Team:BIOTEC Dresden/ProtocolsAHL assay

From 2010.igem.org

(Difference between revisions)
(New page: {{Biotec_Dresden/Header}} <html> <body> <div id="content_prim"> <div id="materials"> <h2>Materials</h2> <ul> <li>Escherichia coli DH5 alpha chemical competent cells</li> <li>1mL SOC (room ...)
(Removing all content from page)
 
(5 intermediate revisions not shown)
Line 1: Line 1:
-
{{Biotec_Dresden/Header}}
 
-
<html>
 
-
<body>
 
-
<div id="content_prim">
 
-
<div id="materials">
 
-
<h2>Materials</h2>
 
-
<ul>
 
-
<li>Escherichia coli DH5 alpha chemical competent cells</li>
 
-
<li>1mL SOC (room temperature) for each reaction</li>
 
-
<li>Ice</li>
 
-
<li>Plasmid DNA</li>
 
-
<li>Heating block</li>
 
-
<li>1.5ml microfuge tube</li>
 
-
<li>LB-agar plate with corresponding antibiotic</li>
 
-
</ul>
 
-
</div>
 
-
<div class="visualClear"></div>
 
-
<div id="procedure">
 
-
<h2>Procedure</h2>
 
-
<ul>
 
-
<li>Chill DNA samples and tubes on ice.
 
-
<li>Place LB-agar plates in <span class="markup temp">37°C</span> incubator to warm.
 
-
<li>Remove chemical competent cells from <span class="markup temp">-80°C</span> freezer and thaw on ice. Alternatively, freshly prepared chemical competent cells may be used immediately.</li>
 
-
<li>Dial a P2 pipetman to either <span class="markup volume">1 or 2μL</span> depending on the salt content of your DNA sample. Use <span class="markup volume">2μL</span> for samples that have been purified in some way.</li>
 
-
<li>Dial a P200 pipetman to <span class="markup volume">50μL</span> or whatever volume of chemical competent cells you want to use; usually <span class="markup volume">20-50μL</span> .</li>
 
-
<li>Pipet <span class="markup volume">1-2μL</span> of DNA sample and add to chemical competent cells. Swirl tip around gently in cells to mix DNA and cells. Do not pipet up and down.</li>
 
-
<li>Place the mix back on <span class="markup temp">ice</span> for <span class="markup time">30 mins</span>.</li>
 
-
<li>Pulse the cells with a heat shock by placing the microfuge tubes in a heating block for <span class="markup time">45 seconds</span> at <span class="markup temp">42°C</span>.</li>
 
-
<li>Place sample on ice for <span class="markup time">2 mins</span> and then add SOC medium. This step should be done as quickly as possible to prevent cells from dying off.</li>
 
-
<li>Chill sample on ice for <span class="markup time">2 mins</span> to permit the cells to recover.</li>
 
-
<li>Transfer eppendorf tube to <span class="markup temp">37°C</span> incubator and shake to promote aeration. Incubate for <span class="markup time">1 hr</span> to permit expression of antibiotic resistance gene.</li>
 
-
<li>Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic.</li>
 
-
</ul>
 
-
</div>
 
-
<h2>Reference</h2>
 
-
<p>adapted from <a href="http://openwetware.org/wiki/Knight:Electroporation">http://openwetware.org/wiki/Knight:Electroporation</a></p>
 
-
</div>
 
-
</body>
 
-
</html>
 
-
 
-
[[Category:BIOTEC Dresden/Protocol|AHL assay]]
 
-
{{Biotec_Dresden/Bottom}}
 

Latest revision as of 02:39, 28 October 2010