[Zbrafish] RE: Zbrafish Digest, Vol 68, Issue 3

Sateesh Reddy via zbrafish%40net.bio.net (by msreddy from hotmail.co.uk)
Fri Jan 7 05:52:59 EST 2011


Hi,
1.. After removing chorion add prechilled 1/2 Ginzburg Fish Ringers solution ( With out Calicium) solution and vortex for 1 min at high speed (If possible please do it on Ice)
2. Spin down for 10min at 100rpm for 5min in 4degree
3. Add 1ml prechilled wash buffer (110mM NaCl, 3.5 mM KCl 2.7mM CaCl2 and 10mM tris/cl pH8.5) and vortex for 1min at high speed for 1min and spein down at high speed for 5min/4degre.
4. Repeat washing step till you observe white cells.
All the best
sat
********************************************
Neurodevelopmetal Genetics Lab,
#512,N-11
College of Biological sciences,
Chungnam National UNiversity,
Daejeon,ROK



 



 

> Date: Thu, 6 Jan 2011 12:03:35 -0500
> From: zbrafish-request from oat.bio.indiana.edu
> Subject: Zbrafish Digest, Vol 68, Issue 3
> To: zbrafish from magpie.bio.indiana.edu
> 
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> Today's Topics:
> 
> 1. How to remove the yolk? (eSAM)
> 2. Re: How to remove the yolk? (Grzegorz Burzynski)
> 3. Re: How to remove the yolk? (Tim Erickson)
> 4. Re: How to remove the yolk? (Bill Trevarrow)
> 
> 
> ----------------------------------------------------------------------
> 
> Message: 1
> Date: Wed, 5 Jan 2011 08:35:52 -0800 (PST)
> From: eSAM <samarut.eric from orange.fr>
> Subject: [Zbrafish] How to remove the yolk?
> To: bionet-organisms-zebrafish from moderators.isc.org
> Message-ID:
> <b1eb96b8-bb71-4593-b94f-2f00f71957c6 from 35g2000prb.googlegroups.com>
> Content-Type: text/plain; charset=ISO-8859-1
> 
> Hello everyone,
> I'm planning to extract protein from whole embryos (from 36 to 72hpf).
> I saw that it is better to work on dechorionated and deyolked
> embryo...
> My question is about how to remove yolk? I saw that by triturating
> embryos in cold Ringer's, it should work...but you start from alive
> embryos? or already fixed?
> Could you give me some tips for this (I guess) easy step.
> Many thanks,
> 
> 
> 
> ------------------------------
> 
> Message: 2
> Date: Wed, 5 Jan 2011 13:27:00 -0500
> From: Grzegorz Burzynski <g.m.burzynski from gmail.com>
> Subject: Re: [Zbrafish] How to remove the yolk?
> To: eSAM <samarut.eric from orange.fr>
> Cc: bionet-organisms-zebrafish from moderators.isc.org
> Message-ID:
> <AANLkTi=Pjg2-MHt04V251hz4__gzhxbbaV8B3788cxZd from mail.gmail.com>
> Content-Type: text/plain; charset="iso-8859-1"
> 
> Easiest way is mechanical dissociation by pipetting embryos up and down
> 10-20 times with your p200 and gentle spin afterwards.
> Sounds like a cave man recipe but it is very efficient.
> 
> Greg
> 
> On Wed, Jan 5, 2011 at 11:35 AM, eSAM <samarut.eric from orange.fr> wrote:
> 
> > Hello everyone,
> > I'm planning to extract protein from whole embryos (from 36 to 72hpf).
> > I saw that it is better to work on dechorionated and deyolked
> > embryo...
> > My question is about how to remove yolk? I saw that by triturating
> > embryos in cold Ringer's, it should work...but you start from alive
> > embryos? or already fixed?
> > Could you give me some tips for this (I guess) easy step.
> > Many thanks,
> >
> > _______________________________________________
> > Zbrafish mailing list
> > Zbrafish from net.bio.net
> > http://www.bio.net/biomail/listinfo/zbrafish
> >
> 
> 
> 
> -- 
> Grzegorz Burzynski, Ph.D.
> Postdoctoral fellow, McCallion lab
> McKusick-Nathans Institute of Genetic Medicine
