Team:Washington/GENERAL PCR PROTOCOL

From 2013.igem.org

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<h1 dir="ltr">General PCR Protocol</h1>
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<p dir="ltr">See also: <a href="http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">PCR_GoTaq</a>
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<p dir="ltr"><a href="http://fg.cns.utexas.edu/fg/protocol__resuspending_PCR_primers.html">Protocol: Resuspending PCR primers</a></p>
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<p dir="ltr">Dilute to 10 times the number on primer vial</p>
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<p dir="ltr">Notes for iGEM 2013 HSB lab:</p>
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<p dir="ltr">We are using the GreenTaq polymerase 2X pre-mixed (<a href="http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>) for PCR. </p>
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<p dir="ltr">Use the protocols below for Green Taq polymerase. </p>
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<p dir="ltr">Use 50uls for PCR insert amplification. Use 10uls for analytical PCRs (screening).</p>
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      <p dir="ltr">25ul 2x Green taq (<a href="http://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>)</p>
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      <p dir="ltr">Total 50ul</p>
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          <p dir="ltr">95 C for 30s</p>
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          <p dir="ltr">95 C for 30s</p>
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          <p dir="ltr">ZZ* C for 30s (important step)</p>
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          <p dir="ltr">72 C for 1min per kb target gene plus 10-15% (important step) </p>
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          <p dir="ltr">Repeat 2-4 34x</p>
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          <p dir="ltr">72 C for 5 to 10min</p>
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          <p dir="ltr">4 C for forever (don&rsquo;t forget to turn the machine off the next day)</p>
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<a href="https://2013.igem.org/Team:Washington"><img class="thumbnail" src='https://static.igem.org/mediawiki/2013/1/1b/Team_Washington2013_banner_final.jpg' width= "965"/></A>
+
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+
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+
-
<a href="https://2012.igem.org/Main_Page"><img src='https://static.igem.org/mediawiki/igem.org/c/c8/IGEM_desat.png' align="right" width= "70"/></a>
+
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+
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+
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+
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+
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+
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+
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+
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<nav id="igemuwmenu-wrap">  
+
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+
-
<ul id="igemuwmenu">
+
-
 
+
-
<li><a href="https://2013.igem.org/Team:Washington">Home</a>
+
<ul>
<ul>
-
<li><a href="https://2013.igem.org/Team:Washington">UW 2013</a></li>
+
  <li dir="ltr">
-
<li><a href="https://2012.igem.org/Team:Washington">UW 2012</a></li>
+
    <p dir="ltr">Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 degrees C higher than XX or YY, whichever is lower. The temperature doesn&rsquo;t have to be exact (within 5 degrees or so).</p>
-
<li><a href="https://2011.igem.org/Team:Washington">UW 2011</a></li>
+
  </li>
-
<li><a href="https://2010.igem.org/Team:Washington">UW 2010</a></li>
+
-
<li><a href="https://2009.igem.org/Team:Washington">UW 2009</a></li>
+
-
<li><a href="https://2008.igem.org/Team:University_of_Washington">UW 2008</a></li>
+
-
<li><a href="https://2013.igem.org/Main_Page">iGEM Homepage</a></li>
+
</ul>
</ul>
-
</li>
+
<br />
-
 
