Team:HKUST/Protocols/Enzyme digestion

From 2009.igem.org

(Difference between revisions)
(New page: 4. Enzyme digestion Purpose: To cut the sequence with specific restriction enzyme Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal...)
 
(4 intermediate revisions not shown)
Line 1: Line 1:
-
4. Enzyme digestion
+
<html xmlns="http://www.w3.org/1999/xhtml">
-
  Purpose: To cut the sequence with specific restriction enzyme
+
<head>
-
  Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal Alkaline Phosphatase, for vector digestion and DNA to be ligated)
+
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" />
-
  Procedure:  
+
<link href="http://igem2009hkust.fileave.com/wiki/template/12092009/style.css " rel="stylesheet" type="text/css" />
-
   a.Add sufficient water to make it a 20 μL reaction.
+
<title>Salt and Soap template</title>
-
   b.Add 1 μg DNA, 2 μL buffer in total, 0.5 μL each restriction enzyme.
+
</head>
-
   c.Vortex and then spin down.
+
 
-
   d.Keep it in 37 °C incubator for one hour. (If needed, incubate for 1.5-2hours)
+
<bodyxx>
-
   e.If the substrate DNA is vector or pieces to be ligated, add 0.5 ul (for 20ul reaction) CIP after incubated for 1.5-2 hours, and then incubate at 37 °C for another 30mins.
+
<div id="containerxx">
-
   f.Check the result by gel electrophoresis.
+
<div id="headerxx">
-
  Tips:  
+
-
  •Restriction enzymes are easy to be denatured, so do not leave it at room temperature.
+
</div>
-
  •If two restriction enzymes must be added with different buffer, digest the DNA with respective restriction enzyme sequentially. Incubate for one hour after each enzyme added.
+
<div id="borderxx">
 +
<div id="mainxx">
 +
<div id="leftxx">
 +
<div id="menuxx">
 +
<ul>
 +
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li>
 +
 
 +
<b>
 +
<span style="color:green">
 +
<li>Main Parts</li>
 +
</span>
 +
</b>
 +
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li>
 +
 
 +
<b>
 +
<span style="color:green">
 +
<li>Resources</li>
 +
</span>
 +
</b>
 +
 
 +
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li>
 +
</ul>
 +
</div>
 +
<div id="menubottom">
 +
<ul>
 +
<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li>
 +
<li><a href="https://2009.igem.org/Team:Biosafety">Biosafety</a></li>
 +
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li>
 +
</ul>
 +
</div>
 +
</div>
 +
<div id="rightxx">
 +
<div class="contentlist"> <h3>a</h3>
 +
</div>
 +
<div class="contentxx">
 +
 
 +
<p>Enzyme digestion (vector, insert) </p>
 +
 
 +
<p> Purpose: To cut the sequence with specific restriction enzyme</p>
 +
Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal Alkaline Phosphatase, for vector digestion and DNA to be ligated)<br> <br>
 +
 
 +
<p>Procedure: </p>
 +
   a.Add sufficient water to make it a 20 μL reaction. <br>
 +
   b.Add 1 μg DNA, 2 μL buffer in total, 0.5 μL each restriction enzyme.<br>
 +
   c.Vortex and then spin down.<br>
 +
   d.Keep it in 37 °C incubator for one hour. (If needed, incubate for 1.5-2hours)<br>
 +
   e.If the substrate DNA is vector or pieces to be ligated, add 0.5 ul (for 20ul reaction) CIP after incubated for 1.5-2 hours, and then incubate at 37 °C for another 30mins.<br>
 +
   f.Check the result by gel electrophoresis.<br>
 +
<p> Tips: </p>
 +
A. Restriction enzymes are easy to be denatured, so do not leave it at room temperature.<br>
 +
B. If two restriction enzymes must be added with different buffer, digest the DNA with respective restriction enzyme sequentially. Incubate for one hour after each enzyme added.</p>
 +
<ul>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel
 +
electrophoresis</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to
 +
yeast</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA
 +
extraction</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li>
 +
 
 +
</ul>
 +
 +
</div>
 +
<div class="productxx">
 +
 +
<div class="clear"></div>
 +
</div>
 +
</div>
 +
<div class="clear"></div>
 +
</div>
 +
</div>
 +
<div id="footerxx">
 +
<div id="copyright">
 +
<span> iGEM 2009 <br /> </span>
 +
</div>
 +
<div id="payment"><img src="http://igem2009hkust.fileave.com/wiki/template/12092009/images/HKUSTLogo.jpg" alt="HKUST" /></div>
 +
</div>
 +
<div id="footerend"></div>
 +
</div>
 +
</bodyxx>
 +
</html>

Latest revision as of 09:55, 20 October 2009

Salt and Soap template

a

Enzyme digestion (vector, insert)

Purpose: To cut the sequence with specific restriction enzyme

Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal Alkaline Phosphatase, for vector digestion and DNA to be ligated)

Procedure:

a.Add sufficient water to make it a 20 μL reaction.
b.Add 1 μg DNA, 2 μL buffer in total, 0.5 μL each restriction enzyme.
c.Vortex and then spin down.
d.Keep it in 37 °C incubator for one hour. (If needed, incubate for 1.5-2hours)
e.If the substrate DNA is vector or pieces to be ligated, add 0.5 ul (for 20ul reaction) CIP after incubated for 1.5-2 hours, and then incubate at 37 °C for another 30mins.
f.Check the result by gel electrophoresis.

Tips:

A. Restriction enzymes are easy to be denatured, so do not leave it at room temperature.
B. If two restriction enzymes must be added with different buffer, digest the DNA with respective restriction enzyme sequentially. Incubate for one hour after each enzyme added.

HKUST

Retrieved from "http://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"