[Arabidopsis] soil sterilization

Felipe Aceituno via arab-gen%40net.bio.net (by criptoendolito from gmail.com)
Wed Apr 8 12:16:28 EST 2009


Hi Vera and everyone
I used to work in a soil microbiology lab. There, they sterilize the
soil by gamma radiation, and it worked pretty well
Hope its an useful info

Felipe Flores Aceituno, MSc
Laboratorio de Biotecnologia
Departamento de Ingeneria Química y Bioprocesos
Facultad de Ingeniería
Pontificia Universidad Catolica de Chile

2009/4/7  <arab-gen-request from oat.bio.indiana.edu>:
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> Today's Topics:
>
>   1. Semi Solid Attempt 2 (Holt, Ben F. III)
>   2. Postdoc researcher position available immediately (Eric Lam)
>   3. PosDoc position (Hideo Matsumura)
>   4. eoxn/intron boundry prediction (chunnu)
>   5. Soil sterelization  (Vera Nunes)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Sat, 4 Apr 2009 18:00:48 -0500
> From: "Holt, Ben F. III" <benholt from ou.edu>
> Subject: [Arabidopsis] Semi Solid Attempt 2
> To: Arabidopsis Listserv <arab-gen from magpie.bio.indiana.edu>
> Message-ID: <C5FD50D2.8515%benholt from ou.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hi everyone,
>
> Apparently there were problems with the last attachment - I went back and checked the message I sent and the attachment was there, so I conclude that the listserv is either blocking these in general or just didn't like the one I sent.
>
> Anyway, I have attached it here again and I have pasted the two protocols below - sorry, but the pasted protocols below are not very pretty. I had to choose between making them look nice or watching the Final Four.
>
> Go Heels!
>
> Ben
>
>
> Additional Protocols
>
> Note: Carbenicillin reduces satellite
> formation.
>
> Caution: Make sure that the rotor was set
> at room temperature for at least 2 h before
> adding the bottles. Rotors at 4°C will
> cause solidification of agar.
>
> 5.1 Expansion of Plasmid cDNA Libraries
> Semi-solid amplification of primary cDNA transformants minimizes representational
> biases that can occur during the expansion of plasmid cDNA libraries (25). Life
> Technologies has further modified this protocol such that the amplification is done at
> 30°C, helping to stabilize unstable clones (26).
>
> Caution: Bottles of semi-solid agar containing suspended colonies must be handled
> gently and incubated without disturbance. Rough handling or bumping of the
> incubator will cause micro colonies to fall out of solution. Incubators that have fans
> can cause colonies to fall out of solution.
>
> 1.
> Prepare 2.0 L of 2X LB. Remove 175 ml of the 2X LB to make the 2X LB
> Glycerol.
> 2.
> Place a large stir bar and 1.35 g SeaPrep® agarose into each of four 500-ml
> autoclavable bottles. Place bottles on stir plates. With the stir plate turned on,
> add 450 ml of 2X LB to each bottle (avoid the formation of large clumps of
> agarose).
> 3.
> Autoclave these bottles of 2X LB agarose for 30 min.
> 4.
> Cool bottles in 37°C water bath for ~2 h until the medium reaches 37°C.
> 5.
> After the medium reaches 37°C, add carbenicillin to 50 "g/ml (preferred
> antibiotic) or add ampicillin to 200 "g/ml. Mix on stir plate.
> 6.
> To each bottle add 4 %&105 to 6 %&105 primary cDNA transformants (colonies
> from original library), and mix thoroughly on a stir plate for 2 min. Tighten caps.
> 7.
> Place bottles in an ice-water bath (0°C) such that the water in the bath is at the
> same level as the medium in the bottle. Incubate for 1 h in the ice bath.
> 8.
> Gently remove bottles from the ice bath and wipe the water off the outside of
> the bottles. Loosen bottle caps. Place these bottles in a gravity flow incubator
> set at 30°C.
> 9.
> Incubate these bottles for 40-45 h without disturbance. Place a centrifuge rotor
> at room temperature the morning of harvest.
> 10.
> Pour contents of bottles into centrifuge bottles and centrifuge at 10,400 * g for
> 20 min at room temperature
> 11.