+
<p dir="ltr">Analytical PCR with Green Taq polymerase:</p>
-
<!--
+
<ol start="3">
-
<li>
+
  <li dir="ltr">
-
<a </a> main menu on plastic degradation
+
    <p dir="ltr">Setup PCR Reaction</p>
 +
  </li>
 +
  <ol>
 +
    <li dir="ltr">
 +
      <p dir="ltr">1uL Template</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">2ul dI H2O</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">1ul 10uM forward primer (Tm XX C)*</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">1ul 10uM reverse Primer (Tm YY C)*</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">5ul 2x Green taq (<a href="http://www.promega.com/%7E/media/Files/Resources/Protocols/Product%20Information%20Sheets/G/GoTaq%20DNA%20Polymerase%20M300.pdf">GoTaq</a>)</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">Total 10uls</p>
 +
    </li>
 +
  </ol>
 +
  <li dir="ltr">
 +
    <p dir="ltr">PCR Reaction Program</p>
 +
  </li>
 +
  <ol>
 +
    <ol>
 +
      <ol>
 +
        <li dir="ltr">
 +
          <p dir="ltr">95 C - 30s</p>
 +
        </li>
 +
        <li dir="ltr">
 +
          <p dir="ltr">95 C - 10s</p>
 +
        </li>
 +
        <li dir="ltr">
 +
          <p dir="ltr">ZZ* C - 10s (important step)</p>
 +
        </li>
 +
        <li dir="ltr">
 +
          <p dir="ltr">72 C - 1min per kb target gene plus 10-15% (important step) </p>
 +
        </li>
 +
        <li dir="ltr">
 +
          <p dir="ltr">Repeat b-d 25x</p>
 +
        </li>
 +
        <li dir="ltr">
 +
          <p dir="ltr">72 C - 5 to 10min</p>
 +
        </li>
 +
        <li dir="ltr">
 +
          <p dir="ltr">10 or 4 C - forever</p>
 +
        </li>
 +
      </ol>
 +
    </ol>
 +
  </ol>
 +
</ol>
 +
<br />
 +
<p dir="ltr">Phusion polymerase:</p>
 +
<ol start="5">
 +
  <li dir="ltr">
 +
    <p dir="ltr">Setup Amplification PCR Reaction</p>
 +
  </li>
 +
  <ol>
 +
    <li dir="ltr">
 +
      <p dir="ltr">1uL Template</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">1uL 25mM dNTP's</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">10uL Phusion HF Buffer</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">1.0ul Forward Primer (Tm XX oC)*</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">1.0ul Reverse Primer (Tm YY oC)*</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">0.5uL Phusion polymerase</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">36.5uL diH2O</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">Total 50uls</p>
 +
    </li>
 +
  </ol>
 +
  <li dir="ltr">
 +
    <p dir="ltr">Amplification PCR Reaction Program</p>
 +
  </li>
 +
  <ol>
 +
    <li dir="ltr">
 +
      <p dir="ltr">98 C - 30s</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">98 C - 10s</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">ZZ* C - 10s (important step)</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">72 C - 30s per kb target gene plus 10-15% (important step) </p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">Repeat b-d 29-34x</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">72 C - 5 to 10min</p>
 +
    </li>
 +
    <li dir="ltr">
 +
      <p dir="ltr">10 or 4 C - forever</p>
 +
    </li>
 +
  </ol>
 +
</ol>
<ul>
<ul>
-
 
+
  <li dir="ltr">
-
for the drop down of plastic degradation
+
    <p dir="ltr">Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 5 C lower than XX or YY, whichever is lower. The temperature doesn&rsquo;t have to be exact (within 5 degrees or so).</p>
-
format <li><a href="https://2012.igem.org/Team:Washington/Plastics#Background">Background</a></li>
+
  </li>
-
 
+
-
 
+
</ul>
</ul>
-
</li>
+
<p dir="ltr">Refer for further details to NEB's online protocol for Phusion:</p>
-
-->
+
<p dir="ltr"><a href="http://www.neb.com/nebecomm/products/protocol87.asp">Protocol for a PCR Reaction using Phusion Hot Start DNA Polymerase</a</p>
-
 
+
</strong><br />
-
 
+
-
<li>
+
-
<a href="https://2013.igem.org/Team:Washington/LightSensing">Light Sensing Experiments</a>
+
-
<ul>
+
-
<li><a href="https://2013.igem.org/Team:Washington/LightSensing#Background">Background</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/LightSensing#Cloning">Cloning</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/LightSensing#Systems">Our Systems</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/LightSensing#Methods">Methods</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/LightSensing#Results">Results</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/LightSensing#Sources">Scientific Sources</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/LightSensing#Future">Future Plans</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/LightSensing#Parts">Parts Submitted</a></li>
+
-
</ul>
+
-
</li>
+
-
 
+
-
 
+
-
<li>
+
-
<a href="https://2013.igem.org/Team:Washington/ECOLIGHTTUNE">e.colightTune</a>
+
-
<ul>
+
-
<li><a href="https://2013.igem.org/Team:Washington/ECOLIGHTTUNE#Background">Background</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/ECOLIGHTTUNE#Usage">App</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/ECOLIGHTTUNE#Benefits">Methods</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/ECOLIGHTTUNE#Results">Results Summary</a></li>
+
-
</ul>
+
-
</li>
+
-
 
+
-
<li>
+
-
<a href="https://2013.igem.org/Team:Washington/Outreach">Outreach</a>
+
-
<ul>
+
-
<li><a href="https://2013.igem.org/Team:Washington/Outreach#Activities">Activities</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/Outreach#Pictures">Pictures</a></li>
+
-
</ul>
+
-
</li>
+
-
 
+
-
<li>
+
-
<a href="https://2013.igem.org/Team:Washington/Protocols">Protocols</a>
+
-
<ul>
+
-
<li><a href="https://2013.igem.org/Team:Washington/Protocols#General_Protocols">General</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/Protocols#Cloning_Protocols">Cloning</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/Protocols#Assay">Light assays</a></li>
+
-
</ul>
+
-
</li>
+
-
 
+
-
<li>
+
-
<a href="https://2013.igem.org/Team:Washington/MoreLinks">Links</a>
+
-
<ul>
+
-
<li><a href="https://2013.igem.org/Team:Washington/MoreLinks#Safety">Safety Form</a></li>
+
-
<li><a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2012&group=Washington">Parts Registry</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Washington/MoreLinks#Documentation">Parts Documentation</a></li>
+
-
 
