Team:HKUST/Protocols/Cell lysis

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(New page: 12.Cell lysis Purpose: to lyse E.coli cell to get linearized ligated plasmid. Materials: cultured transformed E.coli colony, phenol, chloroform, ddH2O Procedure: a.Add 40μL ...)
 
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12.Cell lysis
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  Purpose:  to lyse E.coli cell to get linearized ligated plasmid.
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<head>
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   Materials: cultured transformed E.coli colony, phenol, chloroform, ddH2O
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<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" />
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  Procedure:  
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<link href="http://igem2009hkust.fileave.com/wiki/template/12092009/style.css " rel="stylesheet" type="text/css" />
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   a.Add 40μL ddH2O in a 1.5mL microcentrigfuge tube.
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<title>Salt and Soap template</title>
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   b.Use a toothpick to pick a colony and dissolve it in the water.
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   c.Add 20μL phenol and 20μL chloroform.
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   d.Scrape the tube to mix the solution. A well mixed white solution can be seen after this step.
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   e.Centrifuge it for 10mins (13,000 rpm).
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   f.Place the supernatant in another tube.
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   g.Load 10μL to test the result by gel electrophoresis.
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  Tips:
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  •Ignite the fire for sterile environment when transforming the colony.
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  Safety Tips:
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  •Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical.
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<div id="leftxx">
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B 
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<div id="menuxx">
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  •Be careful with the fire, and extinguish it after use.
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<ul>
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li>
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<div class="contentlist"> <h3>a</h3>
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</div>
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<div class="contentxx">
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<p>Cell lysis</p>
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<p> Purpose:  to lyse E.coli cell to get linearized ligated plasmid.</p>
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   Materials: cultured transformed E.coli colony, phenol, chloroform, ddH2O <br> <br>
 +
 
 +
<p>Procedure: </p>
 +
   a.Add 40μL ddH2O in a 1.5mL microcentrigfuge tube. <br>
 +
   b.Use a toothpick to pick a colony and dissolve it in the water. <br>
 +
   c.Add 20μL phenol and 20μL chloroform. <br>
 +
   d.Scrape the tube to mix the solution. A well mixed white solution can be seen after this step. <br>
 +
   e.Centrifuge it for 10mins (13,000 rpm). <br>
 +
   f.Place the supernatant in another tube. <br>
 +
   g.Load 10μL to test the result by gel electrophoresis. <br> <br>
 +
<p> Tips: </p>
 +
Ignite the fire for sterile environment when transforming the colony.<br>
 +
<p> Safety tips: </p>
 +
1. Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical.<br>
 +
2. Be careful with the fire, and extinguish it after use. <br><br>
 +
 
 +
<ul>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel
 +
electrophoresis</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to
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yeast</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA
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extraction</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li>
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</ul>
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</div>
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<div class="productxx">
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<div class="clear"></div>
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<div id="copyright">
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<span> iGEM 2009 <br /> </span>
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</div>
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<div id="payment"><img src="http://igem2009hkust.fileave.com/wiki/template/12092009/images/HKUSTLogo.jpg" alt="HKUST" /></div>
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</div>
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<div id="footerend"></div>
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</div>
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</bodyxx>
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</html>

Latest revision as of 09:57, 20 October 2009

Salt and Soap template

a

Cell lysis

Purpose: to lyse E.coli cell to get linearized ligated plasmid.

Materials: cultured transformed E.coli colony, phenol, chloroform, ddH2O

Procedure:

a.Add 40μL ddH2O in a 1.5mL microcentrigfuge tube.
b.Use a toothpick to pick a colony and dissolve it in the water.
c.Add 20μL phenol and 20μL chloroform.
d.Scrape the tube to mix the solution. A well mixed white solution can be seen after this step.
e.Centrifuge it for 10mins (13,000 rpm).
f.Place the supernatant in another tube.
g.Load 10μL to test the result by gel electrophoresis.

Tips:

Ignite the fire for sterile environment when transforming the colony.

Safety tips:

1. Phenol is a strong toxin, make sure to wear gloves and be careful with this chemical.
2. Be careful with the fire, and extinguish it after use.

HKUST

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