> Pour off the supernatant.
> 12.
> Resuspend the cells in a total of 100 ml 2X LB Glycerol (12.5%). Remove two
> 100-"l aliquots for plating, further analysis, and colony estimate. Cells can be
> filtered through sterile cheesecloth to remove agarose clumps if present.
> 13.
> Subdivide the cells into ~10-ml aliquots and store at -70°C. It is also useful to
> make a number of 1-ml and 100-"l aliquots.
> 14.
> Frozen cells can be used to prepare DNA for GENETRAPPER(&experiments, for
> plating to screen, or can be further amplified in liquid at 30°C to obtain DNA.
> Use 2.5 %&109 cells/100 ml growth medium for further expansion of the library.
> 32
>
>
>
>
> Media
> 2X LB
> Component Amount/liter
>
> 5
>
> Bacto-Tryptone................................................................. 20 g
>
>
> Bacto-Yeast Extract.......................................................... 10 g
> NaCl ................................................................................. 10 g
>
>
> 2X LB Glycerol (12.5%)
> Component Amount/200 ml
>
>
> 2X LB............................................................................ 175 ml
> Glycerol (100%)...............................................................25 ml
>
>
> Filter-sterilize and store for up to 2 months at room temperature.
>
>
>
>
> Amplifying the pCMV-Script® cDNA Library
>
> The primary library may now be plated and screened; however,
> amplification of the library is desirable to produce a large and stable
> quantity of the library. Do not perform more than one round of
> amplification, as slower-growing clones may be significantly
> underrepresented.
>
> Stratagene recommends amplifying plasmid libraries in 500-ml bottles of 2*
> LB agarose§ using the semi-solid amplification method.4, 5 The libraries are
> amplified in suspension which allows for three-dimensional, uniform colony
> growth. This reduces the potential for under-representation of particular
> clones due to the overgrowth of some colonies during the expansion process
> that accompanies direct plating methods.
>
> Note
> Each 500-ml bottle of 2* LB agarose can accommodate ~5 * 105
> primary cfu. To amplify a library of 1 * 106 primary cfu, two
> bottles are necessary.
>
> Note
> Stratagene has determined that the use of SeaPrep® agarose is
> critical to ensure performance (See Additional Materials
> Required).
>
> 1.
> On a heated stir plate, using a large stirbar, combine 1.35g of SeaPrep
> agarose with 450 ml of 2* LB§ in each 500-ml autoclavable bottle.
> Heat and stir until the agarose is in solution.
> 2.
> Autoclave the bottles and stir bars for 30 minutes.
> 3.
> Allow the bottles to cool to 37°C in a water bath (~1 hour).
> 4.
> Transfer the bottles to a stir plate and add 50 *g/ml of kanamycin.
> 5.
> Add up to 5 * 105 cfu/bottle of primary library and stir for several
> minutes.
> Note
> The bottles must be handled very carefully at this stage.
> Avoid swirling or bumping the bottles as this may cause the
> microcolonies to fall out of suspension. The bottles must be
> incubated without disturbance or representation of the
> amplified library may be compromised.
>
> 6.
> Tighten the bottle caps and incubate the bottles for 1 hour in an ice
> water bath (0°C). The water level in the ice bath should be even with
> the media level in the bottle.
> 7.
> Carefully remove the bottles from the ice bath and gently dry the
> bottles. Loosen the bottle caps and incubate for 40-45 hours at 30°C.
> (Incubation at 30°C reduces under-representation of slower growing
> clones.)
> § See Preparation of Media and Reagents.
>
> pCMV-Script® XR cDNA Library Construction Kit
>
>
> 8.
> Pour the contents of the bottles into sterile 250-ml centrifuge bottles
> and spin at 10,000 * g for 20 minutes at room temperature.
> (Equilibrate the rotor to room temperature several hours prior to
> centrifugation. Using rotors stored at 4°C will cause the agar to
> solidify.)
> 9.
> Remove the semi-solid agarose supernatant and resuspend the pellets in
> 25 ml of 2* LB-glycerol (12.5%) (see Preparation of Media and
> Reagents) per 250-ml centrifuge bottle. Remove 100 *l for estimation
> of library titer and further characterization. Pipet the remainder into
> 1-ml aliquots. Store at -80°C.
