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=PublicFTP href="ftp://ftp.modencode.org/pub/">Public Data</a><br> <a class=list id=GenomesLink href="/Genomes.shtml" >Browse Genomes</a><br> <a class=list id=Mine href="http://intermine.modencode.org/query/">modMine</a><br>  <a class=list id=TFLink href="http://www.modencode.org/cgi/w2h.pl?TranscriptionFactor:Voting">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>[login]</span><br> <a class=list id=SteinLink href="/Stein.shtml" >Data Coordinating Center</a><br> <a class=list id=Policy href="/docs/modENCODE_publication_policy.pdf">Publication Policy</a><br>  <a class=list id=Pipeline href="http://submit.modencode.org/submit/" >Data Submission</a><span class=PI>[login]</span><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>LM-PCR</h2> <p> The amplification method was derived from (3). </p> <ul> <li>For blunting DNA ends, add 25 ul <a href="./embryo-extract-preparation-for-ChIP-chip.shtml"> ChIP DNA</a> or 2.5 ul input DNA (diluted to 25 ul with dH2O) to a PCR tube. Include a negative control by preparing a reaction with no DNA. </li> </ul> <table border="1"> <tr> <td>+ Blunting mix (for 1 reaction): </td> </tr> <tr> <td>H2O </td> <td>73 ul </td> </tr> <tr> <td>10X NEB 2 </td> <td>11 ul </td> </tr> <tr> <td>BSA (10 mg/ml) </td> <td>0.5 ul </td> </tr> <tr> <td>dNTP mix (25 mM each) </td> <td>0.4 ul </td> </tr> <tr> <td>T4 DNA polymerase (3U/ul) </td> <td>0.2 ul </td> </tr> </table> <p> Add and mix by pipetting. Incubate at 12°C for 20 min in a PCR machine. Purify DNA on a <a href="http://www.zymoresearch.com" class="external">Zymo column DNA clean up and concentrator</a>. Elute in 25 ul H2O and place on ice. </p> <ul> <li>For ligation of the linkers, prepare ligation mix: </li> </ul> <table border="1"> <tr> <td>+ Ligation Mix (for 1 reaction): </td> </tr> <tr> <td>H2O </td> <td>13 ul </td> </tr> <tr> <td>10X ligase buffer </td> <td>5 ul </td> </tr> <tr> <td <a href="./annealed-linker-ligation.shtml">annealed linkers</a> (15 uM) </td> <td>6.7 ul </td> </tr> <tr> <td>T4 DNA ligase </td> <td>0.5 ul </td> </tr> </table> <p> Add 25 ul to each sample and incubate 12-20 hours at 16°C. </p> <ul> <li>For amplification, purify the ligated DNA with <a href="http://www.zymoresearch.com" class="external">Zymo column</a> and elute in 15 ul H2O. Transfer to PCR tube, and place on ice. </li> </ul> <table border="1"> <tr> <td>+ PCR Mix (for 1 reaction): </td> </tr> <tr> <td>H2O </td> <td>62.7 ul </td> </tr> <tr> <td>10X PCR buffer (no MgCl2) </td> <td>10 ul </td> </tr> <tr> <td>MgCl2 (25 mM) </td> <td>8 ul </td> </tr> <tr> <td>dNTPs (25 mM each) </td> <td>0.8 ul </td> </tr> <tr> <td>oligo Long (40 uM) </td> <td>2.5 ul </td> </tr> <tr> <td>Taq DNA Polymerase (5u/ul) </td> <td>1 ul </td> </tr> <tr> </tr> </table> <p> Add 85 ul PCR Mix and run the following program in a PCR machine: <code> <pre> Step 1: 55°C for 4 min Step 2: 72°C for 5 min Step 3: 95°C for 2 min Step 4: 95°C for 30 s Step 5: 55°C for 30 s Step 6: 72°C for 1 min Go to step 4 for 24-28 times Step 7: 72° C for 4 min Step 8: 4° C </pre> </code> </p> <ul> <li>Purify the amplified DNA with the Qiaquick PCR purification kit and elute in 50 ul dH2O. Load 5 ul of the eluate on a 1.5% agarose gel to check amplification. The size of the amplified DNA should be 200-600 bp. </li> </ul> <p> <img src="./images/LM-PCRex.jpg" width="200" alt="LM-PCRex.jpg" /> </p> <p> Example gel: 1,2 IP, 3 no antibody control, 4,5 IP, 6 no antibody control, 7 LMPCR negative control (no DNA was used in the first step all the other steps carried in parallel to the samples) 8-14 Inputs (I used 3 mg of extract per IP and took 300 ug for input. Then I used 1/20th of the Input for each amplification). </p> <p> Refs: 3) Ren, B. et al. Genome-wide location and function of DNA binding proteins. Science 290, 2306-9 (2000). </p> </div> </td> </tr> </table> <script type="text/javascript"> var gaJsHost = (("https:" == document.location.protocol) ? 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