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 extract preparation for ChIP-chip</h2> <p> Extracts were prepared as described (2) with minor changes. </p> <ol> <li>Resuspend ~ 500 ul of packed embryos in 1.5 ml <a href="./fa-buffer.shtml">FA buffer</a>+ protease inhibitors. Dounce on ice (~ 30 strokes) using a glass dounce homogenizer pestle B. Transfer to a 15 ml tube and bring the volume to 2 ml with with <a href="./fa-buffer.shtml">FA buffer</a>+ protease inhibitors. </li> <li>Using a Branson sonifier microtip, sonicate samples on ice at the following settings: 35% amplitude, 0.9 sec on, 0.1 sec off, 12 pulses for 7 times. After each time, cool samples in dry ice/ethanol bath for 2 seconds. Make sure that the samples are not overheated. </li> <li>Transfer samples to microfuge tubes and spin at 13,000 g for 15 minutes at 4oC. Take the supernatant and discard pellet. Determine the protein concentration of the supernatant by the Bradford method. Continue with the next step or aliquot and snap freeze in liquid nitrogen and store at -80 oC. </li> <li>Add extract corresponding to ~2.2 (or 3.3 mg in 500 ul volume and add 25 ul sarkosyl instead) mg of protein to a microfuge tube and bring the volume to 400 ul with <a href="./fa-buffer.shtml">FA buffer</a> + protease inhibitors . Add 20 ul 20 % sarkosyl solution and spin at 13,000 g for 5 minutes at 4 oC. Transfer the supernatant to a new tube. Remove 10 % of the material and store it at -20 oC until the following day, when it will be used to prepare input DNA. Add 15-20 ug protein A-purified antibody (15 ug of DPY-27 or SDC-3 proteinA purified antibody is sufficient) or 2-5 ug affinity-purified antibody ( 3 ug of the DPY-27 or SDC-3 antibody is sufficient) to the extract and rotate at 4 oC overnight (16-20 h). </li> </ol> <p> Refs: (2) Sharma, V.M., Li, B. & Reese, J.C. SWI/SNF-dependent chromatin remodeling of RNR3 requires TAF(II)s and the general transcription machinery. Genes Dev 17, 502-15 (2003). </p> </div> </td> </tr> </table> <script type="text/javascript"> var gaJsHost = (("https:" == document.location.protocol) ? "https://ssl." : "http://www."); document.write(unescape("%3Cscript src='" + gaJsHost + "google-analytics.com/ga.js' type='text/javascript'%3E%3C/script%3E")); </script> <script type="text/javascript"> var pageTracker = _gat._getTracker("UA-4121742-1"); pageTracker._initData(); pageTracker._trackPageview(); </script> </body> </html>