> 10.
> Perform 6 serial dilutions with 100 *l of the amplified library diluted
> into 900 *l of LB medium. Plate 10 *l of the 10-5 and 10-6 dilutions
> onto selection plates. Amplification of a primary library containing
> 1 * 106 total transformants should result in a stable library of at least
> 1 * 109 total transformants.
> pCMV-Script® XR cDNA Library Construction Kit
>
>
>
>
>
>
> --
> Ben Holt
> Assistant Professor
> University of Oklahoma
> Department of Botany and Microbiology
> GLCH Rm 219
> 770 Van Vleet Oval
> Norman, OK  73019
> Phone (405)325-9018
> FAX   (405)325-7619
> http://www.ou.edu/cas/botany-micro/faculty/holt.html <http://www.ou.edu/cas/botany-micro/faculty/holt.html><http://www.ou.edu/cas/botany-micro/faculty/holt.html>
>
>
> ------------------------------
>
> Message: 2
> Date: Sat, 4 Apr 2009 12:09:44 -0400
> From: Eric Lam <ericl89 from hotmail.com>
> Subject: [Arabidopsis] Postdoc researcher position available
>        immediately
> To: Arabidopsis Network <arab-gen from magpie.bio.indiana.edu>
> Message-ID: <COL101-W44A13115DD9AE5552FCA6FBF860 from phx.gbl>
> Content-Type: text/plain; charset="Windows-1252"
>
>
> A POSTDOCTORAL POSITION is available at the Biotech Center, Rutgers University, in the laboratory of Prof. Eric Lam (http://aesop.rutgers.edu/~lamlab) for a highly motivated scientist interested in studying chromatin organization and stress responses in higher plants.  We have established a set of 277 mapped lines of novel transposants in Arabidopsis with single Ds insertions that allows functional and structural characterization of global epigenetic control in living plants.  Together with the recently established SOLiD NextGen sequencing capability in Rutgers for multiplexed ChIP-seq and small RNA analyses, our unique resource promise to reveal interesting new insights to the complex network of the epigenome and stress responses using Arabidopsis and grasses as paradigms.  The successful applicant will apply these new tools to examine global epigenetic control as a function of development and in response to stresses.
> The ideal applicant should have prior experience with molecular (ChIP assay, PCR, cloning, Northern blotting, etc.) and biochemical (Western blotting, enzyme assay, etc) techniques, advanced microscopy, and/or chemical biology approaches. Knowledge of epigenetic mechanisms and some bioformatics skills are desirable but not required.  If you are interested in this position, please email a cover-letter stating your research interests, a curriculum vitae, and contact information for three references to: Prof. Eric Lam, ericL89 from hotmail.com
>    The Biotech Center at Rutgers University is located in New Brunswick, New Jersey, and enjoys a convenient location close to the famous Jersey Shore and Pocono Mountains while also easily accessible (within 1 hour by car or train) to metropolitan areas such as New York City and Philadelphia.  The Biotech Center at the School of Environmental and Biological Sciences in the Cook Campus is well-equipped with advanced instrumentations for molecular biology, biochemistry and microscopy.  Rutgers University is an equal opportunity employer that strongly encourages underrepresented groups to apply for open positions.
>
> Related Publications:
>
> "Defining the functional network of epigenetic regulators in Arabidopsis thaliana"
> Molecular Plant, EPub online: Apr. 2009; DOI: 10.1093/mp/ssp017
>
> "Genome-wide transposon tagging reveals location-dependent effects on transcription and chromatin organization in Arabidopsis"
> Plant J.,  Published Online: Apr 12 2008; DOI: 10.1111/j.1365-313X.2008.03517.x
>
> "Bax Inhibitor-1 modulates endoplasmic reticulum stress-mediated programmed cell death in Arabidopsis"
> J. Biol. Chem., 2008; 283: 3200-3210.
>
> “DNA hypomethylation reduces homologous pairing of inserted tandem repeat arrays in somatic nuclei of Arabidopsis thaliana”
> Plant J., 2005 Nov;44(4): 531-40.