+
-
</ul>
+
-
</li>
+
-
 
+
-
<li style="border-right:0; box-shadow:0px 0 0;">
+
-
<a href="https://2013.igem.org/Team:Washington/Funding">Funding</a>
+
-
</li>
+
-
 
+
-
</li>
+
-
                                                                                               
+
-
</ul>
+
-
</nav>
+
-
 
+
-
<script type="text/javascript" src="http://code.jquery.com/jquery-latest.min.js"></script>
+
-
<script type="text/javascript">
+
-
    $(function() {
+
-
if ($.browser.msie && $.browser.version.substr(0,1)<7)
+
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{
+
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$('li').has('ul').mouseover(function(){
+
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$(this).children('ul').css('visibility','visible');
+
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}).mouseout(function(){
+
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$(this).children('ul').css('visibility','hidden');
+
-
})
+
-
}
+
-
 
+
-
/* Mobile */
+
-
$('#igemuwmenu-wrap').prepend('<div id="igemuwmenu-trigger">igemuwmenu</div>');
+
-
$("#igemuwmenu-trigger").on("click", function(){
+
-
$("#menu").slideToggle();
+
-
});
+
-
 
+
-
// iPad
+
-
var isiPad = navigator.userAgent.match(/iPad/i) != null;
+
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if (isiPad) $('#igemuwmenu ul').addClass('no-transition');     
+
-
    });
+
-
// selection
+
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+
-
</script>
+
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+
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<script type="text/javascript">
+
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        $("#content").find("a[href='"+location.href+"']").each(function(){
+
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        $(this).find("a").addClass("selected")
+
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        })
+
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</script>
+
-
 
+
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<script type="text/javascript">
+
-
 
+
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  var _gaq = _gaq || [];
+
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  _gaq.push(['_setAccount', 'UA-26265208-1']);
+
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  _gaq.push(['_trackPageview']);
+
-
 
+
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  (function() {
+
-
    var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;
+
-
    ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';
+
-
    var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);
+
-
  })();
+
-
 
+
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</script>
+
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</html>
+
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+
-
 
+
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==''''''==
+
-
 
+
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+
-
 
+
-
<html>
+
-
 
+
-
<header>
+
-
<b>General Digestion Protocol in PCR Tubes</b>
+
-
</header>
+
-
 
+
-
<body>
+
-
 
+
-
<p>
+
-
 
+
-
2013 UW iGEM team uses Xba1 and Pst1 <a href = "https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1=%7B2DAF5F0E-59C4-4A5D-8809-6CEAD6BAE4B4%7D&enzyme2=%7B90EFE184-8442-45F0-8286-04CA22EEFFF2%7D/">combination</a>for most digestions
+
-
<br>
+
-
XbaI and SpeI together causes 50% inverted insertions and XbaI is less expensive than SpeI.
+
-
<br>
+
-
<br>
+
-
<b>Digestion:</b>
+
-
<br>
+
-
10-42.5 uL DNA (200ng/uL = 2ug) Use the highest concentration (>50ng/ul) DNA available.*
+
-
<br>
+
-
DI water to 50 uL (33.5ul for single digest or 32.5 uL for double digest)
+
-
<br>
+
-
5uL of 10X buffer (check http://www.neb.com/nebecomm/EnzymeFinderSearchByName.asp for a list of enzymes and associated buffers) Use CutSmart with all ‘HF’ enzymes and XbaI.<br>
+
-
0.5 uL BSA (If required. Not required for CutSmart. See above link) **<br>
+
-
1uL enzyme 1 (kept on ice or freezer block)<br>
+
-
For double digest add:<br>
+
-
<u>1uL enzyme 2 (kept on ice)***</u><br>
+
-
Total 50ul<br>
+
-
(Enzymes can added last so they go into a 1X buffer concentration)<br>
+
-
<u>Mix the reaction *well*</u> by pipetting up and down a few times (to mix the 50% glycerol).<br>
+
-
<u>Incubate at 37C</u> for the required amount of time, usually 1 hr. Digests overnight work well.<br>
+
-
<br>
+
-
 
+
-
If double digest is required, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp for buffer, temperature, BSA specifications.
+
-
<br><br>
+
-
 
+
-
*1 unit of enzyme is defined as the amount needed to fully digest 1 ug of DNA in a 50 ul reaction in 1 hour. You can calculate how much enzyme and time is needed to do ~10-fold overdigestion.<br>
+
-
**CutSmart contains BSA and is otherwise equivalent to Buffer 4 (except for DTT).<br>
+
-
***Note that the enzymes are stored in 50% glycerol. The total RXN needs to be no more than 5% glycerol to get effective digestion. Therefore, the volume of enzyme you add should be no more than 10% of the total reaction volume. <br>
+
-
****Thawing of buffer goes quickly when partially immersed in water. <br>
+
-
<br>
+
-
See also:<br>
+
-
<a href = "http://bio.freelogy.org/wiki/Restriction_Digestion/">Restriction Digestion - FreeBio</a>
+
-
 
