407ea500906291721v3ef24347m866f1605a9e487f8 from mail.gmail.com>
> Content-Type: text/plain; charset=3DISO-8859-1
> I am a graduate student in Washington University. I have trouble in cloni=
> a ~1.9 kb DNA fragment from Arabidopsis genomic DNA recently. The genomic
> DNA was prepared by CTAB method and was dissolved in H2O. For 25ul PCR
> reaction, I use DNA ~500ng, MgCl2 1.5mM, dNTP 160uM, Taq 2.5U, primer eac=
> 0.2uM, buffer 1x. The PCR program was 94' 5min; 94' 45s, 54' 45s, 72'
> 2min, 20cycle; 72' 5min, 4' for ever.
>> I tried several times, including trying different concentration of DNA,
> MgCl2... However, I never got the expected 1.9kb band? This was my first
> time to clone gene from genomic DNA, I have no idea why it turns out to b=
> so difficult. Did you have any suggestions? Thanks in advance.
> Chuanmei Zhu
> DBBS(plant biology),
> Washington University, St.Louis, MO,USA. 63130.
>> Tsinghua U (B.S.)
> Arab-gen mailing list
>Arab-gen from net.bio.net>http://www.bio.net/biomail/listinfo/arab-gen>> End of Arab-gen Digest, Vol 50, Issue 15
Senior research fellow
dept of crop physiology,
Molecular plant physiology lab,
UAS, GKVK, Bangalore