>
> "Visualizing chromosome structure/organization"
> Annual Rev. of Plant Biology, 2004; 55: 537-554.
>
>
> Prof. Eric Lam
>
> Director, Biotechnology Center for Agriculture
>      and the Environment
> Professor of Plant Biology and Pathology
>
> Rutgers, The State University of New Jersey, U.S.A.
>
>
>
> Email: ericL89 from hotmail.com; Lam from aesop.rutgers.edu
>
>
>
> ------------------------------
>
> Message: 3
> Date: Mon, 06 Apr 2009 19:32:27 +0900
> From: Hideo Matsumura <hideoma from ibrc.or.jp>
> Subject: [Arabidopsis] PosDoc position
> To: <Arab-gen from magpie.bio.indiana.edu>
> Message-ID: <C600094B.6399%hideoma from ibrc.or.jp>
> Content-Type: text/plain;       charset="ISO-2022-JP"
>
> Positions opens for a post-doctoral fellow
>
> Subject:  rice genetics/genomics
> Term: One year starting from July, 2009; subject to extension up to a total
> of three years
>
> Deadline of application: 11 May, 2009
>
> Contact person for further information:
> Dr. Hideo Matsumura  (hideoma from ibrc.or.jp <mailto:bosyu from ibrc.or.jp> )
>
> Applicants should send
>
> 1. Curriculum vitae
> 2. A list of publications (with reprints of recent publications)
> 3. A letter of self introduction
>
> to
>
> Dr. Hideo Matsumura
> Iwate Biotechnology Research Center
> 22-174-4, Narita, Kitakami, 024-0003
> Iwate, Japan
> e-mail for application: hideoma from ibrc.or.jp
>
>  We are seeking a competent post-doctoral scientist who will be in charge of
> rice genetic studies at Iwate Biotechnology Research Center (IBRC).  He/she
> is expected to carry out isolation and characterization of rice genes
> controlling important agronomic traits (cold tolerance, blast disease
> resistance), making full use of the rice genomic information.  Please see
> our Home Page (http://www.ibrc.or.jp/English/rice/main.html).
>
>
>
> ------------------------------------
> Hideo Matsumura
>
> Iwate Biotechnology Research Center
>
> Narita22-174-4
> Kitakami
> Iwate 024-0003
> Japan
>
> Tel +81-197-68-2911
> Fax +81-197-68-3881
> ------------------------------------
>
>
>
>
>
> ------------------------------
>
> Message: 4
> Date: Sun, 5 Apr 2009 08:20:39 -0700 (PDT)
> From: chunnu <sanjaysingh765 from gmail.com>
> Subject: [Arabidopsis] eoxn/intron boundry prediction
> To: bionet-genome-arabidopsis from moderators.isc.org
> Message-ID:
>        <179e037d-f9d6-4b5d-9cb1-18982a48b217 from b7g2000pre.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> hi there,
> i m looking for a eoxn/intron boundry prediction tool especially for
> arabidopsis and oryza.although there are nuber of tools available for
> this but they didn't produce the grphical outputs means they didn't
> show the region of exon/introns on input. can anyone help me regardng
> this.
> regards
> sanjay
>
>
>
> ------------------------------
>
> Message: 5
> Date: Mon, 06 Apr 2009 11:06:37 +0100
> From: Vera Nunes <vnunes from igc.gulbenkian.pt>
> Subject: [Arabidopsis] Soil sterelization
> To: arab-gen from magpie.bio.indiana.edu
> Message-ID: <1239012397.49d9d42d1e3be from webmail.igc.gulbenkian.pt>
> Content-Type: text/plain; charset=UTF-8
>
>
>
> Hello,
>
> First of all thanks a lot for your answers to my last question. Since
> autoclaving the soil it isn't feasible in our lab, does anyone knows another
> method that we could use to sterilize it? Or at least minimize the chances of
> fungal growth?
>
> Thanks!
>
> Vera Nunes
>
>
>
> ------------------------------
>
> _______________________________________________
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> Arab-gen from net.bio.net
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>
> End of Arab-gen Digest, Vol 48, Issue 4
> ***************************************
>



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