Team:UC Davis/protocols/transformation.html

From 2010.igem.org

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                 <li>polypropylene tubes</li>
                 <li>polypropylene tubes</li>
                 <li>ice </li>
                 <li>ice </li>
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                 <li>ligation product</li>
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                 <li>DNA to be transformed; can consist of <a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">ligation product</a>, <a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">miniprep sample</a>, or <a href="https://2010.igem.org/Team:UC_Davis/protocols/hydration.html" class="help">stock parts from the registry</a></li>
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                <li>water bath</li>
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                 <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/luriabroth.html" class="help">luria broth</a></li>
                 <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/luriabroth.html" class="help">luria broth</a></li>
                 <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/agarplates.html" class="help">nutrient agar plates</a></li>
                 <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/agarplates.html" class="help">nutrient agar plates</a></li>
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                <li>Sterilized glass beads </li>
                 </ul><p>
                 </ul><p>
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 +
<a name="extranotes"><h1>Extra Notes</h1></a>
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Do not keep cells out for too long. It is vital that the cells undergo a rapid temperature change (from very cold to very hot suddenly) in order for crack pores in the cell wall.
<a name="procedure"><h1>Procedure</h1></a>
<a name="procedure"><h1>Procedure</h1></a>
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<li>Thaw DH5α cells on ice for 10 minutes. </li>
<li>Thaw DH5α cells on ice for 10 minutes. </li>
<li>Transfer 50μL of DH5α cells to chill, sterile polypropylene tubes. </li>
<li>Transfer 50μL of DH5α cells to chill, sterile polypropylene tubes. </li>
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<li>Transfer 1μL of ligation product into tubes.</li>
+
<li>Transfer 1μL of DNA into the tubes with the cells.</li>
<li>Incubate on ice for 30 minutes.</li>
<li>Incubate on ice for 30 minutes.</li>
<li>Gently transfer tubes to 42°C water bath for 90s (for heat shock) </li>
<li>Gently transfer tubes to 42°C water bath for 90s (for heat shock) </li>
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<li>Incubate at 37°C for 1 hour in a shaker. </li>
<li>Incubate at 37°C for 1 hour in a shaker. </li>
<li>Plate 200μL of each sample on <a href="https://2010.igem.org/Team:UC_Davis/protocols/luriabroth.html" class="help">nutrient agar plates.</a> (Be sure to choose the plates with the antibiotic that your E. Coli is resistant to.) </li>
<li>Plate 200μL of each sample on <a href="https://2010.igem.org/Team:UC_Davis/protocols/luriabroth.html" class="help">nutrient agar plates.</a> (Be sure to choose the plates with the antibiotic that your E. Coli is resistant to.) </li>
 +
<li>Spread the sample evenly by plating with sterilized glass beads. Empty the plate of glass beads afterwards.  </li>
<li>Incubate overnight at 37°C. </li>
<li>Incubate overnight at 37°C. </li>
</ul>
</ul>
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<ul>
<ul>
<li><a href="#materials" class="help">Materials</a></li>
<li><a href="#materials" class="help">Materials</a></li>
 +
<li><a href="#extranotes" class="help">Extra Notes</a></li>
<li><a href="#procedure" class="help">Procedure</a></li>
<li><a href="#procedure" class="help">Procedure</a></li>
<li><a href="#purpose" class="help">Purpose</a></li>
<li><a href="#purpose" class="help">Purpose</a></li>

Latest revision as of 17:38, 13 September 2010

E. Coli Transformation

Materials

You will need:

Extra Notes

Do not keep cells out for too long. It is vital that the cells undergo a rapid temperature change (from very cold to very hot suddenly) in order for crack pores in the cell wall.

Procedure
  • Thaw DH5α cells on ice for 10 minutes.
  • Transfer 50μL of DH5α cells to chill, sterile polypropylene tubes.
  • Transfer 1μL of DNA into the tubes with the cells.
  • Incubate on ice for 30 minutes.
  • Gently transfer tubes to 42°C water bath for 90s (for heat shock)
  • Transfer tubes back on ice for 2 minutes.
  • Add 800μL of luria broth into each tube.
  • Incubate at 37°C for 1 hour in a shaker.
  • Plate 200μL of each sample on nutrient agar plates. (Be sure to choose the plates with the antibiotic that your E. Coli is resistant to.)
  • Spread the sample evenly by plating with sterilized glass beads. Empty the plate of glass beads afterwards.
  • Incubate overnight at 37°C.

Purpose

To insert plasmids into E. Coli.

References
  • Notes by David Larsen

We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!

We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)

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