The modENCODE Project</title-seeking> <script type="text/javascript" src="js/yahoo-dom-event.js"></script> <script type="text/javascript" src="js/modencode.js"> </script> </head> <body vlink='black' link='black' onload=setActiveLink('Introduction')> <table width="100%"> <tr valign=top> <td> <a href="http://www.modencode.org/"> <img src="/img/modENCODE_logo_small.png" border=0 alt="modENCODE home"/></a> <div class=Links> <h4>RESOURCES</h4> <a class=list id=GenomesLink href="/Genomes.shtml" >Browse Genomes</a><br> <a class=list id=Mine href="http://intermine.modencode.org/query/">modENCODE-mine</a><br> <a class=list id=VoteLink href="/Vote.shtml" >TF Priority List</a><br> <a class=list id=ProtocolsLink href="/Protocols.shtml" >Protocols</a><br> <a class=list id=ReagentsLink href="/Reagents.shtml" >Reagents</a><br> <a class=list id=WIKILink href="http://wiki.modencode.org/project/">WIKI</a> <span class=PI>[restricted access] </span><br> <a class=list id=SteinLink href="/Stein.shtml" >Data Coordinating Center</a><br> <a class=list id=FAQLink href="http://wiki.modencode.org/cgi-bin/w2h.pl?FAQ">FAQ</a><br> <a class=list id=ContactLink href="mailto:help@modencode.org" >Contact us</a><br> <a class=list id=IntroductionLink href="/index.shtml" >Home</a> </div> <div class=Links> <h4><span>PROJECTS</span></h4> <span class=list><i>C. elegans</i></span><br> <a class=list id=WaterstonLink href="/Waterston.shtml" >The Transcriptome</a><br> <a class=list id=LiebLink href="/Lieb.shtml" >Chromatin Function</a><br> <a class=list id=HenikoffLink href="/Henikoff.shtml" >Histone Variants</a><br> <a class=list id=SnyderLink href="/Snyder.shtml" >Regulatory Elements</a><br> <a class=list id=PianoLink href="/Piano.shtml" >The 3' UTRome</a> <br> <span class=list><i>D. melanogaster</i></span><br> <a class=list id=CelnikerLink href="/Celniker.shtml" >The Transcriptome</a><br> <a class=list id=WhiteLink href="/White.shtml" >Regulatory Elements</a><br> <a class=list id=KarpenLink href="/Karpen.shtml" >Chromosomal Proteins</a><br> <a class=list id=LaiLink href="/Lai.shtml" >Small and microRNAs</a><br> <a class=list id=MacAlpineLink href="/MacAlpine.shtml" > Origins of Replication</a> </div> <div class=Links> <h4>EXTERNAL LINKS</h4>  <a class=list href="http://www.genome.gov/modencode/" >modENCODE@NHGRI</a><br> <a class=list href="http://www.flybase.org">FlyBase</a><br> <a class=list href="http://www.wormbase.org">WormBase</a><br>  <a class=list href="http://www.flymine.org/" >FlyMine</a><br> </div> </td> <td align=left> <table width="96%" align=center cellpadding=5 cellspacing=0> <tr> <td colspan=4> <tr > <td valign=center align=right bgcolor=#5f5050 width=10% > <font color=white size=2 face=helvetica> <b>Browse Genomes:</h4> <td valign=center align=left bgcolor=#5f5050 width=35%>  <a href="http://modencode.oicr.on.ca/cgi-bin/gbrowse/worm" target="_blank"> <img src="/img/wgb_s.png" alt="C.elegans"/></a>  <a href="http://modencode.oicr.on.ca/cgi-bin/gbrowse/fly" target="_blank"> <img src="/img/dgb_s.png" alt="D.melanogaster"/></a> <td valign=center align=right bgcolor=#ad8d8c width=10%> <font color=white size=2 face=helvetica> <b>mine modENCODE: <td bgcolor=#ad8d8c width=15%> <a href="http://intermine.modencode.org/query/"> <img src="/img/modmine_s.png" alt="query!!"/></a> </td></tr> </table> <div id="main">  <h2>Embryo collection/fixing for ChIP-chip</h2> <ol> <li>Grow worms on 10-20 10 cm plates. After starving on plates, add concentrated <em>E Coli</em> (HB101) grown to saturation, then resuspended in <a href="./m9-buffer.shtml">M9 Buffer</a>+20%glycerol at equal weight:volume) to obtain as many adults with embryos. Bleach to obtain embryos as described previously 1. </li> <li>Transfer embryos in <a href="./m9-buffer.shtml">M9 Buffer</a> to plates to hatch overnight or in <a href="./m9-buffer.shtml">M9 Buffer</a> shaking overnight to obtain L1s. Wash L1s and put into 500 ml liquid media (S medium+1X PSN antibiotics and nystatin) and grow at 20 oC in an incubator shaking at 230 rpm (2.8 lt Fernbach flasks perform the best). Add concentrated <em>E Coli</em> for food as necessary during growth. It takes around 2.5-3 days to obtain adults with embryos. </li> <li>Collect adults (by filtration over 35uM nitex membrane) and transfer to 50 ml conical tubes. To 5-10 ml of worms add 40 ml of <a href="./bleaching-solution.shtml">Bleaching Solution</a> and mix for approximately 8-12 minutes while monitoring bleaching as described (1). </li> <li>Spin down embryos at 3000 g for 3 minutes. Resuspend pellet (embryos) in 50 ml <a href="./m9-buffer.shtml">M9 Buffer</a> and spin 3000 g for 3 minutes. Repeat this wash 2 more times. </li> <li>Resuspend embryos in 47 ml <a href="./m9-buffer.shtml">M9 Buffer</a> and add 2.8 ml 37% formaldehyde solution. Place on a shaker (50-100 rpm) and incubate for 30 minutes at room temperature (RT, between 22-26 oC). </li> <li>Wash embryos with once with 50 ml 100 mM Tris pH 7.5 (to quench excess formaldehyde), 2 times with 50 ml <a href="./m9-buffer.shtml">M9 Buffer</a>, and once with 10 ml FA buffer supplemented with protease inhibitors (Calbiochem Cat# 539131, Protease Inhibitor Cocktail Set I). </li> </ol> <p> Collect embryos in microfuge tubes by centrifugation at 13,000 g for 10s Discard supernatant and store the embryo pellet at -80 oC. </p> <p> Refs: 1) Stiernagle, T. Maintenance of C. elegans. in WormBook (ed. Community, T.C.e.R.) (WormBook doi/10.1895/wormbook.1.8.1, <a href="http://www.wormbook.org" class="external">http://www.wormbook.org</a>, February 11, 2006). </p> </div> </td> </tr> </table> <script type="text/javascript"> var gaJsHost = (("https:" == document.location.protocol) ? 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