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>IP</h2> <ol> <li>Take 20 ul of proteinA-sepharose beads (Amersham Biosciences) per ChIP sample and wash with 4 times with 1 ml FA buffer. Spin at 2500 g for 2 min to collect the beads. After the washes, suspend the beads in one bed volume of FA buffer. </li> <li>Add 40 ul of the bead slurry to each ChIP sample and continue to rotate at 4 oC for 2 h. </li> <li>Wash beads at room temperature by adding 1 ml of each of the following buffers and incubating on a nutator (or a rotator). Collect beads by spinning for 1-2 minutes at 2500 g. </li> </ol> <p> 2 times FA buffer for 5 minutes 1 time FA-1 M NaCl for 5 minutes. After this wash, transfer beads to new tubes with the next wash buffer. 1 time FA-500 mM NaCl for 10 minutes 1 time TEL buffer for 10 min 2 times TE for 5 min </p> <ol> <li>To elute the immunocomplexes, add 150 ul Elution Buffer and place the tube in a 65 oC heat block for 15 min. Vortex briefly every 5 min. Spin down the beads at 2500 g for 2 min and transfer the supernatant to a new tube. Repeat elution and combine supernatants. </li> <li>Thaw the input samples set aside the previous day and add 2 ul 10 mg/ml RnaseA. Digest at room temperature for 1-2 hours. Then add 250 ul Elution Buffer. Add 2 ul of 10 mg/ml proteinase K (stock in water) to each sample and input. Incubate for 1-2 hours at 55 oC , then transfer to 65 oC for 12-20 h to reverse crosslinks. </li> <li>Purify the DNA with Qiaquick PCR purification kit (Qiagen). Elute with 50 ul H2O. Run 5 ul of the input DNA on a 1.5% agarose gel to check the extent of shearing. Most of the DNA fragments should be 200-800 bp. </li> </ol> <p> From here continue with LM-PCR amplification of the material or move to locus specific qPCR. </p> </div> </td> </tr> </table> <script type="text/javascript"> var gaJsHost = (("https:" == document.location.protocol) ? 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