+
-
<br><br><br>
+
-
Diagnostic Digest: use 10-20 uL Total
+
-
<br>
+
-
 
+
-
<table border="1">
+
-
<tr>
+
-
<td>15 ul</td>
+
-
<td>25 ul</td>
+
-
</tr>
+
-
<tr>
+
-
<td>10-20 uL of DNA<br>
+
-
2.5uL 10x Buffer<br>
+
-
(0.5uL of BSA)<br>
+
-
0.5 - 10 uL of Enz 1<br>
+
-
0.5 - 10 uL of Enz 2<br>
+
-
Fill to 25 uL with H20 <br>
+
-
25 uL Total</td>
+
-
<td>11 uL of DNA<br>
+
-
1.5 uL of buffer 10x<br>
+
-
(0.5 uL of BSA)<br>
+
-
0.5 - 1.0 uL of Enz 1<br>
+
-
0.5 - 1.0 uL of Enz 2<br>
+
-
15 uL Total</td>
+
-
</tr>
+
-
</table>
+
-
 
+
-
 
+
-
</p>
+
-
 
+
</body>
</body>
-
 
</html>
</html>

Latest revision as of 04:31, 27 September 2013


Untitled Document

General PCR Protocol

See also: PCR_GoTaq

Resuspending primers:

Protocol: Resuspending PCR primers


Working Stock:

(serves as a backup)

Dilute to 10 times the number on primer vial

10ul fwd primer 90ul DI water

10ul rev primer 90ul DI water


Notes for iGEM 2013 HSB lab:

We are using the GreenTaq polymerase 2X pre-mixed (GoTaq) for PCR.

Use the protocols below for Green Taq polymerase.

Use 50uls for PCR insert amplification. Use 10uls for analytical PCRs (screening).

If PCRing insert use 1ng/ul DNA concentration.


Amplification PCR with Green Taq polymerase:

  1. Setup Amplification PCR Reaction

    1. 1uL Template DNA

    2. 22ul deionized (dI) H2O

    3. 1ul 10uM forward primer (Tm XX C)*

    4. 1ul 10uM reverse Primer (Tm YY C)*

    5. 25ul 2x Green taq (GoTaq)

    6. Total 50ul

  2. Amplification PCR Reaction Program

        1. 95 C for 30s

        2. 95 C for 30s

        3. ZZ* C for 30s (important step)

        4. 72 C for 1min per kb target gene plus 10-15% (important step)

        5. Repeat 2-4 34x

        6. 72 C for 5 to 10min

        7. 4 C for forever (don’t forget to turn the machine off the next day)

  • Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 2 degrees C higher than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).


Analytical PCR with Green Taq polymerase:

  1. Setup PCR Reaction

    1. 1uL Template

    2. 2ul dI H2O

    3. 1ul 10uM forward primer (Tm XX C)*

    4. 1ul 10uM reverse Primer (Tm YY C)*

    5. 5ul 2x Green taq (GoTaq)

    6. Total 10uls

  2. PCR Reaction Program

        1. 95 C - 30s

        2. 95 C - 10s

        3. ZZ* C - 10s (important step)

        4. 72 C - 1min per kb target gene plus 10-15% (important step)

        5. Repeat b-d 25x

        6. 72 C - 5 to 10min

        7. 10 or 4 C - forever


Phusion polymerase:

  1. Setup Amplification PCR Reaction

    1. 1uL Template

    2. 1uL 25mM dNTP's

    3. 10uL Phusion HF Buffer

    4. 1.0ul Forward Primer (Tm XX oC)*

    5. 1.0ul Reverse Primer (Tm YY oC)*

    6. 0.5uL Phusion polymerase

    7. 36.5uL diH2O

    8. Total 50uls

  2. Amplification PCR Reaction Program

    1. 98 C - 30s

    2. 98 C - 10s

    3. ZZ* C - 10s (important step)

    4. 72 C - 30s per kb target gene plus 10-15% (important step)

    5. Repeat b-d 29-34x

    6. 72 C - 5 to 10min

    7. 10 or 4 C - forever

  • Tms of all primers must first be calculated after primer design. Annealing temperature (represented by ZZ above) should be 5 C lower than XX or YY, whichever is lower. The temperature doesn’t have to be exact (within 5 degrees or so).

Refer for further details to NEB's online protocol for Phusion:

Protocol for a PCR Reaction using Phusion Hot Start DNA Polymerase

Retrieved from "http://2013.igem.org/Team:Washington/GENERAL_PCR_PROTOCOL"