> Johns Hopkins University School of Medicine
> BRB Room 446
> 733 N. Broadway
> Baltimore, MD, 21205-1832
> USA
> 
> Tel: (410)-502-7533
> Fax: (410)-502-5677
> email: burza from jhmi.edu
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> ------------------------------
> 
> Message: 3
> Date: Wed, 5 Jan 2011 11:08:12 -0800
> From: Tim Erickson <ericksti from ohsu.edu>
> Subject: Re: [Zbrafish] How to remove the yolk?
> To: eSAM <samarut.eric from orange.fr>
> Cc: "bionet-organisms-zebrafish from moderators.isc.org"
> <bionet-organisms-zebrafish from moderators.isc.org>
> Message-ID: <381EEBC3-13D1-483D-883D-56FB51D2159D from ohsu.edu>
> Content-Type: text/plain; charset="us-ascii"
> 
> Hi,
> I'd recommend checking out this paper for an excellent yolk removal protocol
> http://www.biomedcentral.com/1471-213X/6/1
> <http://www.biomedcentral.com/1471-213X/6/1>
> Regards,
> Tim Erickson
> 
> On Jan 5, 2011, at 8:35 AM, eSAM wrote:
> 
> Hello everyone,
> I'm planning to extract protein from whole embryos (from 36 to 72hpf).
> I saw that it is better to work on dechorionated and deyolked
> embryo...
> My question is about how to remove yolk? I saw that by triturating
> embryos in cold Ringer's, it should work...but you start from alive
> embryos? or already fixed?
> Could you give me some tips for this (I guess) easy step.
> Many thanks,
> 
> _______________________________________________
> Zbrafish mailing list
> Zbrafish from net.bio.net<mailto:Zbrafish from net.bio.net>
> http://www.bio.net/biomail/listinfo/zbrafish
> 
> Timothy Erickson
> Postdoctoral Researcher
> Nicolson Lab
> Oregon Hearing Research Center
> and Vollum Institute
> Oregon Health and Sciences University
> 3181 SW Sam Jackson Pk. Rd. MRB816-L474
> Phone-(503)494-2928
> Fax-(503)494-9612
> 
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> ------------------------------
> 
> Message: 4
> Date: Wed, 5 Jan 2011 11:49:38 -0800
> From: Bill Trevarrow <trevarro from uoneuro.uoregon.edu>
> Subject: Re: [Zbrafish] How to remove the yolk?
> To: bionet-organisms-zebrafish from moderators.isc.org
> Message-ID: <800BE872-52D8-4F21-BAC5-2916623CBE8B from uoneuro.uoregon.edu>
> Content-Type: text/plain; charset=us-ascii
> 
> Hi Eric,
> 
> I have done this with live embryos.
> The yolk dissolves in the cold ringers and the embryos will settle out (pelleting at 1G).
> If the embryos are fixed the yolk will stay in a lump, not separate well from the embryos, not dissolve well, and will settle out like the embryos. 
> 
> 
> 
> On Jan 5, 2011, at 8:35 AM, eSAM wrote:
> 
> > Hello everyone,
> > I'm planning to extract protein from whole embryos (from 36 to 72hpf).
> > I saw that it is better to work on dechorionated and deyolked
> > embryo...
> > My question is about how to remove yolk? I saw that by triturating
> > embryos in cold Ringer's, it should work...but you start from alive
> > embryos? or already fixed?
> > Could you give me some tips for this (I guess) easy step.
> > Many thanks,
> > 
> > _______________________________________________
> > Zbrafish mailing list
> > Zbrafish from net.bio.net
> > http://www.bio.net/biomail/listinfo/zbrafish
> 
> Bill Trevarrow, Ph.D.
> Thoren Aquatics
> trevarro from uoneuro.uoregon.edu
> Tel: (541) 844-9054
> Fax: (541) 683-0996
> 
> 
> 
> 
> ------------------------------
> 
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> End of Zbrafish Digest, Vol 68, Issue 3
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